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Detection of alkali metal ions in DNA crystals using state-of-the-art X-ray diffraction experiments

Detection of alkali metal ions in DNA crystals using state-of-the-art X-ray diffraction experiments 1208–1215 Nucleic Acids Research, 2001, Vol. 29, No. 5 © 2001 Oxford University Press Detection of alkali metal ions in DNA crystals using state-of-the-art X-ray diffraction experiments 1 5 Valentina Tereshko, Christopher J. Wilds, George Minasov , Thaza P. Prakash , 5 2,3 4 5 Martin A. Maier ,Andrew Howard , Zdzislaw Wawrzak , Muthiah Manoharan and Martin Egli* Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235, USA, Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, IL 60611, USA, Department of Biological, Chemical and Physical Sciences, Illinois Institute of Technology, Chicago, IL 60616, USA, 3 4 IMCA-CAT, Sector 17 and DND-CAT Synchrotron Research Center, Sector 5, Advanced Photon Source, Argonne National Laboratory, Argonne, IL 60439, USA and Department of Medicinal Chemistry, Isis Pharmaceuticals Inc., Carlsbad, CA 92008, USA Received October 3, 2000; Revised and Accepted January 4, 2001 ABSTRACT structures of oligodeoxynucleotides at atomic resolution 2+ 2+ revealed the positions of Mg and Ca ions that account for The observation of light metal ions in nucleic acids the neutralization of between 50 and 100% of the negative crystals is generally a fortuitous event. Sodium ions charges of phosphate groups (7–12). in particular are notoriously difficult to detect Conversely, there are less than a dozen structures currently because their X-ray scattering contributions are deposited in the Nucleic Acid Database (13) that feature an +… virtually identical to those of water and Na O ordered Na ion and in most of these the ion displays octahedral distances are only slightly shorter than strong coordination geometry. For a list of these structures see hydrogen bonds between well-ordered water mole- Table S1 (Supplementary Material available online). The limited cules. We demonstrate here that replacement of Na number of examples attests to the difficulties of reliably detecting + + + by K ,Rb or Cs and precise measurements of + Na ions with less regular coordination geometry. Thus, anomalous differences in intensities provide a whether a particular peak in Fourier electron density maps particularly sensitive method for detecting alkali ends up as a water molecule or a Na ion is often a matter of metal ion-binding sites in nucleic acid crystals. Not personal judgment and the decision to place an ion, although it only can alkali metal ions be readily located in such may be chemically reasonable, lacks solid experimental + + structures, but the presence of Rb or Cs also allows evidence in many cases. The atomic resolution crystal structure structure determination by the single wavelength of a parallel-stranded G-tetraplex constitutes an exception in anomalous diffraction technique. Besides allowing this respect (14). Since monovalent cations affect the stability of the G-tetrad in a crucial manner, the interpretation that elec- identification of high occupancy binding sites, the tron density peaks in and between the planes formed by four combination of high resolution and anomalous guanines represent sodium ions is unchallenged. diffraction data established here can also pinpoint + + Replacement of Na or K by heavier alkali ions in crystalli- binding sites that feature only partial occupancy. zation of a nucleic acid fragment or soaking crystals in Conversely, high resolution of the data alone does solutions of the heavier alkali ions may be helpful for locating not necessarily allow differentiation between water + + sites occupied by Na or K . For example, crystals of a B-DNA and partially ordered metal ions, as demonstrated dodecamer grown from solutions supplemented with either with the crystal structure of a DNA duplex deter- rubidium or cesium chloride in combination with difference mined to a resolution of 0.6 Å. Fourier synthesis revealed coordination of a single Rb in the minor groove (15). Keeping in mind the differences in ionic INTRODUCTION radius and relative binding affinities of alkali metal ions for + + + + double-stranded DNA (Cs >Rb >K >Na ;16),we may Despite