Intramolecular H-bonds govern the recognition of a flexible peptide by an antibody

Intramolecular H-bonds govern the recognition of a flexible peptide by an antibody Abstract Molecular recognition is a fundamental event at the core of essentially every biological process. In particular, intermolecular H-bonds have been recognized as key stabilizing forces in antibody–antigen interactions resulting in exquisite specificity and high affinity. Although equally abundant, the role of intramolecular H-bonds is far less clear and not universally acknowledged. Herein, we have carried out a molecular-level study to dissect the contribution of intramolecular H-bonds in a flexible peptide for the recognition by an antibody. We show that intramolecular H-bonds may have a profound, multifaceted and favorable effect on the binding affinity by up to 2 kcal mol−1 of free energy. Collectively, our results suggest that antibodies are fine tuned to recognize transiently stabilized structures of flexible peptides in solution, for which intramolecular H-bonds play a key role. antibody–antigen interaction, molecular dynamics simulations, molecular recognition, transient folding, unstructured peptide Immunoglobulins (antibodies) are capable to exquisitely recognize an unlimited number of different molecular entities including proteins, peptides and small molecules all termed antigens (1). In addition to their key role in the immune system, antibodies have been widely employed as detection reagents in biological studies, therapeutic agents in the clinical stage, or biosensors in biotechnological devices for a long time (2–4). In order to improve the efficiency in their therapeutic use, antibodies with even higher affinity and selectivity are desired (5, 6). To that end, two basic approaches are taken, one that focuses on the antibody itself and a second one that relies on the preparation of sophisticated antigens eliciting antibodies with more favourable properties using rational design, random mutagenesis or a combination of both (7, 8). But how are those antibodies obtained on the first place? Short peptides are often employed to elicit antibodies through immunization procedures (9) such as in vaccines and in various protein-related detection reagents (10, 11). Intriguingly, short peptides generally do not display a stable secondary structure in solution (12, 13), raising the question of how antibody matures to recognize such flexible antigens. Despite the flexible nature of these antigens in solution, different antibodies recognizing the same peptide exhibit comparable antibody–peptide interaction contacts, and the bound peptide the same secondary structure conformation (14–16). The structural convergence of peptide–antibody complexes seems to contradict the nature of peptide antigens in solution, reported to be flexible and unstructured (17–20). Previously, it was hypothesized that the formation of intramolecular hydrogen bonds (H-bonds) could stabilize certain conformations of the peptide and therefore contributes to the affinity of the peptide–antibody complex (14, 21), although to the best of our knowledge, no formal demonstration of it has been reported to date. Herein, we have addressed the relationship between the structural ensemble of peptide antigens in solution, the thermodynamic properties upon interaction to a specific antibody, and the structure in the bound form. In particular, we have focused on the question of whether intramolecular H-bonds may influence the binding affinity of a flexible peptide to an antibody. By employing a model system composed of the antibody E38035-4B08-222 (heretofore 4B08) and the peptide DINYYTSEP (pep1) derived from N-terminal region of CC chemokine receptor 5 (CCR5), we revealed the role of the peptide’s intramolecular hydrogen bonds in binding to the antibody. Molecular dynamics (MD) simulations, high-resolution X-ray crystallography and calorimetry demonstrated that intramolecular H-bonds indeed play an critical role in at least two important stages of the binding coordinate: the conformation of the peptide in solution and the stabilization of the complex. Materials and Methods Immunization and cloning of 4B08 The peptide employed in this study corresponded to residues 10–19 of the human protein CCR5 (Uniprot entry number P51681) of sequence DINYYTSEP that we termed pep1. This peptide was chemically synthesized with an NH2-Mini-PEG3 at the N-terminal (Peptide Institute, Osaka, Japan). Pep1 was conjugated with Keyhole limpet hemocyanin (KLH, Thermo Fisher Scientific) using glutaraldehyde. Female MRL/lpr mice (SLC, Shizuoka, Japan) were immunized intraperitoneally (i.p.) with 80 µg of Pep1-KLH in Freund's Complete Adjuvant. Second immunization was also performed i.p. using 80 µg of Pep1-KLH in Freund’s incomplete adjuvant and final immunization was performed i.p. with 80 µg of Pep1-KLH in saline at 2-week intervals. Additionally, the mice were administered 50 µg of wild-type budded baculovirus subcutaneously for adjuvant effects at all the step of immunization. Spleen cells were isolated 3 days after the last immunization and fused with sp2/0-Ag14 myeloma cells by conventional methods (22). Hybridoma culture supernatants were screened for antibody production by the enzyme-linked immunosorbent assay with pep1 coated 96-well microtiter plates. To select hybridomas secreting high-affinity antibodies against pep1, the cultures were analyzed using a Biacore3000 (GE healthcare) with pep1 immobilized on a carboxymethyl CM5 sensor chip (GE healthcare). The selected cells were isolated by limiting dilution to establish monoclonal hybridoma cell lines producing antibodies against pep1. Preparation of expression vector The DNA sequence of the single-chain fragment variable (scFv) region of antibody E38035-4B08-222 (heretofore 4B08) was subcloned into pRA2 vector (23). Restriction sites for the insertion were NcoI and EagI (heavy chain variable region) or EcoRV and SacII (light chain variable region). A flexible linker composed of four repeating units of the sequence GGGGS was introduced between the heavy and light chains. The pelB leader preceded the N-terminus and a His6 tag was introduced at the C-terminus to facilitate protein expression. Expression of recombinant scFv Escherichia coli strain BL21(DE3) carrying the expression vector of scFv 4B08 was grown overnight at 28°C, 170 rpm in 3 ml of Luria-Bertani medium. The cells were diluted into 1 L of Luria-Bertani medium and cultured at 28°C and 140 rpm until the OD600 reached a value of 0.6–0.8. At that point isopropyl β-D-1-thiogalactopyranoside was added to the cell culture to a final concentration of 0.5 mM and the cell culture was left overnight at 28°C. Cells were harvested by centrifugation at 7,000 × g for 20 min at 4°C. The cell pellet was resuspended in 40 ml of TRIS buffer (20 mM Tris, 500 mM NaCl, pH 8.0) supplemented with 5 mM imidazole, and the cells lysed with a ultrasonic cell-disrupting UD-201 instrument (TOMY, Japan). The cell lysate was subsequently centrifuged at 4°C, 7,000 × g for 20 min. The supernatant was discarded, and the collected pellet was washed with 20 ml of buffer supplemented with 1% (v/v) Triton X-100 for 30 min, followed by equivalent washing steps with acetone and deionized water, respectively. Refolding and purification of scFv Inclusion bodies were incubated overnight in 20 ml of solubilization buffer composed of 6 M guanidinium chloride, 20 mM TRIS, 500 mM NaCl, at pH 8.0 and 5 mM imidazole. The suspension was centrifuged at 40,000 × g for 30 min at 4°C and the supernatant filtered and loaded onto an affinity column of Ni-NTA agarose (Qiagen) equilibrated with solubilization buffer. The bound fraction to the column was first washed with buffer containing 20 mM imidazole and subsequently eluted with buffer containing 100 and 500 mM imidazole. The eluted scFv was diluted to 10 µM and refolded by stepwise dialysis in TRIS buffer with decreasing concentrations of guanidinium chloride (3 M to 0 M) (24). Refolded 4B08 was concentrated using an Ni-NTA agarose column; the scFv-loaded column was washed with TRIS buffer and scFv was eluted with TRIS buffer containing 500 mM imidazole. Further purification was conducted by size exclusion chromatography (AKTA purifier with Hiload 16/600 superdex 75 pg, GE healthcare) with TRIS buffer at a flow rate of 1.0 ml min−1 at 4°C. Peptides Synthetic peptides were purchased from Medical & Biological Laboratories Co., Ltd. (Nagoya, Japan) or Scrum Inc. (Tokyo, Japan). Peptides had no modifications, and purity was always greater than 95%. The peptides were used without further purification. Concentration of peptide was determined by its absorbance at 280 nm. The sequences of the peptides employed were: pep1, DINYYTSEP; N3A, DIAYYTSEP; Y4A, DINAYTSEP; Y5A, DINYATSEP; T6A, DINYYASEP; S7A, DINYYTAEP; E8A, DINYYTSAP; P9A, DINYYTSEA. Isothermal titration calorimetry (ITC) Thermodynamic parameters of the peptide–antibody interactions were determined in an iTC200 nano-calorimeter instrument (Malvern). The antibody was dialyzed overnight in phosphate buffered saline (PBS composed of 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.76 mM KH2PO4, and pH 7.4). Peptides were dissolved in the same PBS buffer employed to dialyze the scFv. The concentration of 4B08 (in the cell) and peptide (in the syringe) was 9–11 μM and 100–120 μM, except for the experiments employing peptides E8A and T6A. In the titration with E8A, the concentrations of 4B08 and peptide were increased to 30 μM and 320 μM, respectively. For the experiment with mutant T6A, the concentration of antibody and peptide were 40 μM and 500 μM, respectively. Titrations were carried out at 25°C with a reference power of 5 μcal s−1 and a stirring rate of 1,000 rpm. Each experiment consisted of a single 0.5-μL injection of peptide followed by 18 additional injections of 2.0 μL each with an interval between injections of 180 s. Thermodynamic parameters were calculated with the program ORIGIN 7.0 (OriginLab) using a single-site binding model. Crystallization The antibody 4B08 dialyzed overnight in MES buffer (10 mM MES, 30 mM NaCl, pH 5.9) at final concentration of 100 μM was mixed with peptide solubilized in the same MES buffer in which scFv was dialyzed overnight at a final concentration of 200 μM. Crystals of the peptide–antibody complex were obtained by mixing equal volumes of protein solution (2 μl) and crystallization solution [1.8–2.4 M (NH4)2SO4, 0.1 M Bis-Tris, pH 5.9–6.5] at 20°C by the hanging-drop vapor diffusion method. Well-developed crystals of the complexes between antibody and pep1 or T6A were harvested from the wells containing 1.8 M (NH4)2SO4, 0.1 M Bis-Tris at pH 6.5, and those of the complex between N3A and 4B08 were harvested from the solution containing 2.2 M (NH4)2SO4, 0.1 M Bis-Tris at pH 6.2. The crystals were briefly transferred to the same solution supplemented with 30% glycerol prior to storage in liquid N2 until data collection. Data collection and refinement Diffraction data were collected at beamlines BL5A and AR-NE3A both at the Photon Factory (Tsukuba, Japan) under cryogenic conditions (100 K). Data were indexed, integrated with MOSFLM (25), and scaled with SCALA or AIMLESS (26). The structures of the complex between pep1 and scFv 4B08 were determined by the method of molecular replacement as described previously for an anti-P-cadherin antibody (27) with PHASER (28). Refinement was performed with COOT (29) and REFMAC5 (30). Structural validation was performed with COOT and PROCHECK (31). Data collection and refinement statistics are summarized in Table I. Although four antibody–peptide complexes were found in the asymmetric unit of the complexes between antibody and pep1 or T6A, only three were used for further analysis. The fourth copy showed essentially the same similar structural features to the other three ones but dynamic disorder in the peptide precluded unambiguous characterization. Table I. Data collection and refinement statisticsa Data collection  4B08 + pep1  4B08 + N3A  4B08 + T6A  Space group  P 1 21 1  P 2 21 21  P 1 21 1  Unit cell            a, b, c (Å)  80.5, 73.0, 87.0  61.6, 81.9, 114.2  80.5, 72.9, 86.5      α, β, γ (°)  90.0, 114.3, 90.0  90.0, 90.0, 90.0  90.0, 114.2, 90.0  Resolution (Å)  33.1–1.35  37.3–1.96  33.1–1.35  Wavelength  1.000  1.000  1.000  Observations  723,496 (103,749)  309,459 (15,814)  853,946 (97,855)  Unique reflections  193,517 (27,587)  40,361 (2,555)  197,862 (27,517)  Rmerge. (%)  8.3 (51.9)  12.9 (42.5)  8.1 (39.2)  CC1/2  0.995 (0.759)  0.997 (0.939)  0.996 (0.843)  I/σI  9.7 (2.6)  10.0 (3.7)  10.6 (2.7)  Multiplicity  3.7 (3.8)  7.7 (6.2)  4.3 (3.6)  Completeness (%)  96.5 (94.4)  95.6 (87.1)  99.1 (94.9)  Refinement statistics        Resolution (Å)  33.1–1.35  37.3–1.96  33.1–1.35  Rwork/Rfree (%)b  13.6/18.4  20.3/25.8  13.5/17.3  No. atoms (protein)  7,554  3,742  7,330  No. atoms (peptide)  322c  134  272c  No. atoms (solvent)  888  364  952  No. atoms (other)  62  40  40  B-factor (protein) (Å2)  14.6  16.3  16.8  B-factor (peptide) (Å2)  20.1  15.1  26.8  B-factor (solvent) (Å2)  23.9  21.9  26.0  B-factor (other) (Å2)  30.9  37.7  32.3  Ramachandran plot            Preferred regions (%)  89.9  90.0  90.2      Allowed regions (%)  9.6  9.5  9.3      Outliers (%)  0.5  0.5  0.5  RMSD bond (Å)  0.014  0.013  0.014  RMSD angle (°)  1.76  1.55  1.71  PDB entry code  5YD3  5YD5  5YD4  Data collection  4B08 + pep1  4B08 + N3A  4B08 + T6A  Space group  P 1 21 1  P 2 21 21  P 1 21 1  Unit cell            a, b, c (Å)  80.5, 73.0, 