significant advances in the X-ray analysis of 3D structures take this observation as an indication that Na can occupy a of DNA and RNA over the past two decades, reliable detection more or less identical site as Rb . In a similar fashion, binding of light metal ions surrounding nucleic acid molecules remains of K below a so-called AA platform within a domain of group a challenge. Owing to their regular coordination geometry, 2+ 2+ I intron RNA was confirmed by soaking crystals in thallium earth alkali metal ions such as Mg and Ca can often be acetate or cesium hydroxide and examining the corresponding located in Fourier electron density maps. Examples include region of difference electron density maps for changes in crystal structures of small and medium size RNAs determined at both relatively low (1–4) and high (5,6) resolutions. Crystal intensity (17). *To whom correspondence should be addressed. Tel: +1 615 343 8070; Fax: +1 615 343 6707; Email: martin.egli@vanderbilt.edu Correspondence may also be addressed to Muthiah Manoharan. Tel: +1 760 603 2381; Fax: +1 760 929 0036; Email: mmanohar@isisph.com Nucleic Acids Research, 2001, Vol. 29, No. 5 1209 MATERIALS AND METHODS Oligonucleotide synthesis and purification To investigate DNA–ion interactions in crystals we chose A-DNA decamers of sequence GCGTATACGC. This particular DNA sequence was used previously to analyze the conformations of DNA–RNA chimeras (21), DNA hydration (22) and the structural origins of the enhanced RNA affinity (23,24) and exonuclease resistance (25) displayed by 2′-O-modified oligonucleotide analogs. Four DNA decamers with a single 2′-O-aminooxy- ethyl (AOE)-, 2′-O-methyl-3′-methylenephosphonate (MEP)-, 2′-O-fluoroethyl (FET)- or 2′-O-methyl-[tri(oxyethyl)] (TOE)- thymine in place of T6 were included in the crystallization experiments. We found that chemical modification of the T6 sugar moiety in this particular A-DNA decamer leads to crystals of exceptional quality (22). The conformational features of the 2′-O-substituent in TOE were reported before (23) and more detailed accounts of the syntheses, structures and hydration for the other modifications will be described elsewhere. The Z-DNA hexamer CGCGCG was synthesized on the 3 µ mol scale (Pharmacia Expedite), deprotected following established procedures and purified by HPLC. All other oligonucleotides were purified by HPLC and ion exchange chromatography to ≥98%. Figure 1. Idealized curves for the real (f′) and imaginary (f′′) anomalous Crystallizations scattering components of alkali and earth alkali metal ions as a function of X-ray energy (taken from 29). Data for elements P, S and the CuKα wavelength Three different ion concentrations were used to grow crystals (1.5418 Å) are included for comparison. The energy E and the corresponding + + + + with Na ,K ,Rb or Cs : 40 (low salt), 230 (medium salt) and wavelength λ of absorption edges are shown together with the color code for 450 mM (high salt) (Table 1). Hanging drops (4 µ l) containing each ion/element. The wavelength of the beam at the Advanced Photon Source 1 mM DNA, 40 mM alkali metal ions (chloride salt), 20 mM ID beamlines can easily be tuned within the 2–0.66 Å (third harmonic) range. Absorption edges below 6 keV correspond to X-ray wavelengths >2 Å, which alkaline cacodylate buffer, pH 6.0, 6 mM spermine and 10 mM present difficulties for use in crystallography. Among alkali metal ions only the MgCl were equilibrated against 1 ml 35% 2-methyl-2,4- + 2+ K absorption edges of Rb and Sr , located near the right-hand side border, are pentanediol (MPD). Low salt crystals appeared within 2 weeks suitable for use in high energy SAD experiments. The anomalous signals of 2+ + 2+ + and were used for data collection [Mg-MEP (22) and Mg-TOE Ba and Cs as well as of Ca and K can be used at low energy. 2+ (23); only Mg and spermine were located]. The phase diagram technique (26,27) was used to grow medium and high To date there are no reports that exploit the anomalous salt crystals. Thus, 1 (medium salt) and 3 µ l(high salt) 1 M scattering contributions of alkali metal ions to determine their alkali chloride solutions, respectively, were added to locations in nucleic acid crystals. All these ions with the untouched droplets left from low salt crystallizations. Initially exception of Na exhibit significant anomalous effects (Fig. 1). crystals in the droplets dissolved, but appeared again within In combination with a tunable X-ray synchrotron source, the 2 weeks after the MPD concentration in the reservoir was 2+ use of anomalous effects for locating metal ions promises to be raised to 40%. Ba -containing crystals of AOE were grown much more sensitive than difference Fourier synthesis along from 4 µ l hanging drops containing 1 mM DNA, 20 mM with monitoring of the R value (18) during the ensuing sodium cacodylate, pH 6.0, 2 mM spermine and 5 mM BaCl , free refinement cycles. Moreover, the presence of anomalously equilibrated against 1 ml 35% MPD. Z-DNA crystals were + + + scattering K ,Rb or Cs ions offers the opportunity to deter- grown from sitting droplets that contained 1.5 mM DNA, mine nucleic acid crystal structures by the single wavelength 20 mM rubidium cacodylate, pH 7.0, and 10 mM spermine and anomalous diffraction (SAD) technique. were equilibrated against a reservoir of 20% MPD. Here we present protocols for the determination of alkali X-ray data collection and processing metal ion-binding sites in nucleic acid crystals and demon- strate that the anomalous diffraction components of Rb and Crystals suitable for data collection were mounted in nylon Cs are suitable for SAD-type structure determination. In the loops and frozen and stored in liquid nitrogen. Data sets with 2+ first application of the SAD technique for crystallographic crystals containing Ba were collected on an in-house rotating structure determination, diffraction data of a protein–peptide anode generator/image plate set-up (Table 1). All other data complex were phased using a single iodine atom (19). The use collections were conducted at the Advanced Photon Source, of SAD to locate sulfur atoms present in almost all proteins using the insertion device beamlines (ID-B) of the DND and shows promise as a general method for solving the phase IMCA collaborative access teams, located at sectors 5 and 17, problem of native crystals of macromolecules (20). Use of the respectively. Both are equipped with 165 mm MARCCD SAD technique successfully demonstrated here for locating detectors. Data to the maximum resolution limits of the alkali metal ions in crystals of oligodeoxynucleotides should individual crystals were collected at wavelengths <1 Å. To be of general applicability with DNA and RNA. improve completeness and to avoid overloads in the lower 1210 Nucleic Acids Research, 2001, Vol. 29, No. 5 Table 1. Data collection and refinement statistics of A-DNA decamers GCGTATACGC in space group P2 2 2 1 1 1 Compound Ba-AOE Cs-MEP Cs-TOE Rb-FET Rb-TOE K-MEP K-TOE Na-TOE Na-FET Z-DNA Salt form high medium high medium high medium medium medium Unit cell a, Å 24.99 24.82 24.87 24.82 25.16 24.65 25.13 25.13 24.96 18.32 b, Å 44.57 44.33 44.68 45.26 44.83 44.61 44.53 44.53 43.57 30.68 c, Å 45.12 44.82 44.69 43.97 44.82 44.16 45.13 45.13 45.40 42.49 a 2 B factor (Å) DNA 21 8 13 1216 2515 1519 6 Site 1 – 10 15 15 25 30 33 – – – Site2 35(Ba) 2535 3040 3532 2150(Mg) – Site 3 – 60 65 69 70 35 34 25 33 – High resolution X-ray data λ, Å 1.5418 0.8151 0.9500 0.9500 0.9500 0.8151 0.8151 0.8151 0.6630 D, Å 1.70 1.06 1.05 1.05 1.30 1.30 1.30 1.45 0.60 N ref 6913 22 132 19 840 22 802 12 230 12 024 12 024 8695 57 623 % 97.5 97.2 95.1 99.8 99.5 98.9 98.9 99.7 96.0 R 0.068 0.057 0.041 0.046 0.044 0.041 0.041 0.058 0.062 merge R 19.0/21.2 15.0/16.9 16.1/17.8 15.4/17.0 16.5/19.0 20.2/24.6 16.1/17.9 16.1/18.1 15.9/19 16.0/18.5 work/free Anomalous X-ray data λ, Å 1.5418 1.6531 1.7970 0.8151 0.8151 1.6513 D, Å 2.50 2.0 2.0 1.50 1.50 2.0 % 97.5 99.3 98.7 98.8 99.1 98.4 R 0.065 0.082 0.079 0.059 0.044 0.092 merge ∆ F/F 0.0469 0.0784 0.0384 0.0620 0.021 0.019 PP 1.20 1.68 1.35 2.66 – – FOM 0.33 0.41 0.35 0.51 – – Refinement with ion occupancy equal to 1. R factor = Σ |F(hkl) – F(hkl) |/Σ F(hkl) . R = Σ Σ |I(hkl) – <I(hkl)>|/Σ Σ <I(hkl) >. λ, D, the wavelength and resolution of the X-ray data; N ref, %, the hkl o c hkl o merge hkl i i hkl i i number of unique reflections and completeness. R factors based on 2.0 Å anomalous data. ∆ F/F =Σ <|F(+)| – |F(–)|>/<1/2(|F(+)| + |F(–)|>. PP, FOM, phasing power and figure of merit. hkl resolution