87.0  61.6, 81.9, 114.2  80.5, 72.9, 86.5      α, β, γ (°)  90.0, 114.3, 90.0  90.0, 90.0, 90.0  90.0, 114.2, 90.0  Resolution (Å)  33.1–1.35  37.3–1.96  33.1–1.35  Wavelength  1.000  1.000  1.000  Observations  723,496 (103,749)  309,459 (15,814)  853,946 (97,855)  Unique reflections  193,517 (27,587)  40,361 (2,555)  197,862 (27,517)  Rmerge. (%)  8.3 (51.9)  12.9 (42.5)  8.1 (39.2)  CC1/2  0.995 (0.759)  0.997 (0.939)  0.996 (0.843)  I/σI  9.7 (2.6)  10.0 (3.7)  10.6 (2.7)  Multiplicity  3.7 (3.8)  7.7 (6.2)  4.3 (3.6)  Completeness (%)  96.5 (94.4)  95.6 (87.1)  99.1 (94.9)  Refinement statistics        Resolution (Å)  33.1–1.35  37.3–1.96  33.1–1.35  Rwork/Rfree (%)b  13.6/18.4  20.3/25.8  13.5/17.3  No. atoms (protein)  7,554  3,742  7,330  No. atoms (peptide)  322c  134  272c  No. atoms (solvent)  888  364  952  No. atoms (other)  62  40  40  B-factor (protein) (Å2)  14.6  16.3  16.8  B-factor (peptide) (Å2)  20.1  15.1  26.8  B-factor (solvent) (Å2)  23.9  21.9  26.0  B-factor (other) (Å2)  30.9  37.7  32.3  Ramachandran plot            Preferred regions (%)  89.9  90.0  90.2      Allowed regions (%)  9.6  9.5  9.3      Outliers (%)  0.5  0.5  0.5  RMSD bond (Å)  0.014  0.013  0.014  RMSD angle (°)  1.76  1.55  1.71  PDB entry code  5YD3  5YD5  5YD4  aStatistical values given in parenthesis refer to the highest resolution bin (see also Figs 1 and 5). bRfree was calculated as Rwork taking 2–4% of data not included in the refinement. cThe greater number of modeled atoms of pep1 with respect to T6A reflect the existence of alternative conformations. Table I. Data collection and refinement statisticsa Data collection  4B08 + pep1  4B08 + N3A  4B08 + T6A  Space group  P 1 21 1  P 2 21 21  P 1 21 1  Unit cell            a, b, c (Å)  80.5, 73.0, 87.0  61.6, 81.9, 114.2  80.5, 72.9, 86.5      α, β, γ (°)  90.0, 114.3, 90.0  90.0, 90.0, 90.0  90.0, 114.2, 90.0  Resolution (Å)  33.1–1.35  37.3–1.96  33.1–1.35  Wavelength  1.000  1.000  1.000  Observations  723,496 (103,749)  309,459 (15,814)  853,946 (97,855)  Unique reflections  193,517 (27,587)  40,361 (2,555)  197,862 (27,517)  Rmerge. (%)  8.3 (51.9)  12.9 (42.5)  8.1 (39.2)  CC1/2  0.995 (0.759)  0.997 (0.939)  0.996 (0.843)  I/σI  9.7 (2.6)  10.0 (3.7)  10.6 (2.7)  Multiplicity  3.7 (3.8)  7.7 (6.2)  4.3 (3.6)  Completeness (%)  96.5 (94.4)  95.6 (87.1)  99.1 (94.9)  Refinement statistics        Resolution (Å)  33.1–1.35  37.3–1.96  33.1–1.35  Rwork/Rfree (%)b  13.6/18.4  20.3/25.8  13.5/17.3  No. atoms (protein)  7,554  3,742  7,330  No. atoms (peptide)  322c  134  272c  No. atoms (solvent)  888  364  952  No. atoms (other)  62  40  40  B-factor (protein) (Å2)  14.6  16.3  16.8  B-factor (peptide) (Å2)  20.1  15.1  26.8  B-factor (solvent) (Å2)  23.9  21.9  26.0  B-factor (other) (Å2)  30.9  37.7  32.3  Ramachandran plot            Preferred regions (%)  89.9  90.0  90.2      Allowed regions (%)  9.6  9.5  9.3      Outliers (%)  0.5  0.5  0.5  RMSD bond (Å)  0.014  0.013  0.014  RMSD angle (°)  1.76  1.55  1.71  PDB entry code  5YD3  5YD5  5YD4  Data collection  4B08 + pep1  4B08 + N3A  4B08 + T6A  Space group  P 1 21 1  P 2 21 21  P 1 21 1  Unit cell            a, b, c (Å)  80.5, 73.0, 87.0  61.6, 81.9, 114.2  80.5, 72.9, 86.5      α, β, γ (°)  90.0, 114.3, 90.0  90.0, 90.0, 90.0  90.0, 114.2, 90.0  Resolution (Å)  33.1–1.35  37.3–1.96  33.1–1.35  Wavelength  1.000  1.000  1.000  Observations  723,496 (103,749)  309,459 (15,814)  853,946 (97,855)  Unique reflections  193,517 (27,587)  40,361 (2,555)  197,862 (27,517)  Rmerge. (%)  8.3 (51.9)  12.9 (42.5)  8.1 (39.2)  CC1/2  0.995 (0.759)  0.997 (0.939)  0.996 (0.843)  I/σI  9.7 (2.6)  10.0 (3.7)  10.6 (2.7)  Multiplicity  3.7 (3.8)  7.7 (6.2)  4.3 (3.6)  Completeness (%)  96.5 (94.4)  95.6 (87.1)  99.1 (94.9)  Refinement statistics        Resolution (Å)  33.1–1.35  37.3–1.96  33.1–1.35  Rwork/Rfree (%)b  13.6/18.4  20.3/25.8  13.5/17.3  No. atoms (protein)  7,554  3,742  7,330  No. atoms (peptide)  322c  134  272c  No. atoms (solvent)  888  364  952  No. atoms (other)  62  40  40  B-factor (protein) (Å2)  14.6  16.3  16.8  B-factor (peptide) (Å2)  20.1  15.1  26.8  B-factor (solvent) (Å2)  23.9  21.9  26.0  B-factor (other) (Å2)  30.9  37.7  32.3  Ramachandran plot            Preferred regions (%)  89.9  90.0  90.2      Allowed regions (%)  9.6  9.5  9.3      Outliers (%)  0.5  0.5  0.5  RMSD bond (Å)  0.014  0.013  0.014  RMSD angle (°)  1.76  1.55  1.71  PDB entry code  5YD3  5YD5  5YD4  aStatistical values given in parenthesis refer to the highest resolution bin (see also Figs 1 and 5). bRfree was calculated as Rwork taking 2–4% of data not included in the refinement. cThe greater number of modeled atoms of pep1 with respect to T6A reflect the existence of alternative conformations. MD simulations MD simulations were performed using the NAMD 2.9 package (32) with the CHARMM22 force field (33) and the CMAP backbone energy correction (34). The initial coordinates of the WT peptide and the variants were linear conformations where all the phi/psi angles were set to 180° and the N-terminal and C-terminal residues were capped with acetyl and N-methyl amide groups, respectively. These isolated linear peptide structures were solvated with TIP3P water (35) in a rectangular box such that the minimum distance to the edge of the box is 12 Å under periodic boundary conditions. Sodium or chloride ions were included to neutralize the protein charge, then further ions were added corresponding to a salt solution of concentration 0.14 M. The time step was set to 2 fs throughout the simulations. A cut-off distance of 10 Å for Coulomb and van der Waals interactions was employed. Coulomb interactions were evaluated through the particle mesh Ewald method (36). The systems were first energy minimized by the conjugate gradient method (5,000 steps) and were gradually heated from 0 K to 298 K over 50 ps. Subsequently, the equilibration and production runs were performed five times for each peptide in the NPT ensembles at 298 K for 200 ns (1 μs in total). Coordinate frames were saved every 10 ps from the trajectory. Results High-resolution crystal structure of the antibody–peptide complex The high-resolution crystal structure of the complex of the scFv antibody 4B08 with pep1 was determined at 1.35 Å resolution (Fig. 1, Table II). The structures of the antibody–peptide complexes present in the asymmetric unit were mostly identical to each other as indicated by the low root-mean square deviation (RMSD) values (RMSD4B08 = 0.6 ± 0.2 Å and RMSDpep1 = 0.4 ± 0.2 Å), the number and distance of the nine intermolecular and four water-mediated H-bonds and the interaction surface (995 ± 31 Å2) were very similar in all the copies in the asymmetric unit (Fig. 1c, d, Supplementary Tables SI and SII). The conformation of the peptide bound to the antibody differs from the helical conformation observed in a 27-long peptide that included the sequence of pep1 (and presumably better representing the structure of that region in CCR5), which may explain the lack of binding activity of this antibody for the full-length protein. Table II. Thermodynamic parameters of binding of pep1 and mutants to scFv (see also Fig. 2)a Peptide  KD (µM)  Ratio  ΔG (kcal mol−1)  ΔH (kcal mol−1)  −TΔS (kcal mol−1)  n  pep1  0.48 ± 0.08  n.a.  −8.6 ± 0.1  −17.2 ± 0.8  8.6 ± 0.7  0.94 ± 0.05  N3A  1.3 ± 0.3  2.7  −8.1 ± 0.1  −13.7 ± 0.3  5.7 ± 0.2  1.09 ± 0.05  Y4A  0.71 ± 0.27  1.5  −8.4 ± 0.2  −18.4 ± 2.1  9.9 ± 2.3  1.02 ± 0.14  Y5A  1.1 ± 0.5  2.3  −8.2 ± 0.3  −12.5 ± 0.7  4.3 ± 0.4  1.02 ± 0.02  T6A  13 ± 2.7  27  −6.7 ± 0.1  −8.2 ± 0.7  1.5 ± 0.8  0.90 ± 0.04  S7A  n.d.  >100  n.d.  n.d.  n.d.  n.d.  E8A  3.5 ± 0.6  7.3  −7.5 ± 0.1  −14.8 ± 0.6  7.3 ± 0.7  0.87 ± 0.05  P9A  n.d.  >100  n.d.  n.d.  n.d.  n.d.  Peptide  KD (µM)  Ratio  ΔG (kcal mol−1)  ΔH (kcal mol−1)  −TΔS (kcal mol−1)  n  pep1  0.48 ± 0.08  n.a.  −8.6 ± 0.1  −17.2 ± 0.8  8.6 ± 0.7  0.94 ± 0.05  N3A  1.3 ± 0.3  2.7  −8.1 ± 0.1  −13.7 ± 0.3  5.7 ± 0.2  1.09 ± 0.05  Y4A  0.71 ± 0.27  1.5  −8.4 ± 0.2  −18.4 ± 2.1  9.9 ± 2.3  1.02 ± 0.14  Y5A  1.1 ± 0.5  2.3  −8.2 ± 0.3  −12.5 ± 0.7  4.3 ± 0.4  1.02 ± 0.02  T6A  13 ± 2.7  27  −6.7 ± 0.1  −8.2 ± 0.7  1.5 ± 0.8  0.90 ± 0.04  S7A  n.d.  >100  n.d.  n.d.  n.d.  n.d.  E8A  3.5 ± 0.6  7.3  −7.5 ± 0.1  −14.8 ± 0.6  7.3 ± 0.7  0.87 ± 0.05  P9A  n.d.  >100  n.d.  n.d.  n.d.  n.d.  aExperiments were performed at 25°C. Table II. Thermodynamic parameters of binding of pep1 and mutants to scFv (see also Fig. 2)a Peptide  KD (µM)  Ratio  ΔG (kcal mol−1)  ΔH (kcal mol−1)  −TΔS (kcal mol−1)  n  pep1  0.48 ± 0.08  n.a.  −8.6 ± 0.1  −17.2 ± 0.8  8.6 ± 0.7  0.94 ± 0.05  N3A  1.3 ± 0.3  2.7  −8.1 ± 0.1  −13.7 ± 0.3  5.7 ± 0.2  1.09 ± 0.05  Y4A  0.71 ± 0.27  1.5  −8.4 ± 0.2  −18.4 ± 2.1  9.9 ± 2.3  1.02 ± 0.14  Y5A  1.1 ± 0.5  2.3  −8.2 ± 0.3  −12.5 ± 0.7  4.3 ± 0.4  1.02 ± 0.02  T6A  13 ± 2.7  27  −6.7 ± 0.1  −8.2 ± 0.7  1.5 ± 0.8  0.90 ± 0.04  S7A  n.d.  >100  n.d.  n.d.  n.d.  n.d.  E8A  3.5 ± 0.6  7.3  −7.5 ± 0.1  −14.8 ± 0.6  7.3 ± 0.7  0.87 ± 0.05  P9A  n.d.  >100  n.d.  n.d.  n.d.  n.d.  Peptide  KD (µM)  Ratio  ΔG (kcal mol−1)  ΔH (kcal mol−1)  −TΔS (kcal mol−1)  n  pep1  0.48 ± 0.08  n.a.  −8.6 ± 0.1  −17.2 ± 0.8  8.6 ± 0.7  0.94 ± 0.05  N3A  1.3 ± 0.3  2.7  −8.1 ± 0.1  −13.7 ± 0.3  5.7 ± 0.2  1.09 ± 0.05  Y4A  0.71 ± 0.27  1.5  −8.4 ± 0.2  −18.4 ± 2.1  9.9 ± 2.3  1.02 ± 0.14  Y5A  1.1 ± 0.5  2.3  −8.2 ± 0.3  −12.5 ± 0.7  4.3 ± 0.4  1.02 ± 0.02  T6A  13 ± 2.7  27  −6.7 ± 0.1  −8.2 ± 0.7  1.5 ± 0.8  0.90 ± 0.04  S7A  n.d.  >100  n.d.  n.d.  n.d.  n.d.  E8A  3.5 ± 0.6  7.3  −7.5 ± 0.1  −14.8 ± 0.6  7.3 ± 0.7  0.87 ± 0.05  P9A  n.d.  >100  n.d.  n.d.  n.d.  n.d.  aExperiments were performed at 25°C. Fig. 1 View largeDownload slide X-ray crystal structure of the complex between pep1 and scFv antibody 4B08. (a) and (b) Structure of the critical CDR region of the antibody when in complex with pep1, represented here in two orientations differing by 90°. The heavy and light chains of the antibody are shown in dark and light gray, respectively. The peptide is depicted with orange sticks. The blue mesh corresponds to the sigma-A weighted 2fo-fc electron density map at 1.35 Å resolution (σ = 1). (c) Detailed interaction map at the contact interface between peptide and antibody. Carbon atoms corresponding to pep1 and 4B08 are depicted in orange and dark gray, respectively. Water molecules are represented by blue circles. The panel was prepared with LigPlot+ (54). (d) Detailed three-dimensional view of the intermolecular H-bond network between the peptide and antibody. Peptide and key residues of the antibody are depicted with orange and gray sticks, respectively. (e) Intramolecular H-bond network within the peptide. See also Table I, Supplementary Fig. S1 and Tables SI–SIII. Fig. 1 View largeDownload slide X-ray crystal structure of the complex between pep1 and scFv antibody 4B08. (a) and (b) Structure of the critical CDR region of the antibody when in complex with pep1, represented here in two orientations differing by 90°. The heavy and light chains of the antibody are shown in dark and light gray, respectively. The peptide is depicted with orange sticks. The blue mesh corresponds to the sigma-A weighted 2fo-fc electron density map at 1.35 Å resolution (σ = 1). (c) Detailed interaction map at the contact interface between peptide and antibody. Carbon atoms corresponding to pep1 and 4B08 are depicted in orange and dark gray, respectively. Water molecules are represented by blue circles. The panel was prepared with LigPlot+ (54). (d) Detailed three-dimensional view of the intermolecular H-bond network between the peptide and antibody. Peptide and key residues of the antibody are depicted with orange and gray sticks, respectively. (e) Intramolecular H-bond network within the peptide. See also Table I, Supplementary Fig. S1 and Tables SI–SIII. From the number of intermolecular H-bonds, B-factor analysis (BSA; Supplementary Fig. S1) and frequent disorder of Asp1 of pep1 in the electron density, it was suggested that the binding contribution of the C-terminal region (residues 6–9) of the peptide carries more weight than that of the N-terminal region (residues 1–5, see below for a more comprehensive validation by site-directed mutagenesis). Residues Ser 7 and Pro9 of the peptide contributed with the greatest BSA (106 ± 1 Å2 and 134 ± 3 Å2, respectively) and number of polar interactions (four H-bonds, and one H-bond and one salt bridge, respectively). In contrast, residues Asn3 and Thr6 contributed the least to BSA of the complex (14 ± 6 Å2 and 18 ± 1 Å2, respectively) not engaging in polar interactions with the antibody. Nonetheless, these two residues participate in the four intramolecular H-bonds encountered in the peptide (Fig. 1e, Supplementary Table SIII). Two intramolecular H-bonds were formed between the carbonyl group of the side-chain of Asn3 and the hydrogen of the main-chain nitrogen atom of residues Tyr4 and Tyr5. The other two intramolecular H-bonds were contributed the side chain hydroxyl of Thr6, which engaged the main-chain carbonyl and the carboxylic acid group of Glu8. The combination of minimal intermolecular interaction with the antibody and the formation of intramolecular H-bonds exclusively by these two residues in the complex was thus a unique opportunity to isolate the contribution of this ubiquitous class of H-bonds in the recognition of a peptide by an antibody. Energetic contributions to binding The ITC technique was employed to dissect the thermodynamic basis of binding of pep1 to 4B08 and to determine the energetic contributions of