bins, separate data sets were measured for the low 1I0N (Rb-TOE), 1I0O (K-MEP), 1I0P (K-TOE), 1I0Q (Na- and high resolution ranges in each case. Anomalous data were TOE), 1I0G (Na-FET) and 1I0T (Z-DNA). collected at a wavelength of 0.8151 Å for Rb -containing crystals (third harmonic range) and in the low energy range RESULTS AND DISCUSSION (≥1.54 Å) for all other crystals (Fig. 1). All data were integrated and scaled either in the DENZO/SCALEPACK or HKL2000 Overall strategy suites (28) and selected crystal data and data collection parameters In order to explore the feasibility of using the anomalous are summarized in Table 1. diffraction of alkali metal ions to locate their binding sites in nucleic acid crystals and to provide phase information via SAD, Structure determination, Patterson and electron density we conducted an extensive set of X-ray diffraction experiments at map calculation and refinement the Advanced Photon Source. The study included a variety of Heavy atom searches, Patterson map as well as electron A-form DNA decamer crystals for which multiple data sets at density map calculations and SAD phasing were performed different wavelengths were collected so as to optimize both the with the program CNS (29) and maps were displayed with the magnitude of the anomalous signal and the resolution program TURBO FRODO (30). For all structures initial refine- 2+ (Table 1). The structures of a Ba -form crystal (strong anomalous ments were performed with CNS. To calculate the R values free signal) and of the Z-DNA duplex (resolution 0.6 Å) were used (18) 10% of the data were set aside prior to the refinements. All as ‘anomalous diffraction’ and ‘high resolution’ references. anisotropic refinements with high resolution data were Anomalous data were collected in the low energy range for conducted with the program SHELX-97 (31). Selected refine- + + 2+ + K ,Cs and Ba or, in the case of Rb , at the K absorption edge ment parameters for all structures based on high resolution (Fig. 1). Preliminary diffraction experiments were also conducted data are listed in Table 1. 2+ with decamer crystals containing Sr and confirmed the useful- ness of this ion for SAD-based structure determination (data Coordinates not shown). While anomalous data were typically collected to Final coordinates and structure factors for all structures were resolutions of ∼2 Å, data to high resolution (up to 1.05 Å) were deposited in the Protein Data Bank (PDB). PDB codes: 1I0F also collected at a shorter wavelength for each crystal (Table (Ba-AOE), 1I0J (Cs-MEP), 1I0K (Cs-TOE), 1I0M (Rb-FET), 1). The benefits of atomic resolution data for analyzing ion and Nucleic Acids Research, 2001, Vol. 29, No. 5 1211 solvent interactions in crystals are obvious and an approach that combines the precision of high resolution with the sensi- tivity of anomalous diffraction can be expected to be superior to all previous crystallographic methods to locate alkali metal ions. In addition, to potentially gain an insight into both low and high occupancy DNA binding sites for these metal ions their concentrations in the crystallization solutions were varied (see Materials and Methods). SAD phasing Data collection at the appropriate wavelengths with DNA crystals containing alkali or earth alkali metal ions demonstrates the merits of some of these ions for phasing (Table 1). The heavy atom search procedure in the program CNS was applied to anomalous difference Patterson maps calculated at 2.5, 2.0 and 2+ + 1.5 Å based on data sets for DNA crystals containing Ba ,Cs and Rb , respectively. An example of an anomalous Patterson map is depicted in Figure S1 (Supplementary Material avail- able online). One ion-binding site was located both in Cs-TOE (medium salt) and Ba-AOE crystals (sites 1 and 2, respec- tively, Fig. 2A). Ions at both sites 1 and 2 were located in Cs- MEP and Rb-FET crystals grown under high salt conditions. In the Harker sections peak 1 was twice as high as peak 2. Based on the sites determined in the Patterson maps, SAD phasing was then performed using data to 2.5, 2.0 and 1.8 Å resolution 2+ + + for Ba ,Cs and Rb , respectively. Values for phasing power and figure of merit are listed in Table 1. However, the heavy atom search procedure was not successful in the case of the Rb-TOE (medium salt) and K-MEP (high salt) structures. In order to find additional binding sites the above SAD phases were used in combination with the anomalous differ- ence structure factors to compute anomalous difference Fourier and double-difference (or log likelihood gradient) maps. Examples of anomalous difference Fourier and double- difference maps for the Rb-FET structure are depicted in Figure 2A. In this manner a third ion-binding site was retrieved in the Rb-FET and Cs-MEP structures (site 3). Moreover, a second site (site 2) previously absent in the Cs-TOE structure was also found in this fashion. The resulting phases were subsequently improved by density modification. 2+ As expected, Ba present in the reference structure proved Figure 2. Maps calculated at 1.8 Å resolution with SAD phases for the Rb-FET structure and superimposed on the final structure (selected DNA residues are very potent for phasing and a single site allowed structure numbered). (A) The anomalous difference Fourier (blue) and double-difference determination via SAD. Similarly, Cs - (high and medium salt (red) maps contoured at the 4σ level. Ion-binding sites 1 and 2 (shown with forms) and Rb -form (high salt form) crystals were used for their symmetry mates, designated by lower case letters) were located in the SAD-type phasing. Electron density for two thirds of the anomalous difference Patterson map and used for phasing. Ion-binding site 3 was found at this stage and is characterized by a significantly greater peak duplex was clearly visible with the Ba-AOE, Cs-MEP and Rb- height than the remaining peaks in the double-difference density map. (B)Stereo FET structures and a portion of the Rb-FET experimental diagram of the density-modified electron density map contoured at the 1σ level electron density map is depicted in Figure 2B. and superimposed on the central TAT portion of the structure. DNA atoms are colored yellow, red, cyan, orange and green for carbon, oxygen, nitrogen, Ion-binding sites and coordination modes phosphorus and fluorine, respectively. Electron density for the FET substituent of T6 is clearly visible in this map. Our experiments reveal up to three alkali metal ion-binding sites in the A-DNA decamer crystals (Fig. 2A). Sites 1 and 3 are exclusively occupied by alkali ions, whereas site 2 can accommodate both alkali and earth alkali metal ions (Fig. 3). the minor groove–terminal base pair interface formed by three Site 1 involves phosphate groups from three adjacent duplexes neighboring duplexes (Fig. 6). Summaries of the distances to and the coordination modes for individual alkali metal ions are DNA atoms and water molecules for individual alkali ions at depicted in Figure 4. For a superposition of this binding site sites 1, 2 and 3 are given in Tables S2–S4, respectively. from seven different structures see Figure S2. Site 2 is located All analyzed duplexes exhibit a kink of between 12° and 15° in the major groove and comprises three adjacent base pairs (Fig. 5). Sites 3 and 1 are fairly closely spaced and at the into the major groove at the T4pA5 step and ions bound at former ions are engaged in direct contact to backbone atoms at site 2 stabilize this kink (Fig. 3). Numerical details for helical 1212 Nucleic Acids Research, 2001, Vol. 29, No. 5 Figure 3. (A) Superposition of A-DNA decamer duplexes crystallized in the presence of different alkali or earth alkali metal ions. The bottom halves of duplexes were used in the superposition (r.m.s.d. <0.5 Å). The view depicts binding site 2 in the major groove common for all ions and demonstrates similar kinks for duplexes at the T4pA5 base-pair steps. Binding site 1 involving phosphate groups is exclusively occupied by alkali metal ions and is shown along with symmetry- related sites (1a and 1b). DNA duplexes listed in Table 1 are drawn as stick models with color codes indicated. The low salt Mg-MEP (21) and Mg-TOE (22) forms 2+ 2+ were also included in the superposition and are colored red and magenta, respectively. Mg and Ba coordinated in the major groove are shown as spheres with larger radius. Alkali metal ions are drawn as smaller spheres. (B) Schematic of the major groove of the A-DNA decamer with sequence GCGTATACGC. A green circle indicates ions binding at the G3pT4 