individual residues from Asn3 to Pro9 (Fig. 2, Table II, Supplementary Fig. S2). The dissociation constant (KD) of the complex between pep1 and 4B08 was determined from the binding isotherm by non-linear regression (KD = 480 nM). The affinity of the peptide for the antibody was driven by a large change of enthalpy (ΔH = −17.2 ± 0.8 kcal mol−1) and opposed by an unfavourable change of entropy (−TΔS = 8.7 ± 0.7 kcal mol−1). The thermodynamic signature is consistent with the binding of a flexible peptide to its target, since the large increase in non-covalent interactions (favourable enthalpy change) was opposed by a substantial entropic-loss resulting from the combined loss of conformational dynamics and translational and rotational degrees of freedom. Fig. 2 View largeDownload slide Search for hot-spot residues. (a)–(h) Characterization of the binding of pep1 (and Ala-scan variants) to scFv 4B08 by ITC. The antibody was titrated with peptide at 25°C. In each panel, the upper plot corresponds to the titration kinetics, whereas the lower plot represents the integrated binding isotherms. Molar ratio refers to the relative concentration of peptide-to-protein in the cell. The binding enthalpy (ΔH) and the dissociation constant (KD) were obtained by non-linear regression of the integrated data to a one-site binding model with the program ORIGIN. The results are given in Table I. Fig. 2 View largeDownload slide Search for hot-spot residues. (a)–(h) Characterization of the binding of pep1 (and Ala-scan variants) to scFv 4B08 by ITC. The antibody was titrated with peptide at 25°C. In each panel, the upper plot corresponds to the titration kinetics, whereas the lower plot represents the integrated binding isotherms. Molar ratio refers to the relative concentration of peptide-to-protein in the cell. The binding enthalpy (ΔH) and the dissociation constant (KD) were obtained by non-linear regression of the integrated data to a one-site binding model with the program ORIGIN. The results are given in Table I. Substitution of each individual residue (Asn3 to Pro9) to alanine ablates the side chain from the β-carbon and further, thus revealing the relative contribution of each side-chain to the binding constant and its energetic consequences (Fig. 2a–h, Table II). The separate replacement of the residues with the highest contribution to BSA and intermolecular interactions, Ser7 and Pro9, with Ala (S7A and P9A) completely abrogated the engagement of the peptide to the antibody. The substitution of Glu8 also in the C-terminal region of the peptide with Ala (E8A) reduced the affinity for the antibody with respect to pep1 by c.a. 7-fold (ΔΔG = 1.1 ± 0.2 kcal mol−1) accompanied by a decrease in the enthalpy change (ΔΔH = 2.4 ± 1.4 kcal mol−1). The replacement of residues engaging in intramolecular H-bonds in the peptide Asn3 and Thr6 with Ala (N3A and T6A) was also examined. The ablation of the side chain of Asn3 reduced moderately the affinity of the modified peptide for the antibody by c.a. 2.5-fold, in line with what was observed for other residues of the N-terminal region (Y4A, Y5A). In contrast to N3A, the substitution of Thr6 with Ala had a large and negative effect on the affinity, which plunged by 27-fold (ΔΔG = 1.9 ± 0.2 kcal mol−1). In thermodynamic terms, the poor binding ability of T6A is explained by a remarkable loss of favourable enthalpy with respect to the unchanged peptide (ΔΔH = 9.0 ± 1.5 kcal mol−1), suggesting a significant erosion in the strength of the interactions in the antibody–peptide complex. This effect was somehow unexpected given the little contribution of Thr6 to BSA and the absence of direct intermolecular interactions with the antibody. The representation of the thermodynamic data for all the substitutions examined (Supplementary Fig. S2) shows a moderate correlation between the loss of affinity and the change of enthalpy, suggesting that, in general, lower affinity resulted from weaker non-covalent interactions. As expected, the changes of enthalpy and entropy are fully correlated by the well-known entropy–enthalpy compensation effect (37). Behaviour of peptides in solution (MD simulations) Because we aimed at producing a complete picture of the behaviour of the peptide not only in complex with the antibody but also in solution (unbound state), we performed MD simulations and focused our attention on the four intramolecular H-bonds observed in the crystal structure (Fig. 3). A total simulation time of 1 μs for each peptide was analyzed, divided into five independent runs of 200 ns each. The results obtained in illustrative runs are given in the accompanying movies and graphs, showing large fluctuations in the conformation of the peptides and lacking a stable secondary structure, both observations consistent with the expected behavior of a flexible peptide in solution (Supplementary Fig. S3, Movies S1–3). Fig. 3 View largeDownload slide Characterization of intramolecular H-bonds in the unbound form of the peptide. (a) Schematic representation of the four intramolecular bonds under investigation (#1, #2, #3, and #4) as they appear in the bound form of pep1 (protein not shown). (b) Distance probability plot between heavy atoms participating in intramolecular H-bonds during MD simulations of the unbound peptide. Data of H-bonds corresponding to #1–2 of N3A, and #3–4 of T6A are not shown because the mutations abrogate those bonds. Distances were determined with UCSF Chimera 1.10.1 (55) and the normalized distribution plots calculated with R (version 3.2.3, https://www.R-project.org/) using kernel density estimation. Orange, green and blue corresponded to the data obtained for pep1, N3A, and T6A, respectively. (c) Fraction of structures showing the indicated H-bonds. An H-bond was considered to appear when the distance between the proton donor and acceptor was less than 3.5 Å (38, 39) as calculated by UCSF Chimera. Restricting the H-bond angle to <150° did not significantly affect the estimation of the fraction of intramolecular H-bonds #1–4. See also Supplementary Figs S2–S4 and Movies S1–S3. Fig. 3 View largeDownload slide Characterization of intramolecular H-bonds in the unbound form of the peptide. (a) Schematic representation of the four intramolecular bonds under investigation (#1, #2, #3, and #4) as they appear in the bound form of pep1 (protein not shown). (b) Distance probability plot between heavy atoms participating in intramolecular H-bonds during MD simulations of the unbound peptide. Data of H-bonds corresponding to #1–2 of N3A, and #3–4 of T6A are not shown because the mutations abrogate those bonds. Distances were determined with UCSF Chimera 1.10.1 (55) and the normalized distribution plots calculated with R (version 3.2.3, https://www.R-project.org/) using kernel density estimation. Orange, green and blue corresponded to the data obtained for pep1, N3A, and T6A, respectively. (c) Fraction of structures showing the indicated H-bonds. An H-bond was considered to appear when the distance between the proton donor and acceptor was less than 3.5 Å (38, 39) as calculated by UCSF Chimera. Restricting the H-bond angle to <150° did not significantly affect the estimation of the fraction of intramolecular H-bonds #1–4. See also Supplementary Figs S2–S4 and Movies S1–S3. The intramolecular H-bond pattern, and their distances, were monitored every 100 ps, resulting in 10,000 independent structures being analyzed (Fig. 3b and c). An H-bond was considered to be present if the distance between the heavy atoms was within the standard cut-off distance of 3.5 Å (38, 39). No significantly different results were obtained when H-bonds were restricted to angles less than 150° (Supplementary Fig. S4). Because of the inherent flexibility of the peptide, the frequency of intramolecular H-bonds in solution was very low (≤ 3%) except for the intramolecular H-bond #3, which was detected repeatedly in about 25% of the trajectories of pep1, and half that value in N3A. Hydrogen bonds linking the atom γO of the side chain of residue i of Ser or Thr to the main chain NH group of the residue i + 2 are termed ST turn, and are often found in protein structures (40). This H-bond is not present in T6A, since the substitution of Thr6 by Ala removes the necessary atom γO from the side chain. The distance between the heavy atoms comprising the H-bonds also undergo large fluctuations, except for the distance between the atoms participating in H-bond #3 (Supplementary Fig. S5). To evaluate structural differences among the peptides in solution, the dihedral angles of residues Ile2 to Glu8 were calculated. Three-dimensional comparative Ramachandran plots are shown in Fig. 4a and b and the conventional bi-dimensional plots in Supplementary Fig. S6. The three peaks appearing in these plots correspond to the combination of phi and psi angles of the major secondary structure elements (α-helix, −60°/−45°; β-sheet, −135°/135°; left-handed helix, 60°/45°). As revealed from the examination of the Ramachandran plots, the secondary structure profile of pep1 and T6A differ significantly except in the last residue Glu8. In particular, a greater α-helical content was observed in residues Ile2 to Thr6 of the peptide T6A with respect to pep1. This region was stabilized by three intramolecular H-bonds different from those examined earlier and formed between main chain atoms of Asp1/Tyr5, Ile2/Thr6 and Asn3/Ser7 (Fig. 4c). These three intramolecular H-bonds appeared in 42–44% of the trajectories in T6A, but only 2–4% in pep1. The effect on the conformation of N3A was somehow intermediate between pep1 and T6A, appearing in 19–26% of the trajectories. Therefore, the loss of the side chain of Asn, and especially of Thr6, increased the formation of other intramolecular H-bonds in solution that favoured α-helical conformations. Fig. 4 View largeDownload slide Secondary structure of unbound pep1, N3A, and T6A in solution. (a) Ramachandran probability plot of a representative residue (Tyr4) obtained from MD simulations. The dihedral angle was calculated with UCSF Chimera, and the normalized distribution plots calculated with R using kernel density estimation. The arrows point at three key regions of the phi/psi angle plot, i.e. α-helix, β-sheet, and left-handed helix. The colors orange, green and blue corresponds to pep1, N3A and T6A, respectively. (b) Ramachandran probability plots for other residues of the peptide using the same color scheme. The asterisk in Asn3 and Thr6 indicates that each of these residues is changed to Ala in N3A and T6A, respectively. (c) Fraction of intramolecular H-bonds between main-chain atoms of the indicated residues. These bonds represent mainly helical conformations, especially α-helix. An H-bond was considered to appear when the distance between the proton donor and acceptor was less than 3.5 Å as calculated by UCSF Chimera. Fig. 4 View largeDownload slide Secondary structure of unbound pep1, N3A, and T6A in solution. (a) Ramachandran probability plot of a representative residue (Tyr4) obtained from MD simulations. The dihedral angle was calculated with UCSF Chimera, and the normalized distribution plots calculated with R using kernel density estimation. The arrows point at three key regions of the phi/psi angle plot, i.e. α-helix, β-sheet, and left-handed helix. The colors orange, green and blue corresponds to pep1, N3A and T6A, respectively. (b) Ramachandran probability plots for other residues of the peptide using the same color scheme. The asterisk in Asn3 and Thr6 indicates that each of these residues is changed to Ala in N3A and T6A, respectively. (c) Fraction of intramolecular H-bonds between main-chain atoms of the indicated residues. These bonds represent mainly helical conformations, especially α-helix. An H-bond was considered to appear when the distance between the proton donor and acceptor was less than 3.5 Å as calculated by UCSF Chimera. Crystal structures of N3A and T6A in complex with 4B08 Crystal structures of 4B08 in complex with N3A or with T6A were determined 1.96 Å and 1.35 Å resolution, respectively (Fig. 5a–d, Table I). The complex between N3A and the antibody was crystallized in spacegroup P22121 containing two copies in the asymmetric unit that were essentially identical to each other (RMSD of antibody or N3A < 0.12 Å). This structure was also very similar to that of the complex with pep1 (RMSD4B08 = 0.5 ± 0.1 Å; RMSDpeptide = 0.3 ± 0.2 Å). We note that the RMSD values between these two different crystal structures were smaller than the values found for copies within the same asymmetric unit in the complex of 4B08 with pep1 (see above) (Fig. 5e). Despite their overall similarity, the rotamers of residues Tyr4 and Tyr5 adopted a different conformation that we attributed to the loss of intramolecular H-bonds caused by the Asn to Ala substitution and to differences in the crystal packing forces. In this complex, Tyr5 engaged in CH–π interactions with Ala3, and Tyr4-Tyr5 displayed π–π stacking interactions. Although no significant differences of the BSA in the critical C-terminal region were observed, the re-arrangement of the Tyr residues resulted in the loss of one of the nine intermolecular H-bonds between the N3A peptide and the antibody. Specifically, H-bonds between Tyr5 and Asn61 and between two bridging water molecules are lost, although it is partially compensated by a new water-mediated H-bond network (Supplementary Table SI). Fig. 5 View largeDownload slide X-ray crystal structure of N3A and T6A in complex with antibody. (a) X-ray crystal structure of N3A bound to 4B08. The heavy and light chains of the antibody are shown in dark and light gray, respectively. The peptide (green) and key residues of the antibody are depicted with sticks. Intermolecular H-bonds between the peptide and the protein are indicated. (b) Same crystal structure showing only the intramolecular H-bonds of the peptide. (c) and (d) Equivalent structure of T6A (blue) bound to 4B08. (e) Overlay of the main-chain trace of pep1 (orange), N3A (green), and T6A (blue) bound to 4B08 (light gray). (f) Similarity plot between the conformation of the unbound peptide in the MD simulations and the structure bound to the antibody. The RMSD values were calculated with VMD version 1.9.2 (56) and the normalized distribution plots calculated with R using kernel density estimation. The arrow highlights a region of high structural similarity to the peptide in the bound form, which is populated by pep1 but not by the modified peptides. (g) Number of similarity events between the structures of the peptide in the unbound form (MD simulations) and bound to antibody form (X-ray) at different timescales. The RMSD at which an event is recorded was set to a cutoff value of >2.0 Å. The RMSD values were calculated with VMD using the coordinates of Cα. A close-up view of the data corresponding to the longest time-scale (>1 ns) is also shown. See also Table I and Supplementary Figs S1 and S5 and Tables SI–SIII. Fig. 5 View largeDownload slide X-ray crystal structure of N3A and T6A in complex with antibody. (a) X-ray crystal structure of N3A bound to 4B08. The heavy and light chains of the antibody are shown in dark and light gray, respectively. The peptide (green) and key residues of the antibody are depicted with sticks. Intermolecular H-bonds between the peptide and the protein are indicated. (b) Same crystal structure showing only the intramolecular H-bonds of the peptide. (c) and (d) Equivalent structure of T6A (blue) bound to 4B08. (e) Overlay of the main-chain trace of pep1 (orange), N3A (green), and T6A (blue) bound to 4B08 (light gray). (f) Similarity plot between the conformation of the unbound peptide in the MD simulations and the structure bound to the antibody. The RMSD values were calculated with VMD version 1.9.2 (56) and the normalized distribution plots calculated with R using kernel density estimation. The arrow highlights a region of high structural similarity to the peptide in the bound form, which is populated by pep1 but not by the modified peptides. (g) Number of similarity events between the structures of the peptide in the unbound form (MD simulations) and bound to antibody form (X-ray) at different timescales. The RMSD at which an event is recorded was set to a cutoff value of >2.0 Å. The RMSD values were calculated with VMD using the coordinates of Cα. A close-up view of the data corresponding to the longest time-scale (>1 ns) is also shown. See also Table I and Supplementary Figs S1 and S5 and Tables SI–SIII. The complex between T6A and 4B08 crystallized in the same spacegroup than that of pep1. As in the examples from above, the complexes of 4B08 with T6A were essentially indistinguishable to each other when compared with either copies within the asymmetric unit (RMSD4B08 = 0.6 ± 0.1 Å; RMSDT6A = 0.4 ± 0.2 Å) or when compared with the complex containing the unmodified pep1 (RMSD4B08 = 0.4 ± 0.3 Å; RMSDpeptide = 0.3 ± 0.2 Å). Similarly, the values of BSA, and the number and distances of the intermolecular and water-mediated H-bonds were well conserved with respect to the complex with pep1, except for the intramolecular H-bonds #3 and #4 eliminated upon substitution of the side chain of Thr6. When the bound conformations of the peptides were compared with the ensemble in solution, a complex landscape emerged. The conformation of the peptide bound to the antibody only represented a minor fraction of the ensemble in solution. The vast majority of unbound conformations of the unmodified and modified peptides appeared in the region where the RMSD with respect to the bound form was > 2 Å, as expected from a flexible peptide (Fig. 5f). However, we also observed a small fraction of conformations with low RMSD (< 2 Å) for pep1 epitope and to a lesser degree for N3A. Moreover, the number of occurrences and the duration of these low RMSD structures increased steeply from T6A to N3A to pep1 (Fig. 5g). When the comparison of the solution ensemble with the bound form is restricted to only the N-terminal region, or the C-terminal region, opposite trends are identified for pep1 with respect to T6A (Supplementary Fig. S7). On the one hand, the conformation of the N-terminal of the peptide in solution and the bound form is more similar for T6A. On the other hand, when comparing the C-terminal region (critical for binding), pep1 displays the largest population of homolog conformers. The peptide N3A shows intermediate behaviour. Collectively, these data suggest that the recognition of the peptide is at least in part favoured by a conformational selection mechanism resulting from a critical intramolecular H-bond. Discussion Although intramolecular H-bonds are often found in peptides bound to antibodies (21, 41, 42), there have not been comprehensive studies addressing their influence in the dynamics of the peptide in solution and the structure and thermodynamics in the bound state. We selected the model peptide pep1 corresponding to the N-terminal region of CCR5 to explore the intimate details of intramolecular H-bonds in the recognition by an antibody. Our approach encompassed MD simulations, binding thermodynamics and high-resolution X-ray crystallography, revealing a multifaceted influence of this important class of H-bonds in the structural dynamics of the peptide in solution and in the affinity for the antibody. The results indicate that a critical intramolecular H-bond is significantly populated in solution despite the intrinsic flexibility of the peptide, exerting a strong influence in the change of enthalpy in the bound state that strengthened the binding constant. To mimic the solution structures of the pep1, we have resorted to MD simulations employing the CHARMM22/CMAP force field. Recent benchmarks showed that, during MD simulations with the CHARMM22/CMAP force field, an unstructured peptide is sometimes biased towards α-helical conformations (43). Although the higher tendency of N3A and T6A toward helical conformations might have been influenced by the force field, simulations of the wild type pep1 rarely sampled helical conformations at the corresponding region (Fig. 4). Therefore, the formation of the intramolecular hydrogen bond by the side chain of Thr6, the key hydrogen bond in this study, was independent from force field bias. Current empirical force fields are not fully free from bias to a varying extent (44–46). However, considering the consistency of our experimental measurements, and the general absence of force field bias, we believe our conclusions are relevant to the general mechanism of peptide–antibody interactions. Based on our observations, we have elaborated the diagram shown in Fig. 6 explaining the recognition of a flexible peptide by an anti-peptide antibody and the role of its intramolecular H-bonds. In solution, a critical intramolecular H-bond like that between Thr6 and the main chain of Glu8 transiently stabilizes a conformation, which could form a well populated (i.e. lowest energy) state in the unbound peptide ensemble. Such a well populated and stabilized unbound peptide conformation would be thus recognized by an antibody with higher probability. The region where the critical intramolecular H-bond interaction is established becomes essential for an overall binding process. In our particular example, this region comprises residues of the C-terminal of the peptide. Without the critical #3 intramolecular H-bond, the peptide could not form the populated states of the C-terminal region (Supplementary Fig. S7b), and the antibody would have less opportunities to mature recognizing the C-terminal conformation of the peptide antigen. Moreover, additional intramolecular H-bonds in the critical epitope and in the neighbouring region further favour the energetic component of the binding reaction by increasing the enthalpic component and increasing the overall affinity. It will be interesting to investigate whether the peptide containing the substitution T6A, displaying a significant difference in the Ramachandran probability plot in solution with respect to pep1 (Fig. 4), would elicit antibodies recognizing the new conformation of the peptide. Fig. 6 View largeDownload slide Multifaceted contribution of intramolecular H-bonds for the binding of a flexible peptide to an antibody. The model represents the free energy profile of the peptide along the reaction coordinate. In the unbound form, the peptide appears as a flexible ensemble of different conformations. Intramolecular H-bonds make a modest but critical contribution favouring conformations that resemble the structure in the bound state. When bound to the antibody, the peptide is greatly stabilized by these intramolecular H-bonds, bringing a neat favourable change of enthalpy. Intramolecular H-bonds in the vicinity of the hot-spot region (C-terminal region) make a stronger contribution than those in the N-terminal region. Fig. 6 View largeDownload slide Multifaceted contribution of intramolecular H-bonds for the binding of a flexible peptide to an antibody. The model represents the free energy profile of the peptide along the reaction coordinate. In the unbound form, the peptide appears as a flexible ensemble of different conformations. Intramolecular H-bonds make a modest but critical contribution favouring conformations that resemble the structure in the bound state. When bound to the antibody, the peptide is greatly stabilized by these intramolecular H-bonds, bringing a neat favourable change of enthalpy. Intramolecular H-bonds in the vicinity of the hot-spot region (C-terminal region) make a stronger contribution than those in the N-terminal region. A recent computational study has suggested that intrinsically disordered proteins adopt bound conformations before binding to its binding partner (47). Similar transient conformations have been proposed in more general associations involving globular proteins (48, 49). In line with these observations, we have shown that intramolecular hydrogen bonds also have general implications for the molecular mechanisms of peptide (and possibly protein) association. We would like to stress that this mechanism is different to that presented as pre-organization (50–53), since the competent structure of pep1 in solution is transiently stabilized by the intramolecular H-bond, and the influence in binding occurs mostly in the bound state. Collectively, our study established a role for intramolecular H-bonds in antibody–antigen recognition. Conclusions Anti-peptide antibodies recognize their flexible peptide-antigens, even in the absence of defined secondary or tertiary structures in solution. However, it is reasonable to assume that some type of structure should guide their selection and antibody maturation. Our study addresses this question to an unprecedented level of molecular detail. Herein, we have demonstrated that the epitope region of a model flexible peptide in solution sporadically adopts the structure of the bound state, being stabilized by intramolecular H-bonds prior to binding. This result suggests that structures transiently stabilized by intramolecular H-bonds would be one of the critical factors to take into consideration in the preparation of antigens for rational antibody production. Supplementary Data Supplementary Data are available at JB Online. Acknowledgements We thank the staff of the Photon Factory (Tsukuba, Japan) for excellent technical support. Access to beamlines BL5A and AR-NE3A was granted by the Photon Factory Advisory Committee (Proposal nos. 2013G378, and 2014G190). We acknowledge access to the super-computing facilities at the Human Genome Center of The University of Tokyo (http://sc.hgc.jp/shirokane.html). We are grateful to Dr. Eugenio Vazquez (Santiago de Compostela, Spain) for insightful discussions. Funding This work was supported by the Funding program for world-leading Innovative R&D on Science and Technology (FIRST) from JSPS, the Platform for Drug Discovery, Informatics, and Structural Life Science (MEXT), JSPS Grants-in-Aid for Scientific Research 25249115 (K.T.) and 15K06962 (J.M.M.C.). Conflict of Interest None declared. References 1 Tsumoto K., Caaveiro J.M.M. ( 2016) Antigen-antibody binding. eLS . doi: 10.1002/9780470015902.a0001117.pub3 2 Diamantis N., Banerji U. ( 2016) Antibody-drug conjugates-an emerging class of cancer treatment. Br. J. Cancer  114, 362– 367 Google Scholar CrossRef Search ADS PubMed  3 Towbin H., Staehelin T., Gordon J. ( 1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets - procedure and some applications. Proc. Natl. Acad. Sci. USA  76, 4350– 4354 Google Scholar CrossRef Search ADS   4 Kitago Y., Kaneko M.K., Ogasawara S., Kato Y., Takagi J. 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Chem.  25, 1605– 1612 Google Scholar CrossRef Search ADS PubMed  56 Humphrey W., Dalke A., Schulten K. ( 1996) VMD: visual molecular dynamics. J. Mol. Graph. Model  14, 33– 38 Google Scholar CrossRef Search ADS   Abbreviations Abbreviations CCR5 C-C chemokine receptor type 5 ITC isothermal titration calorimetry MD molecular dynamics RMSD root-mean square deviation scFv single-chain fragment variable © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Biochemistry Oxford University Press