step. No ion was found at the chemically identical G13pA14 step. Asterisks indicate 2′-O-modified nucleotides. Figure 5. Major groove solvation around GpTpA portions. (A) G3pT4pA5: Mg-MEP and Mg-TOE (low salt). (B) G3pT4pA5: Ba-AOE. (C) G3pT4pA5: consensus geometry of site 2 in the Cs-MEP and Rb-FET (high salt) as well as Figure 4. Coordination geometry of alkali metal ions bound at site 1, involving in Cs-, Rb-, K- and Na-TOE (medium salt) crystals. The water drawn with an phosphate groups from three adjacent duplexes. (A) Cs-MEP, Rb-FET (high open circle is absent in all medium salt forms. (D) Hydration of the chemically salt), Cs-TOE and Rb-TOE (medium salt) structures. (B) K-MEP (high salt) equivalent G13pT14pA15 portion. Residues from strands one and two are and K-TOE (medium salt) structures. Ordered ions (green) are arranged in the drawn with solid and open bonds, respectively, C1′ atoms are black and ion–DNA plane defined by P10, P15 and P20 (A and B) and ion–DNA (green) and ion–water (green) and ion–water contacts (red) are represented by dashed lines. contacts (red) are represented by dashed lines. (C)Na-TOE and Na-FET structures (medium salt). (D) Mg-MEP (low salt). No ordered ions were found in the structures depicted in (C) and (D). P P distances are in Å and correspond to average values with standard deviations in parentheses (A and C). Water the nature of the bound ion. The packing mode of decamers in molecules are red and open circles in (B) are waters absent in K-MEP due to the orthorhombic lattice involves stacking of terminal base limited resolution. pairs from two duplexes into the minor groove of a third. The observed metal ions either stabilize lattice interactions (sites 1 parameters of all duplexes are depicted in Figure S3. Interest- and 3) or the kink of duplexes (site 2), the latter likely being ingly, the magnitude of the kink appears to be independent of caused by the particular packing mode. Thus, each duplex is in Nucleic Acids Research, 2001, Vol. 29, No. 5 1213 + + Figure 6. Coordination geometry of Cs and Rb at binding site 3 in the Cs-MEP and Rb-FET (high salt) structures. Residues from three adjacent duplexes at the minor groove–terminal base pair interface are drawn with solid, gray and open bonds and are numbered. Ion–DNA and ion–water contacts are drawn with green and red dashed lines, respectively. An ion bound at site 1 and the water molecule (W) corresponding to the peak indicated by red arrows and lines in Figures 7 and 8, respectively, are included. In the Rb-, K- and Na-TOE (medium salt) structures, the peak at site 3 was found to be shifted from its posi- tion in the Cs-MEP and Rb-FET structures (see Table S4).. Figure 8. Dependence of ‘solvent’ peak height in electron density maps of selected DNA decamers as a function of X-ray data resolution, nature of alkali metal ion and ion concentration. Blue, cyan and green arrows represent ions coordinated at sites 1, 2 and 3, respectively, and red arrows represent hydration site W (Fig. 6). + + lower the occupancies for K and Rb . On the other hand, site 2 appears to be ideal for Cs , independent of the ion concentration, while the geometric constraints of the binding site may not be + + ideal for Rb and K . Based on an analysis of ion–ligand distances, site 3 appears to be well suited for coordination by + + Cs and Rb . However, the high B factors of these ions (Table 1) plus the fact that they were not visible in Patterson maps suggest relatively low occupancies. The reduced occupancies at site 3comparedwithsite 1are probably duetothe different numbers of phosphates present at the two sites (three versus one at sites 1 and 3, respectively). These data are in line with the observation that sites 1 and 3 are exclusively occupied by Figure 7. Average distances between alkali metal ions and oxygen atoms alkali metal ions and that site 2 is optimal for the larger alkali (DNA or water) plotted as a function of ion type for high and medium salt concentrations. The black solid line indicates ideal coordination distances for and all earth alkali metal ions. Taken together, it appears that individual alkali metal ions. Open circles indicate coordination spheres that analyses of ion–ligand distances in DNA crystals can give lack water molecules compared