Intramolecular H-bonds govern the recognition of a flexible peptide by an antibody

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved
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0021-924X
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1756-2651
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10.1093/jb/mvy032
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Abstract

Abstract Molecular recognition is a fundamental event at the core of essentially every biological process. In particular, intermolecular H-bonds have been recognized as key stabilizing forces in antibody–antigen interactions resulting in exquisite specificity and high affinity. Although equally abundant, the role of intramolecular H-bonds is far less clear and not universally acknowledged. Herein, we have carried out a molecular-level study to dissect the contribution of intramolecular H-bonds in a flexible peptide for the recognition by an antibody. We show that intramolecular H-bonds may have a profound, multifaceted and favorable effect on the binding affinity by up to 2 kcal mol−1 of free energy. Collectively, our results suggest that antibodies are fine tuned to recognize transiently stabilized structures of flexible peptides in solution, for which intramolecular H-bonds play a key role. antibody–antigen interaction, molecular dynamics simulations, molecular recognition, transient folding, unstructured peptide Immunoglobulins (antibodies) are capable to exquisitely recognize an unlimited number of different molecular entities including proteins, peptides and small molecules all termed antigens (1). In addition to their key role in the immune system, antibodies have been widely employed as detection reagents in biological studies, therapeutic agents in the clinical stage, or biosensors in biotechnological devices for a long time (2–4). In order to improve the efficiency in their therapeutic use, antibodies with even higher affinity and selectivity are desired (5, 6). To that end, two basic approaches are taken, one that focuses on the antibody itself and a second one that relies on the preparation of sophisticated antigens eliciting antibodies with more favourable properties using rational design, random mutagenesis or a combination of both (7, 8). But how are those antibodies obtained on the first place? Short peptides are often employed to elicit antibodies through immunization procedures (9) such as in vaccines and in various protein-related detection reagents (10, 11). Intriguingly, short peptides generally do not display a stable secondary structure in solution (12, 13), raising the question of how antibody matures to recognize such flexible antigens. Despite the flexible nature of these antigens in solution, different antibodies recognizing the same peptide exhibit comparable antibody–peptide interaction contacts, and the bound peptide the same secondary structure conformation (14–16). The structural convergence of peptide–antibody complexes seems to contradict the nature of peptide antigens in solution, reported to be flexible and unstructured (17–20). Previously, it was hypothesized that the formation of intramolecular hydrogen bonds (H-bonds) could stabilize certain conformations of the peptide and therefore contributes to the affinity of the peptide–antibody complex (14, 21), although to the best of our knowledge, no formal demonstration of it has been reported to date. Herein, we have addressed the relationship between the structural ensemble of peptide antigens in solution, the thermodynamic properties upon interaction to a specific antibody, and the structure in the bound form. In particular, we have focused on the question of whether intramolecular H-bonds may influence the binding affinity of a flexible peptide to an antibody. By employing a model system composed of the antibody E38035-4B08-222 (heretofore 4B08) and the peptide DINYYTSEP (pep1) derived from N-terminal region of CC chemokine receptor 5 (CCR5), we revealed the role of the peptide’s intramolecular hydrogen bonds in binding to the antibody. Molecular dynamics (MD) simulations, high-resolution X-ray crystallography and calorimetry demonstrated that intramolecular H-bonds indeed play an critical role in at least two important stages of the binding coordinate: the conformation of the peptide in solution and the stabilization of the complex. Materials and Methods Immunization and cloning of 4B08 The peptide employed in this study corresponded to residues 10–19 of the human protein CCR5 (Uniprot entry number P51681) of sequence DINYYTSEP that we termed pep1. This peptide was chemically synthesized with an NH2-Mini-PEG3 at the N-terminal (Peptide Institute, Osaka, Japan). Pep1 was conjugated with Keyhole limpet hemocyanin (KLH, Thermo Fisher Scientific) using glutaraldehyde. Female MRL/lpr mice (SLC, Shizuoka, Japan) were immunized intraperitoneally (i.p.) with 80 µg of Pep1-KLH in Freund's Complete Adjuvant. Second immunization was also performed i.p. using 80 µg of Pep1-KLH in Freund’s incomplete adjuvant and final immunization was performed i.p. with 80 µg of Pep1-KLH in saline at 2-week intervals. Additionally, the mice were administered 50 µg of wild-type budded baculovirus subcutaneously for adjuvant effects at all the step of immunization. Spleen cells were isolated 3 days after the last immunization and fused with sp2/0-Ag14 myeloma cells by conventional methods (22). Hybridoma culture supernatants were screened for antibody production by the enzyme-linked immunosorbent assay with pep1 coated 96-well microtiter plates. To select hybridomas secreting high-affinity antibodies against pep1, the cultures were analyzed using a Biacore3000 (GE healthcare) with pep1 immobilized on a carboxymethyl CM5 sensor chip (GE healthcare). The selected cells were isolated by limiting dilution to establish monoclonal hybridoma cell lines producing antibodies against pep1. Preparation of expression vector The DNA sequence of the single-chain fragment variable (scFv) region of antibody E38035-4B08-222 (heretofore 4B08) was subcloned into pRA2 vector (23). Restriction sites for the insertion were NcoI and EagI (heavy chain variable region) or EcoRV and SacII (light chain variable region). A flexible linker composed of four repeating units of the sequence GGGGS was introduced between the heavy and light chains. The pelB leader preceded the N-terminus and a His6 tag was introduced at the C-terminus to facilitate protein expression. Expression of recombinant scFv Escherichia coli strain BL21(DE3) carrying the expression vector of scFv 4B08 was grown overnight at 28°C, 170 rpm in 3 ml of Luria-Bertani medium. The cells were diluted into 1 L of Luria-Bertani medium and cultured at 28°C and 140 rpm until the OD600 reached a value of 0.6–0.8. At that point isopropyl β-D-1-thiogalactopyranoside was added to the cell culture to a final concentration of 0.5 mM and the cell culture was left overnight at 28°C. Cells were harvested by centrifugation at 7,000 × g for 20 min at 4°C. The cell pellet was resuspended in 40 ml of TRIS buffer (20 mM Tris, 500 mM NaCl, pH 8.0) supplemented with 5 mM imidazole, and the cells lysed with a ultrasonic cell-disrupting UD-201 instrument (TOMY, Japan). The cell lysate was subsequently centrifuged at 4°C, 7,000 × g for 20 min. The supernatant was discarded, and the collected pellet was washed with 20 ml of buffer supplemented with 1% (v/v) Triton X-100 for 30 min, followed by equivalent washing steps with acetone and deionized water, respectively. Refolding and purification of scFv Inclusion bodies were incubated overnight in 20 ml of solubilization buffer composed of 6 M guanidinium chloride, 20 mM TRIS, 500 mM NaCl, at pH 8.0 and 5 mM imidazole. The suspension was centrifuged at 40,000 × g for 30 min at 4°C and the supernatant filtered and loaded onto an affinity column of Ni-NTA agarose (Qiagen) equilibrated with solubilization buffer. The bound fraction to the column was first washed with buffer containing 20 mM imidazole and subsequently eluted with buffer containing 100 and 500 mM imidazole. The eluted scFv was diluted to 10 µM and refolded by stepwise dialysis in TRIS buffer with decreasing concentrations of guanidinium chloride (3 M to 0 M) (24). Refolded 4B08 was concentrated using an Ni-NTA agarose column; the scFv-loaded column was washed with TRIS buffer and scFv was eluted with TRIS buffer containing 500 mM imidazole. Further purification was conducted by size exclusion chromatography (AKTA purifier with Hiload 16/600 superdex 75 pg, GE healthcare) with TRIS buffer at a flow rate of 1.0 ml min−1 at 4°C. Peptides Synthetic peptides were purchased from Medical & Biological Laboratories Co., Ltd. (Nagoya, Japan) or Scrum Inc. (Tokyo, Japan). Peptides had no modifications, and purity was always greater than 95%. The peptides were used without further purification. Concentration of peptide was determined by its absorbance at 280 nm. The sequences of the peptides employed were: pep1, DINYYTSEP; N3A, DIAYYTSEP; Y4A, DINAYTSEP; Y5A, DINYATSEP; T6A, DINYYASEP; S7A, DINYYTAEP; E8A, DINYYTSAP; P9A, DINYYTSEA. Isothermal titration calorimetry (ITC) Thermodynamic parameters of the peptide–antibody interactions were determined in an iTC200 nano-calorimeter instrument (Malvern). The antibody was dialyzed overnight in phosphate buffered saline (PBS composed of 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.76 mM KH2PO4, and pH 7.4). Peptides were dissolved in the same PBS buffer employed to dialyze the scFv. The concentration of 4B08 (in the cell) and peptide (in the syringe) was 9–11 μM and 100–120 μM, except for the experiments employing peptides E8A and T6A. In the titration with E8A, the concentrations of 4B08 and peptide were increased to 30 μM and 320 μM, respectively. For the experiment with mutant T6A, the concentration of antibody and peptide were 40 μM and 500 μM, respectively. Titrations were carried out at 25°C with a reference power of 5 μcal s−1 and a stirring rate of 1,000 rpm. Each experiment consisted of a single 0.5-μL injection of peptide followed by 18 additional injections of 2.0 μL each with an interval between injections of 180 s. Thermodynamic parameters were calculated with the program ORIGIN 7.0 (OriginLab) using a single-site binding model. Crystallization The antibody 4B08 dialyzed overnight in MES buffer (10 mM MES, 30 mM NaCl, pH 5.9) at final concentration of 100 μM was mixed with peptide solubilized in the same MES buffer in which scFv was dialyzed overnight at a final concentration of 200 μM. Crystals of the peptide–antibody complex were obtained by mixing equal volumes of protein solution (2 μl) and crystallization solution [1.8–2.4 M (NH4)2SO4, 0.1 M Bis-Tris, pH 5.9–6.5] at 20°C by the hanging-drop vapor diffusion method. Well-developed crystals of the complexes between antibody and pep1 or T6A were harvested from the wells containing 1.8 M (NH4)2SO4, 0.1 M Bis-Tris at pH 6.5, and those of the complex between N3A and 4B08 were harvested from the solution containing 2.2 M (NH4)2SO4, 0.1 M Bis-Tris at pH 6.2. The crystals were briefly transferred to the same solution supplemented with 30% glycerol prior to storage in liquid N2 until data collection. Data collection and refinement Diffraction data were collected at beamlines BL5A and AR-NE3A both at the Photon Factory (Tsukuba, Japan) under cryogenic conditions (100 K). Data were indexed, integrated with MOSFLM (25), and scaled with SCALA or AIMLESS (26). The structures of the complex between pep1 and scFv 4B08 were determined by the method of molecular replacement as described previously for an anti-P-cadherin antibody (27) with PHASER (28). Refinement was performed with COOT (29) and REFMAC5 (30). Structural validation was performed with COOT and PROCHECK (31). Data collection and refinement statistics are summarized in Table I. Although four antibody–peptide complexes were found in the asymmetric unit of the complexes between antibody and pep1 or T6A, only three were used for further analysis. The fourth copy showed essentially the same similar structural features to the other three ones but dynamic disorder in the peptide precluded unambiguous characterization. Table I. Data collection and refinement statisticsa Data collection  4B08 + pep1  4B08 + N3A  4B08 + T6A  Space group  P 1 21 1  P 2 21 21  P 1 21 1  Unit cell            a, b, c (Å)  80.5, 73.0, 87.0  61.6, 81.9, 114.2  80.5, 72.9, 86.5      α, β, γ (°)  90.0, 114.3, 90.0  90.0, 90.0, 90.0  90.0, 114.2, 90.0  Resolution (Å)  33.1–1.35  37.3–1.96  33.1–1.35  Wavelength  1.000  1.000  1.000  Observations  723,496 (103,749)  309,459 (15,814)  853,946 (97,855)  Unique reflections  193,517 (27,587)  40,361 (2,555)  197,862 (27,517)  Rmerge. (%)  8.3 (51.9)  12.9 (42.5)  8.1 (39.2)  CC1/2  0.995 (0.759)  0.997 (0.939)  0.996 (0.843)  I/σI  9.7 (2.6)  10.0 (3.7)  10.6 (2.7)  Multiplicity  3.7 (3.8)  7.7 (6.2)  4.3 (3.6)  Completeness (%)  96.5 (94.4)  95.6 (87.1)  99.1 (94.9)  Refinement statistics        Resolution (Å)  33.1–1.35  37.3–1.96  33.1–1.35  Rwork/Rfree (%)b  13.6/18.4  20.3/25.8  13.5/17.3  No. atoms (protein)  7,554  3,742  7,330  No. atoms (peptide)  322c  134  272c  No. atoms (solvent)  888  364  952  No. atoms (other)  62  40  40  B-factor (protein) (Å2)  14.6  16.3  16.8  B-factor (peptide) (Å2)  20.1  15.1  26.8  B-factor (solvent) (Å2)  23.9  21.9  26.0  B-factor (other) (Å2)  30.9  37.7  32.3  Ramachandran plot            Preferred regions (%)  89.9  90.0  90.2      Allowed regions (%)  9.6  9.5  9.3      Outliers (%)  0.5  0.5  0.5  RMSD bond (Å)  0.014  0.013  0.014  RMSD angle (°)  1.76  1.55  1.71  PDB entry code  5YD3  5YD5  5YD4  Data collection  4B08 + pep1  4B08 + N3A  4B08 + T6A  Space group  P 1 21 1  P 2 21 21  P 1 21 1  Unit cell            a, b, c (Å)  80.5, 73.0, 87.0  61.6, 81.9, 114.2  80.5, 72.9, 86.5      α, β, γ (°)  90.0, 114.3, 90.0  90.0, 90.0, 90.0  90.0, 114.2, 90.0  Resolution (Å)  33.1–1.35  37.3–1.96  33.1–1.35  Wavelength  1.000  1.000  1.000  Observations  723,496 (103,749)  309,459 (15,814)  853,946 (97,855)  Unique reflections  193,517 (27,587)  40,361 (2,555)  197,862 (27,517)  Rmerge. (%)  8.3 (51.9)  12.9 (42.5)  8.1 (39.2)  CC1/2  0.995 (0.759)  0.997 (0.939)  0.996 (0.843)  I/σI  9.7 (2.6)  10.0 (3.7)  10.6 (2.7)  Multiplicity  3.7 (3.8)  7.7 (6.2)  4.3 (3.6)  Completeness (%)  96.5 (94.4)  95.6 (87.1)  99.1 (94.9)  Refinement statistics        Resolution (Å)  33.1–1.35  37.3–1.96  33.1–1.35  Rwork/Rfree (%)b  13.6/18.4  20.3/25.8  13.5/17.3  No. atoms (protein)  7,554  3,742  7,330  No. atoms (peptide)  322c  134  272c  No. atoms (solvent)  888  364  952  No. atoms (other)  62  40  40  B-factor (protein) (Å2)  14.6  16.3  16.8  B-factor (peptide) (Å2)  20.1  15.1  26.8  B-factor (solvent) (Å2)  23.9  21.9  26.0  B-factor (other) (Å2)  30.9  37.7  32.3  Ramachandran plot            Preferred regions (%)  89.9  90.0  90.2      Allowed regions (%)  9.6  9.5  9.3      Outliers (%)  0.5  0.5  0.5  RMSD bond (Å)  0.014  0.013  0.014  RMSD angle (°)  1.76  1.55  1.71  PDB entry code  5YD3  5YD5  5YD4  aStatistical values given in parenthesis refer to the highest resolution bin (see also Figs 1 and 5). bRfree was calculated as Rwork taking 2–4% of data not included in the refinement. cThe greater number of modeled atoms of pep1 with respect to T6A reflect the existence of alternative conformations. Table I. Data collection and refinement statisticsa Data collection  4B08 + pep1  4B08 + N3A  4B08 + T6A  Space group  P 1 21 1  P 2 21 21  P 1 21 1  Unit cell            a, b, c (Å)  80.5, 73.0, 87.0  61.6, 81.9, 114.2  80.5, 72.9, 86.5      α, β, γ (°)  90.0, 114.3, 90.0  90.0, 90.0, 90.0  90.0, 114.2, 90.0  Resolution (Å)  33.1–1.35  37.3–1.96  33.1–1.35  Wavelength  1.000  1.000  1.000  Observations  723,496 (103,749)  309,459 (15,814)  853,946 (97,855)  Unique reflections  193,517 (27,587)  40,361 (2,555)  197,862 (27,517)  Rmerge. (%)  8.3 (51.9)  12.9 (42.5)  8.1 (39.2)  CC1/2  0.995 (0.759)  0.997 (0.939)  0.996 (0.843)  I/σI  9.7 (2.6)  10.0 (3.7)  10.6 (2.7)  Multiplicity  3.7 (3.8)  7.7 (6.2)  4.3 (3.6)  Completeness (%)  96.5 (94.4)  95.6 (87.1)  99.1 (94.9)  Refinement statistics        Resolution (Å)  33.1–1.35  37.3–1.96  33.1–1.35  Rwork/Rfree (%)b  13.6/18.4  20.3/25.8  13.5/17.3  No. atoms (protein)  7,554  3,742  7,330  No. atoms (peptide)  322c  134  272c  No. atoms (solvent)  888  364  952  No. atoms (other)  62  40  40  B-factor (protein) (Å2)  14.6  16.3  16.8  B-factor (peptide) (Å2)  20.1  15.1  26.8  B-factor (solvent) (Å2)  23.9  21.9  26.0  B-factor (other) (Å2)  30.9  37.7  32.3  Ramachandran plot            Preferred regions (%)  89.9  90.0  90.2      Allowed regions (%)  9.6  9.5  9.3      Outliers (%)  0.5  0.5  0.5  RMSD bond (Å)  0.014  0.013  0.014  RMSD angle (°)  1.76  1.55  1.71  PDB entry code  5YD3  5YD5  5YD4  Data collection  4B08 + pep1  4B08 + N3A  4B08 + T6A  Space group  P 1 21 1  P 2 21 21  P 1 21 1  Unit cell            a, b, c (Å)  80.5, 73.0, 87.0  61.6, 81.9, 114.2  80.5, 72.9, 86.5      α, β, γ (°)  90.0, 114.3, 90.0  90.0, 90.0, 90.0  90.0, 114.2, 90.0  Resolution (Å)  33.1–1.35  37.3–1.96  33.1–1.35  Wavelength  1.000  1.000  1.000  Observations  723,496 (103,749)  309,459 (15,814)  853,946 (97,855)  Unique reflections  193,517 (27,587)  40,361 (2,555)  197,862 (27,517)  Rmerge. (%)  8.3 (51.9)  12.9 (42.5)  8.1 (39.2)  CC1/2  0.995 (0.759)  0.997 (0.939)  0.996 (0.843)  I/σI  9.7 (2.6)  10.0 (3.7)  10.6 (2.7)  Multiplicity  3.7 (3.8)  7.7 (6.2)  4.3 (3.6)  Completeness (%)  96.5 (94.4)  95.6 (87.1)  99.1 (94.9)  Refinement statistics        Resolution (Å)  33.1–1.35  37.3–1.96  33.1–1.35  Rwork/Rfree (%)b  13.6/18.4  20.3/25.8  13.5/17.3  No. atoms (protein)  7,554  3,742  7,330  No. atoms (peptide)  322c  134  272c  No. atoms (solvent)  888  364  952  No. atoms (other)  62  40  40  B-factor (protein) (Å2)  14.6  16.3  16.8  B-factor (peptide) (Å2)  20.1  15.1  26.8  B-factor (solvent) (Å2)  23.9  21.9  26.0  B-factor (other) (Å2)  30.9  37.7  32.3  Ramachandran plot            Preferred regions (%)  89.9  90.0  90.2      Allowed regions (%)  9.6  9.5  9.3      Outliers (%)  0.5  0.5  0.5  RMSD bond (Å)  0.014  0.013  0.014  RMSD angle (°)  1.76  1.55  1.71  PDB entry code  5YD3  5YD5  5YD4  aStatistical values given in parenthesis refer to the highest resolution bin (see also Figs 1 and 5). bRfree was calculated as Rwork taking 2–4% of data not included in the refinement. cThe greater number of modeled atoms of pep1 with respect to T6A reflect the existence of alternative conformations. MD simulations MD simulations were performed using the NAMD 2.9 package (32) with the CHARMM22 force field (33) and the CMAP backbone energy correction (34). The initial coordinates of the WT peptide and the variants were linear conformations where all the phi/psi angles were set to 180° and the N-terminal and C-terminal residues were capped with acetyl and N-methyl amide groups, respectively. These isolated linear peptide structures were solvated with TIP3P water (35) in a rectangular box such that the minimum distance to the edge of the box is 12 Å under periodic boundary conditions. Sodium or chloride ions were included to neutralize the protein charge, then further ions were added corresponding to a salt solution of concentration 0.14 M. The time step was set to 2 fs throughout the simulations. A cut-off distance of 10 Å for Coulomb and van der Waals interactions was employed. Coulomb interactions were evaluated through the particle mesh Ewald method (36). The systems were first energy minimized by the conjugate gradient method (5,000 steps) and were gradually heated from 0 K to 298 K over 50 ps. Subsequently, the equilibration and production runs were performed five times for each peptide in the NPT ensembles at 298 K for 200 ns (1 μs in total). Coordinate frames were saved every 10 ps from the trajectory. Results High-resolution crystal structure of the antibody–peptide complex The high-resolution crystal structure of the complex of the scFv antibody 4B08 with pep1 was determined at 1.35 Å resolution (Fig. 1, Table II). The structures of the antibody–peptide complexes present in the asymmetric unit were mostly identical to each other as indicated by the low root-mean square deviation (RMSD) values (RMSD4B08 = 0.6 ± 0.2 Å and RMSDpep1 = 0.4 ± 0.2 Å), the number and distance of the nine intermolecular and four water-mediated H-bonds and the interaction surface (995 ± 31 Å2) were very similar in all the copies in the asymmetric unit (Fig. 1c, d, Supplementary Tables SI and SII). The conformation of the peptide bound to the antibody differs from the helical conformation observed in a 27-long peptide that included the sequence of pep1 (and presumably better representing the structure of that region in CCR5), which may explain the lack of binding activity of this antibody for the full-length protein. Table II. Thermodynamic parameters of binding of pep1 and mutants to scFv (see also Fig. 2)a Peptide  KD (µM)  Ratio  ΔG (kcal mol−1)  ΔH (kcal mol−1)  −TΔS (kcal mol−1)  n  pep1  0.48 ± 0.08  n.a.  −8.6 ± 0.1  −17.2 ± 0.8  8.6 ± 0.7  0.94 ± 0.05  N3A  1.3 ± 0.3  2.7  −8.1 ± 0.1  −13.7 ± 0.3  5.7 ± 0.2  1.09 ± 0.05  Y4A  0.71 ± 0.27  1.5  −8.4 ± 0.2  −18.4 ± 2.1  9.9 ± 2.3  1.02 ± 0.14  Y5A  1.1 ± 0.5  2.3  −8.2 ± 0.3  −12.5 ± 0.7  4.3 ± 0.4  1.02 ± 0.02  T6A  13 ± 2.7  27  −6.7 ± 0.1  −8.2 ± 0.7  1.5 ± 0.8  0.90 ± 0.04  S7A  n.d.  >100  n.d.  n.d.  n.d.  n.d.  E8A  3.5 ± 0.6  7.3  −7.5 ± 0.1  −14.8 ± 0.6  7.3 ± 0.7  0.87 ± 0.05  P9A  n.d.  >100  n.d.  n.d.  n.d.  n.d.  Peptide  KD (µM)  Ratio  ΔG (kcal mol−1)  ΔH (kcal mol−1)  −TΔS (kcal mol−1)  n  pep1  0.48 ± 0.08  n.a.  −8.6 ± 0.1  −17.2 ± 0.8  8.6 ± 0.7  0.94 ± 0.05  N3A  1.3 ± 0.3  2.7  −8.1 ± 0.1  −13.7 ± 0.3  5.7 ± 0.2  1.09 ± 0.05  Y4A  0.71 ± 0.27  1.5  −8.4 ± 0.2  −18.4 ± 2.1  9.9 ± 2.3  1.02 ± 0.14  Y5A  1.1 ± 0.5  2.3  −8.2 ± 0.3  −12.5 ± 0.7  4.3 ± 0.4  1.02 ± 0.02  T6A  13 ± 2.7  27  −6.7 ± 0.1  −8.2 ± 0.7  1.5 ± 0.8  0.90 ± 0.04  S7A  n.d.  >100  n.d.  n.d.  n.d.  n.d.  E8A  3.5 ± 0.6  7.3  −7.5 ± 0.1  −14.8 ± 0.6  7.3 ± 0.7  0.87 ± 0.05  P9A  n.d.  >100  n.d.  n.d.  n.d.  n.d.  aExperiments were performed at 25°C. Table II. Thermodynamic parameters of binding of pep1 and mutants to scFv (see also Fig. 2)a Peptide  KD (µM)  Ratio  ΔG (kcal mol−1)  ΔH (kcal mol−1)  −TΔS (kcal mol−1)  n  pep1  0.48 ± 0.08  n.a.  −8.6 ± 0.1  −17.2 ± 0.8  8.6 ± 0.7  0.94 ± 0.05  N3A  1.3 ± 0.3  2.7  −8.1 ± 0.1  −13.7 ± 0.3  5.7 ± 0.2  1.09 ± 0.05  Y4A  0.71 ± 0.27  1.5  −8.4 ± 0.2  −18.4 ± 2.1  9.9 ± 2.3  1.02 ± 0.14  Y5A  1.1 ± 0.5  2.3  −8.2 ± 0.3  −12.5 ± 0.7  4.3 ± 0.4  1.02 ± 0.02  T6A  13 ± 2.7  27  −6.7 ± 0.1  −8.2 ± 0.7  1.5 ± 0.8  0.90 ± 0.04  S7A  n.d.  >100  n.d.  n.d.  n.d.  n.d.  E8A  3.5 ± 0.6  7.3  −7.5 ± 0.1  −14.8 ± 0.6  7.3 ± 0.7  0.87 ± 0.05  P9A  n.d.  >100  n.d.  n.d.  n.d.  n.d.  Peptide  KD (µM)  Ratio  ΔG (kcal mol−1)  ΔH (kcal mol−1)  −TΔS (kcal mol−1)  n  pep1  0.48 ± 0.08  n.a.  −8.6 ± 0.1  −17.2 ± 0.8  8.6 ± 0.7  0.94 ± 0.05  N3A  1.3 ± 0.3  2.7  −8.1 ± 0.1  −13.7 ± 0.3  5.7 ± 0.2  1.09 ± 0.05  Y4A  0.71 ± 0.27  1.5  −8.4 ± 0.2  −18.4 ± 2.1  9.9 ± 2.3  1.02 ± 0.14  Y5A  1.1 ± 0.5  2.3  −8.2 ± 0.3  −12.5 ± 0.7  4.3 ± 0.4  1.02 ± 0.02  T6A  13 ± 2.7  27  −6.7 ± 0.1  −8.2 ± 0.7  1.5 ± 0.8  0.90 ± 0.04  S7A  n.d.  >100  n.d.  n.d.  n.d.  n.d.  E8A  3.5 ± 0.6  7.3  −7.5 ± 0.1  −14.8 ± 0.6  7.3 ± 0.7  0.87 ± 0.05  P9A  n.d.  >100  n.d.  n.d.  n.d.  n.d.  aExperiments were performed at 25°C. Fig. 1 View largeDownload slide X-ray crystal structure of the complex between pep1 and scFv antibody 4B08. (a) and (b) Structure of the critical CDR region of the antibody when in complex with pep1, represented here in two orientations differing by 90°. The heavy and light chains of the antibody are shown in dark and light gray, respectively. The peptide is depicted with orange sticks. The blue mesh corresponds to the sigma-A weighted 2fo-fc electron density map at 1.35 Å resolution (σ = 1). (c) Detailed interaction map at the contact interface between peptide and antibody. Carbon atoms corresponding to pep1 and 4B08 are depicted in orange and dark gray, respectively. Water molecules are represented by blue circles. The panel was prepared with LigPlot+ (54). (d) Detailed three-dimensional view of the intermolecular H-bond network between the peptide and antibody. Peptide and key residues of the antibody are depicted with orange and gray sticks, respectively. (e) Intramolecular H-bond network within the peptide. See also Table I, Supplementary Fig. S1 and Tables SI–SIII. Fig. 1 View largeDownload slide X-ray crystal structure of the complex between pep1 and scFv antibody 4B08. (a) and (b) Structure of the critical CDR region of the antibody when in complex with pep1, represented here in two orientations differing by 90°. The heavy and light chains of the antibody are shown in dark and light gray, respectively. The peptide is depicted with orange sticks. The blue mesh corresponds to the sigma-A weighted 2fo-fc electron density map at 1.35 Å resolution (σ = 1). (c) Detailed interaction map at the contact interface between peptide and antibody. Carbon atoms corresponding to pep1 and 4B08 are depicted in orange and dark gray, respectively. Water molecules are represented by blue circles. The panel was prepared with LigPlot+ (54). (d) Detailed three-dimensional view of the intermolecular H-bond network between the peptide and antibody. Peptide and key residues of the antibody are depicted with orange and gray sticks, respectively. (e) Intramolecular H-bond network within the peptide. See also Table I, Supplementary Fig. S1 and Tables SI–SIII. From the number of intermolecular H-bonds, B-factor analysis (BSA; Supplementary Fig. S1) and frequent disorder of Asp1 of pep1 in the electron density, it was suggested that the binding contribution of the C-terminal region (residues 6–9) of the peptide carries more weight than that of the N-terminal region (residues 1–5, see below for a more comprehensive validation by site-directed mutagenesis). Residues Ser 7 and Pro9 of the peptide contributed with the greatest BSA (106 ± 1 Å2 and 134 ± 3 Å2, respectively) and number of polar interactions (four H-bonds, and one H-bond and one salt bridge, respectively). In contrast, residues Asn3 and Thr6 contributed the least to BSA of the complex (14 ± 6 Å2 and 18 ± 1 Å2, respectively) not engaging in polar interactions with the antibody. Nonetheless, these two residues participate in the four intramolecular H-bonds encountered in the peptide (Fig. 1e, Supplementary Table SIII). Two intramolecular H-bonds were formed between the carbonyl group of the side-chain of Asn3 and the hydrogen of the main-chain nitrogen atom of residues Tyr4 and Tyr5. The other two intramolecular H-bonds were contributed the side chain hydroxyl of Thr6, which engaged the main-chain carbonyl and the carboxylic acid group of Glu8. The combination of minimal intermolecular interaction with the antibody and the formation of intramolecular H-bonds exclusively by these two residues in the complex was thus a unique