with fully occupied sites (high salt Cs-MEP qualitative information about ion occupancy. and Rb-FET; see Tables S2–S4). Anomalous diffraction versus high resolution data Do resolution-dependent Fourier electron density maps and contact with seven ions; three symmetry-related ions each at anomalous maps provide a consistent picture concerning the the type 1 and 3 sites and one ion at site 2. locations of alkali metal ions in the DNA crystals? We examined this question by comparing the putative identities of Occupancies of binding sites peaks in both types of maps (Fig. 8). Accordingly, we found In addition to B-factor refinements (values for individual ions only one case (Cs-MEP, high salt form) where the top three are listed in Table 1), analyses of ion–ligand distances in peaks in the electron density map coincide with the three crystals may furnish insight into the occupancy of a particular site. strongest peaks in the anomalous map, independent of the For example, comparisons of alkali metal ion–ligand distances at resolution of the former. In all other cases the locations of at sites 1–3 as a function of the metal ion concentration demonstrate least two of the peaks identified as ions based on the anoma- that site 1 can contract to accommodate the smaller ions (Fig. 7). lous data deviate from those hinted at as ions based on electron + + + For the high salt Cs ,Rb and K crystal forms the site appears density peak lists (Fig. 8). 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Conversely, the use of Ångstrom resolution. J. Mol. Biol., 273, 171–182. both anomalous diffraction and atomic resolution data for 15. Tereshko,V., Minasov,G. and Egli,M. (1999) A “hydrat-ion” spine in a B-DNA minor groove. J. Am. Chem. Soc., 121, 3590–3595. nucleic acid crystals grown from solutions of different ionic 16. Bleam,M.L., Anderson,C.F. and Record,M.T.,Jr (1980) Relative binding strengths allows a refined understanding of alkali metal ion affinities of monovalent cations for double-stranded DNA. Proc. Natl coordination to DNA and should benefit nucleic acid crystallo- Acad. Sci. USA, 77, 3085–3089. graphy in general. 17. Basu,S., Rambo,R.P., Strauss-Soukup,J., Cate,J.H., Ferré-D’Amaré,A.R., Strobel,S.A. and Doudna,J.A. (1998) A specific monovalent metal ion integral to the AA platform of the RNA tetraloop receptor. Nature Struct. Biol., 5, 986–992. SUPPPLEMENTARY MATERIAL 18. Brünger,A.T. (1992) Free R value: a novel statistical quantity for Supplementary Material is available at NAR Online. assessing the accuracy of crystal structures. Nature, 355, 472–475. 19. Chen,L.Q., Rose,J.P., Breslow,E., Yang,D., Chang,W.R., Furey,W.F., Sax,M. and Wang,B.C. (1991) Crystal structure of a bovine neurophysin-II dipeptide complex at 2.8 Å determined from the single-wavelength ACKNOWLEDGEMENTS anomalous scattering signal of an incorporated iodine atom. Proc. Natl We would like to thank Mr Guillermo Vasquez, Mr Martin Acad. Sci. USA, 88, 4240–4244. 20. Dauter,Z., Dauter,M., de La Fortelle,E., Bricogne,G. and Sheldrick,G.M. Casper and Dr Haoyun An (Isis Pharmaceuticals Inc., (1999) Can anomalous signal of sulfur become a tool for solving protein Carlsbad, CA) for providing the 2′-O-modified AOE, FET and crystal structures? J. Mol. Biol., 289, 83–92. MEP decamers, Dr M.Teplova for help with the crystalliza- 21. Egli,M., Usman,N. and Rich,A. (1993) Conformational influence of the tions and Dr Lucy V.Malinina for discussions. This work was ribose 2′-hydroxyl group: crystal structures of DNA-RNA chimeric duplexes. Biochemistry, 32, 3221–3237. supported by NIH grant GM-55237 (M.E.) and C.J.W. 22. Egli,M., Tereshko,V., Teplova,M., Minasov,G., Joachimiak,A., acknowledges fellowship support by the Natural Sciences and Sanishvili,R., Weeks,C.M., Miller,R., Maier,M.A., An,H., Cook,P.D. and Engineering Research Council of Canada. The DuPont-North- Manoharan,M. (2000) X-ray crystallographic analysis of the hydration of western-Dow Collaborative Access Team Synchrotron A- and B-form DNA at atomic resolution. Biopolymers, 48, 234–252. 23. Tereshko,V., Portmann,S., Tay,E., Martin,P., Natt,F., Altmann,K.