opportunity to isolate the contribution of this ubiquitous class of H-bonds in the recognition of a peptide by an antibody. Energetic contributions to binding The ITC technique was employed to dissect the thermodynamic basis of binding of pep1 to 4B08 and to determine the energetic contributions of individual residues from Asn3 to Pro9 (Fig. 2, Table II, Supplementary Fig. S2). The dissociation constant (KD) of the complex between pep1 and 4B08 was determined from the binding isotherm by non-linear regression (KD = 480 nM). The affinity of the peptide for the antibody was driven by a large change of enthalpy (ΔH = −17.2 ± 0.8 kcal mol−1) and opposed by an unfavourable change of entropy (−TΔS = 8.7 ± 0.7 kcal mol−1). The thermodynamic signature is consistent with the binding of a flexible peptide to its target, since the large increase in non-covalent interactions (favourable enthalpy change) was opposed by a substantial entropic-loss resulting from the combined loss of conformational dynamics and translational and rotational degrees of freedom. Fig. 2 View largeDownload slide Search for hot-spot residues. (a)–(h) Characterization of the binding of pep1 (and Ala-scan variants) to scFv 4B08 by ITC. The antibody was titrated with peptide at 25°C. In each panel, the upper plot corresponds to the titration kinetics, whereas the lower plot represents the integrated binding isotherms. Molar ratio refers to the relative concentration of peptide-to-protein in the cell. The binding enthalpy (ΔH) and the dissociation constant (KD) were obtained by non-linear regression of the integrated data to a one-site binding model with the program ORIGIN. The results are given in Table I. Fig. 2 View largeDownload slide Search for hot-spot residues. (a)–(h) Characterization of the binding of pep1 (and Ala-scan variants) to scFv 4B08 by ITC. The antibody was titrated with peptide at 25°C. In each panel, the upper plot corresponds to the titration kinetics, whereas the lower plot represents the integrated binding isotherms. Molar ratio refers to the relative concentration of peptide-to-protein in the cell. The binding enthalpy (ΔH) and the dissociation constant (KD) were obtained by non-linear regression of the integrated data to a one-site binding model with the program ORIGIN. The results are given in Table I. Substitution of each individual residue (Asn3 to Pro9) to alanine ablates the side chain from the β-carbon and further, thus revealing the relative contribution of each side-chain to the binding constant and its energetic consequences (Fig. 2a–h, Table II). The separate replacement of the residues with the highest contribution to BSA and intermolecular interactions, Ser7 and Pro9, with Ala (S7A and P9A) completely abrogated the engagement of the peptide to the antibody. The substitution of Glu8 also in the C-terminal region of the peptide with Ala (E8A) reduced the affinity for the antibody with respect to pep1 by c.a. 7-fold (ΔΔG = 1.1 ± 0.2 kcal mol−1) accompanied by a decrease in the enthalpy change (ΔΔH = 2.4 ± 1.4 kcal mol−1). The replacement of residues engaging in intramolecular H-bonds in the peptide Asn3 and Thr6 with Ala (N3A and T6A) was also examined. The ablation of the side chain of Asn3 reduced moderately the affinity of the modified peptide for the antibody by c.a. 2.5-fold, in line with what was observed for other residues of the N-terminal region (Y4A, Y5A). In contrast to N3A, the substitution of Thr6 with Ala had a large and negative effect on the affinity, which plunged by 27-fold (ΔΔG = 1.9 ± 0.2 kcal mol−1). In thermodynamic terms, the poor binding ability of T6A is explained by a remarkable loss of favourable enthalpy with respect to the unchanged peptide (ΔΔH = 9.0 ± 1.5 kcal mol−1), suggesting a significant erosion in the strength of the interactions in the antibody–peptide complex. This effect was somehow unexpected given the little contribution of Thr6 to BSA and the absence of direct intermolecular interactions with the antibody. The representation of the thermodynamic data for all the substitutions examined (Supplementary Fig. S2) shows a moderate correlation between the loss of affinity and the change of enthalpy, suggesting that, in general, lower affinity resulted from weaker non-covalent interactions. As expected, the changes of enthalpy and entropy are fully correlated by the well-known entropy–enthalpy compensation effect (37). Behaviour of peptides in solution (MD simulations) Because we aimed at producing a complete picture of the behaviour of the peptide not only in complex with the antibody but also in solution (unbound state), we performed MD simulations and focused our attention on the four intramolecular H-bonds observed in the crystal structure (Fig. 3). A total simulation time of 1 μs for each peptide was analyzed, divided into five independent runs of 200 ns each. The results obtained in illustrative runs are given in the accompanying movies and graphs, showing large fluctuations in the conformation of the peptides and lacking a stable secondary structure, both observations consistent with the expected behavior of a flexible peptide in solution (Supplementary Fig. S3, Movies S1–3). Fig. 3 View largeDownload slide Characterization of intramolecular H-bonds in the unbound form of the peptide. (a) Schematic representation of the four intramolecular bonds under investigation (#1, #2, #3, and #4) as they appear in the bound form of pep1 (protein not shown). (b) Distance probability plot between heavy atoms participating in intramolecular H-bonds during MD simulations of the unbound peptide. Data of H-bonds corresponding to #1–2 of N3A, and #3–4 of T6A are not shown because the mutations abrogate those bonds. Distances were determined with UCSF Chimera 1.10.1 (55) and the normalized distribution plots calculated with R (version 3.2.3, https://www.R-project.org/) using kernel density estimation. Orange, green and blue corresponded to the data obtained for pep1, N3A, and T6A, respectively. (c) Fraction of structures showing the indicated H-bonds. An H-bond was considered to appear when the distance between the proton donor and acceptor was less than 3.5 Å (38, 39) as calculated by UCSF Chimera. Restricting the H-bond angle to <150° did not significantly affect the estimation of the fraction of intramolecular H-bonds #1–4. See also Supplementary Figs S2–S4 and Movies S1–S3. Fig. 3 View largeDownload slide Characterization of intramolecular H-bonds in the unbound form of the peptide. (a) Schematic representation of the four intramolecular bonds under investigation (#1, #2, #3, and #4) as they appear in the bound form of pep1 (protein not shown). (b) Distance probability plot between heavy atoms participating in intramolecular H-bonds during MD simulations of the unbound peptide. Data of H-bonds corresponding to #1–2 of N3A, and #3–4 of T6A are not shown because the mutations abrogate those bonds. Distances were determined with UCSF Chimera 1.10.1 (55) and the normalized distribution plots calculated with R (version 3.2.3, https://www.R-project.org/) using kernel density estimation. Orange, green and blue corresponded to the data obtained for pep1, N3A, and T6A, respectively. (c) Fraction of structures showing the indicated H-bonds. An H-bond was considered to appear when the distance between the proton donor and acceptor was less than 3.5 Å (38, 39) as calculated by UCSF Chimera. Restricting the H-bond angle to <150° did not significantly affect the estimation of the fraction of intramolecular H-bonds #1–4. See also Supplementary Figs S2–S4 and Movies S1–S3. The intramolecular H-bond pattern, and their distances, were monitored every 100 ps, resulting in 10,000 independent structures being analyzed (Fig. 3b and c). An H-bond was considered to be present if the distance between the heavy atoms was within the standard cut-off distance of 3.5 Å (38, 39). No significantly different results were obtained when H-bonds were restricted to angles less than 150° (Supplementary Fig. S4). Because of the inherent flexibility of the peptide, the frequency of intramolecular H-bonds in solution was very low (≤ 3%) except for the intramolecular H-bond #3, which was detected repeatedly in about 25% of the trajectories of pep1, and half that value in N3A. Hydrogen bonds linking the atom γO of the side chain of residue i of Ser or Thr to the main chain NH group of the residue i + 2 are termed ST turn, and are often found in protein structures (40). This H-bond is not present in T6A, since the substitution of Thr6 by Ala removes the necessary atom γO from the side chain. The distance between the heavy atoms comprising the H-bonds also undergo large fluctuations, except for the distance between the atoms participating in H-bond #3 (Supplementary Fig. S5). To evaluate structural differences among the peptides in solution, the dihedral angles of residues Ile2 to Glu8 were calculated. Three-dimensional comparative Ramachandran plots are shown in Fig. 4a and b and the conventional bi-dimensional plots in Supplementary Fig. S6. The three peaks appearing in these plots correspond to the combination of phi and psi angles of the major secondary structure elements (α-helix, −60°/−45°; β-sheet, −135°/135°; left-handed helix, 60°/45°). As revealed from the examination of the Ramachandran plots, the secondary structure profile of pep1 and T6A differ significantly except in the last residue Glu8. In particular, a greater α-helical content was observed in residues Ile2 to Thr6 of the peptide T6A with respect to pep1. This region was stabilized by three intramolecular H-bonds different from those examined earlier and formed between main chain atoms of Asp1/Tyr5, Ile2/Thr6 and Asn3/Ser7 (Fig. 4c). These three intramolecular H-bonds appeared in 42–44% of the trajectories in T6A, but only 2–4% in pep1. The effect on the conformation of N3A was somehow intermediate between pep1 and T6A, appearing in 19–26% of the trajectories. Therefore, the loss of the side chain of Asn, and especially of Thr6, increased the formation of other intramolecular H-bonds in solution that favoured α-helical conformations. Fig. 4 View largeDownload slide Secondary structure of unbound pep1, N3A, and T6A in solution. (a) Ramachandran probability plot of a representative residue (Tyr4) obtained from MD simulations. The dihedral angle was calculated with UCSF Chimera, and the normalized distribution plots calculated with R using kernel density estimation. The arrows point at three key regions of the phi/psi angle plot, i.e. α-helix, β-sheet, and left-handed helix. The colors orange, green and blue corresponds to pep1, N3A and T6A, respectively. (b) Ramachandran probability plots for other residues of the peptide using the same color scheme. The asterisk in Asn3 and Thr6 indicates that each of these residues is changed to Ala in N3A and T6A, respectively. (c) Fraction of intramolecular H-bonds between main-chain atoms of the indicated residues. These bonds represent mainly helical conformations, especially α-helix. An H-bond was considered to appear when the distance between the proton donor and acceptor was less than 3.5 Å as calculated by UCSF Chimera. Fig. 4 View largeDownload slide Secondary structure of unbound pep1, N3A, and T6A in solution. (a) Ramachandran probability plot of a representative residue (Tyr4) obtained from MD simulations. The dihedral angle was calculated with UCSF Chimera, and the normalized distribution plots calculated with R using kernel density estimation. The arrows point at three key regions of the phi/psi angle plot, i.e. α-helix, β-sheet, and left-handed helix. The colors orange, green and blue corresponds to pep1, N3A and T6A, respectively. (b) Ramachandran probability plots for other residues of the peptide using the same color scheme. The asterisk in Asn3 and Thr6 indicates that each of these residues is changed to Ala in N3A and T6A, respectively. (c) Fraction of intramolecular H-bonds between main-chain atoms of the indicated residues. These bonds represent mainly helical conformations, especially α-helix. An H-bond was considered to appear when the distance between the proton donor and acceptor was less than 3.5 Å as calculated by UCSF Chimera. Crystal structures of N3A and T6A in complex with 4B08 Crystal structures of 4B08 in complex with N3A or with T6A were determined 1.96 Å and 1.35 Å resolution, respectively (Fig. 5a–d, Table I). The complex between N3A and the antibody was crystallized in spacegroup P22121 containing two copies in the asymmetric unit that were essentially identical to each other (RMSD of antibody or N3A < 0.12 Å). This structure was also very similar to that of the complex with pep1 (RMSD4B08 = 0.5 ± 0.1 Å; RMSDpeptide = 0.3 ± 0.2 Å). We note that the RMSD values between these two different crystal structures were smaller than the values found for copies within the same asymmetric unit in the complex of 4B08 with pep1 (see above) (Fig. 5e). Despite their overall similarity, the rotamers of residues Tyr4 and Tyr5 adopted a different conformation that we attributed to the loss of intramolecular H-bonds caused by the Asn to Ala substitution and to differences in the crystal packing forces. In this complex, Tyr5 engaged in CH–π interactions with Ala3, and Tyr4-Tyr5 displayed π–π stacking interactions. Although no significant differences of the BSA in the critical C-terminal region were observed, the re-arrangement of the Tyr residues resulted in the loss of one of the nine intermolecular H-bonds between the N3A peptide and the antibody. Specifically, H-bonds between Tyr5 and Asn61 and between two bridging water molecules are lost, although it is partially compensated by a new water-mediated H-bond network (Supplementary Table SI). Fig. 5 View largeDownload slide X-ray crystal structure of N3A and T6A in complex with antibody. (a) X-ray crystal structure of N3A bound to 4B08. The heavy and light chains of the antibody are shown in dark and light gray, respectively. The peptide (green) and key residues of the antibody are depicted with sticks. Intermolecular H-bonds between the peptide and the protein are indicated. (b) Same crystal structure showing only the intramolecular H-bonds of the peptide. (c) and (d) Equivalent structure of T6A (blue) bound to 4B08. (e) Overlay of the main-chain trace of pep1 (orange), N3A (green), and T6A (blue) bound to 4B08 (light gray). (f) Similarity plot between the conformation of the unbound peptide in the MD simulations and the structure bound to the antibody. The RMSD values were calculated with VMD version 1.9.2 (56) and the normalized distribution plots calculated with R using kernel density estimation. The arrow highlights a region of high structural similarity to the peptide in the bound form, which is populated by pep1 but not by the modified peptides. (g) Number of similarity events between the structures of the peptide in the unbound form (MD simulations) and bound to antibody form (X-ray) at different timescales. The RMSD at which an event is recorded was set to a cutoff value of >2.0 Å. The RMSD values were calculated with VMD using the coordinates of Cα. A close-up view of the data corresponding to the longest time-scale (>1 ns) is also shown. See also Table I and Supplementary Figs S1 and S5 and Tables SI–SIII. Fig. 5 View largeDownload slide X-ray crystal structure of N3A and T6A in complex with antibody. (a) X-ray crystal structure of N3A bound to 4B08. The heavy and light chains of the antibody are shown in dark and light gray, respectively. The peptide (green) and key residues of the antibody are depicted with sticks. Intermolecular H-bonds between the peptide and the protein are indicated. (b) Same crystal structure showing only the intramolecular H-bonds of the peptide. (c) and (d) Equivalent structure of T6A (blue) bound to 4B08. (e) Overlay of the main-chain trace of pep1 (orange), N3A (green), and T6A (blue) bound to 4B08 (light gray). (f) Similarity plot between the conformation of the unbound peptide in the MD simulations and the structure bound to the antibody. The RMSD values were calculated with VMD version 1.9.2 (56) and the normalized distribution plots calculated with R using kernel density estimation. The arrow highlights a region of high structural similarity to the peptide in the bound form, which is populated by pep1 but not by the modified peptides. (g) Number of similarity events between the structures of the peptide in the unbound form (MD simulations) and bound to antibody form (X-ray) at different timescales. The RMSD at which an event is recorded was set to a cutoff value of >2.0 Å. The RMSD values were calculated with VMD using the coordinates of Cα. A close-up view of the data corresponding to the longest time-scale (>1 ns) is also shown. See also Table I and Supplementary Figs S1 and S5 and Tables SI–SIII. The complex between T6A and 4B08 crystallized in the same spacegroup than that of pep1. As in the examples from above, the complexes of 4B08 with T6A were essentially indistinguishable to each other when compared with either copies within the asymmetric unit (RMSD4B08 = 0.6 ± 0.1 Å; RMSDT6A = 0.4 ± 0.2 Å) or when compared with the complex containing the unmodified pep1 (RMSD4B08 = 0.4 ± 0.3 Å; RMSDpeptide = 0.3 ± 0.2 Å). Similarly, the values of BSA, and the number and distances of the intermolecular and water-mediated H-bonds were well conserved with respect to the complex with pep1, except for the intramolecular H-bonds #3 and #4 eliminated upon substitution of the side chain of Thr6. When the bound conformations of the peptides were compared with the ensemble in solution, a complex landscape emerged. The conformation of the peptide bound to the antibody only represented a minor fraction of the ensemble in solution. The vast majority of unbound conformations of the unmodified and modified peptides appeared in the region where the RMSD with respect to the bound form was > 2 Å, as expected from a flexible peptide (Fig. 5f). However, we also observed a small fraction of conformations with low RMSD (< 2 Å) for pep1 epitope and to a lesser degree for N3A. Moreover, the number of occurrences and the duration of these low RMSD structures increased steeply from T6A to N3A to pep1 (Fig. 5g). When the comparison of the solution ensemble with the bound form is restricted to only the N-terminal region, or the C-terminal region, opposite trends are identified for pep1 with respect to T6A (Supplementary Fig. S7). On the one hand, the conformation of the N-terminal of the peptide in solution and the bound form is more similar for T6A. On the other hand, when comparing the C-terminal region (critical for binding), pep1 displays the largest population of homolog conformers. The peptide N3A shows intermediate behaviour. Collectively, these data suggest that the recognition of the peptide is at least in part favoured by a conformational selection mechanism resulting from a critical intramolecular H-bond. Discussion Although intramolecular H-bonds are often found in peptides bound to antibodies (21, 41, 42), there have not been comprehensive studies addressing their influence in the dynamics of the peptide in solution and the structure and thermodynamics in the bound state. We selected the model peptide pep1 corresponding to the N-terminal region of CCR5 to explore the intimate details of intramolecular H-bonds in the recognition by an antibody. Our approach encompassed MD simulations, binding thermodynamics and high-resolution X-ray crystallography, revealing a multifaceted influence of this important class of H-bonds in the structural dynamics of the peptide in solution and in the affinity for the antibody. The results indicate that a critical intramolecular H-bond is significantly populated in solution despite the intrinsic flexibility of the peptide, exerting a strong influence in the change of enthalpy in the bound state that strengthened the binding constant. To mimic the solution structures of the pep1, we have resorted to MD simulations employing the CHARMM22/CMAP force field. Recent benchmarks showed that, during MD simulations with the CHARMM22/CMAP force field, an unstructured peptide is sometimes biased towards α-helical conformations (43). Although the higher tendency of N3A and T6A toward helical conformations might have been influenced by the force field, simulations of the wild type pep1 rarely sampled helical conformations at the corresponding region (Fig. 4). Therefore, the formation of the intramolecular hydrogen bond by the side chain of Thr6, the key hydrogen bond in this study, was independent from force field bias. Current empirical force fields are not fully free from bias to a varying extent (44–46). However, considering the consistency of our experimental measurements, and the general absence of force field bias, we believe our conclusions are relevant to the general mechanism of peptide–antibody interactions. Based on our observations, we have elaborated the diagram shown in Fig. 6 explaining the recognition of a flexible peptide by an anti-peptide antibody and the role of its intramolecular H-bonds. In solution, a critical intramolecular H-bond like that between Thr6 and the main chain of Glu8 transiently stabilizes a conformation, which could form a well populated (i.e. lowest energy) state in the unbound peptide ensemble. Such a well populated and stabilized unbound peptide conformation would be thus recognized by an antibody with higher probability. The region where the critical intramolecular H-bond interaction is established becomes essential for an overall binding process. In our particular example, this region comprises residues of the C-terminal of the peptide. Without the critical #3 intramolecular H-bond, the peptide could not form the populated states of the C-terminal region (Supplementary Fig. S7b), and the antibody would have less opportunities to mature recognizing the C-terminal conformation of the peptide antigen. Moreover, additional intramolecular H-bonds in the critical epitope and in the neighbouring region further favour the energetic component of the binding reaction by increasing the enthalpic component and increasing the overall affinity. It will be interesting to investigate whether the peptide containing the substitution T6A, displaying a significant difference in the Ramachandran probability plot in solution with respect to pep1 (Fig. 4), would elicit antibodies recognizing the new conformation of the peptide. Fig. 6 View largeDownload slide Multifaceted contribution of intramolecular H-bonds for the binding of a flexible peptide to an antibody. The model represents the free energy profile of the peptide along the reaction coordinate. In the unbound form, the peptide appears as a flexible ensemble of different conformations. Intramolecular H-bonds make a modest but critical contribution favouring conformations that resemble the structure in the bound state. When bound to the antibody, the peptide is greatly stabilized by these intramolecular H-bonds, bringing a neat favourable change of enthalpy. Intramolecular H-bonds in the vicinity of the hot-spot region (C-terminal region) make a stronger contribution than those in the N-terminal region. Fig. 6 View largeDownload slide Multifaceted contribution of intramolecular H-bonds for the binding of a flexible peptide to an antibody. The model represents the free energy profile of the peptide along the reaction coordinate. In the unbound form, the peptide appears as a flexible ensemble of different conformations. Intramolecular H-bonds make a modest but critical contribution favouring conformations that resemble the structure in the bound state. When bound to the antibody, the peptide is greatly stabilized by these intramolecular H-bonds, bringing a neat favourable change of enthalpy. Intramolecular H-bonds in the vicinity of the hot-spot region (C-terminal region) make a stronger contribution than those in the N-terminal region. A recent computational study has suggested that intrinsically disordered proteins adopt bound conformations before binding to its binding partner (47). Similar transient conformations have been proposed in more general associations involving globular proteins (48, 49). In line with these observations, we have shown that intramolecular hydrogen bonds also have general implications for the molecular mechanisms of peptide (and possibly protein) association. We would like to stress that this mechanism is different to that presented as pre-organization (50–53), since the competent structure of pep1 in solution is transiently stabilized by the intramolecular H-bond, and the influence in binding occurs mostly in the bound state. Collectively, our study established a role for intramolecular H-bonds in antibody–antigen recognition. Conclusions Anti-peptide antibodies recognize their flexible peptide-antigens, even in the absence of defined secondary or tertiary structures in solution. However, it is reasonable to assume that some type of structure should guide their selection and antibody maturation. Our study addresses this question to an unprecedented level of molecular detail. Herein, we have demonstrated that the epitope region of a model flexible peptide in solution sporadically adopts the structure of the bound state, being stabilized by intramolecular H-bonds prior to binding. This result suggests that structures transiently stabilized by intramolecular H-bonds would be one of the critical factors to take into consideration in the preparation of antigens for rational antibody production. Supplementary Data Supplementary Data are available at JB Online. Acknowledgements We thank the staff of the Photon Factory (Tsukuba, Japan) for excellent technical support. Access to beamlines BL5A and AR-NE3A was granted by the Photon Factory Advisory Committee (Proposal nos. 2013G378, and 2014G190). We acknowledge access to the super-computing facilities at the Human Genome Center of The University of Tokyo (http://sc.hgc.jp/shirokane.html). We are grateful to Dr. Eugenio Vazquez (Santiago de Compostela, Spain) for insightful discussions. Funding This work was supported by the Funding program for world-leading Innovative R&D on Science and Technology (FIRST) from JSPS, the Platform for Drug Discovery, Informatics, and Structural Life Science (MEXT), JSPS Grants-in-Aid for Scientific Research 25249115 (K.T.) and 15K06962 (J.M.M.C.). Conflict of Interest None declared. References 1 Tsumoto K., Caaveiro J.M.M. ( 2016) Antigen-antibody binding. eLS . doi: 10.1002/9780470015902.a0001117.pub3 2 Diamantis N., Banerji U. ( 2016) Antibody-drug conjugates-an emerging class of cancer treatment. Br. J. Cancer  114, 362– 367 Google Scholar CrossRef Search ADS PubMed  3 Towbin H., Staehelin T., Gordon J. ( 1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets - procedure and some applications. Proc. Natl. Acad. Sci. USA  76, 4350– 4354 Google Scholar CrossRef Search ADS   4 Kitago Y., Kaneko M.K., Ogasawara S., Kato Y., Takagi J. 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The Journal of BiochemistryOxford University Press

Published: Mar 30, 2018

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