-H. and Research Center at the Advanced Photon Source (Sector 5) is Egli,M. (1998) Structure and stability of DNA duplexes with incorporated supported by E. I. DuPont de Nemours & Co., The Dow Chem- 2′-O-modified RNA analogues. Biochemistry, 37, 10626–10634. ical Company, the National Science Foundation and the State 24. Teplova,M., Minasov,G., Tereshko,V., Inamati,G.B., Cook,P.D., of Illinois. Manoharan,M. and Egli,M. (1999) Crystal structure and improved Nucleic Acids Research, 2001, Vol. 29, No. 5 1215 antisense properties of 2′-O-(2-methoxyethyl)-RNA. Nature Struct. Biol., 27. Fernandez,L.G., Subirana,J.A., Verdaguer,N., Pyshnyi,D., Campos,L. and 6, 535–539. Malinina,L.V. (1997) Structural variability of A-DNA in crystals of the 25. Teplova,M., Wallace,S.T., Minasov,G., Tereshko,V., Symons,A., octamer d(pCpCpCpGpCpGpGpG). J. Biomol. Struct. Dyn., 15, 233–245. Cook,P.D., Manoharan,M. and Egli,M. (1999) Structural origins of the 28. Otwinowski,Z. and Minor,W. (1997) Processing of X-ray diffraction data exonuclease resistance of a zwitterionic RNA. Proc. Natl Acad. Sci. USA, collected in oscillation mode. Methods Enzymol., 276, 307–326. 96, 14240–14245. 29. Brünger,A.T. (1998) Crystallography & NMR System (CNS), Version 0.5. 26. Malinina,L.V., Makhaldiani,V.V., Tereshko,V.A., Zarytova,V.F. and Yale University, New Haven, CT. Ivanova,E.M. (1987) Phase diagrams for DNA crystallization systems. 30. Cambillau,C. and Roussel,A. (1997) Turbo Frodo, Version OpenGL.1. J. Biolmol. Struct. Dyn., 5, 405–433. Université Aix-Marseille II, Marseille, France. 31. Sheldrick,G.M. and Schneider,T.R. (1997) SHELXL: High-resolution refinement. Methods Enzymol., 276, 319–343. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press

Detection of alkali metal ions in DNA crystals using state-of-the-art X-ray diffraction experiments

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1208–1215 Nucleic Acids Research, 2001, Vol. 29, No. 5 © 2001 Oxford University Press Detection of alkali metal ions in DNA crystals using state-of-the-art X-ray diffraction experiments 1 5 Valentina Tereshko, Christopher J. Wilds, George Minasov , Thaza P. Prakash , 5 2,3 4 5 Martin A. Maier ,Andrew Howard , Zdzislaw Wawrzak , Muthiah Manoharan and Martin Egli* Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235, USA, Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, IL 60611, USA, Department of Biological, Chemical and Physical Sciences, Illinois Institute of Technology, Chicago, IL 60616, USA, 3 4 IMCA-CAT, Sector 17 and DND-CAT Synchrotron Research Center, Sector 5, Advanced Photon Source, Argonne National Laboratory, Argonne, IL 60439, USA and Department of Medicinal Chemistry, Isis Pharmaceuticals Inc., Carlsbad, CA 92008, USA Received October 3, 2000; Revised and Accepted January 4, 2001 ABSTRACT structures of oligodeoxynucleotides at atomic resolution 2+ 2+ revealed the positions of Mg and Ca ions that account for The observation of light metal ions in nucleic acids the neutralization of between 50 and 100% of the negative crystals is generally a fortuitous event. Sodium ions charges of phosphate groups (7–12). in particular are notoriously difficult to detect Conversely, there are less than a dozen structures currently because their X-ray scattering contributions are deposited in the Nucleic Acid Database (13) that feature an +… virtually identical to those of water and Na O ordered Na ion and in most of these the ion displays octahedral distances are only slightly shorter than strong coordination geometry. For a list of these structures see hydrogen bonds between well-ordered water mole- Table S1 (Supplementary Material available online). The limited cules. We demonstrate here that replacement of Na number of examples attests to the difficulties of reliably detecting + + + by K ,Rb or Cs and precise measurements of + Na ions with less regular coordination geometry. Thus, anomalous differences in intensities provide a whether a particular peak in Fourier electron density maps particularly sensitive method for detecting alkali ends up as a water molecule or a Na ion is often a matter of metal ion-binding sites in nucleic acid crystals. Not personal judgment and the decision to place an ion, although it only can alkali metal ions be readily located in such may be chemically reasonable, lacks solid experimental + + structures, but the presence of Rb or Cs also allows evidence in many cases. The atomic resolution crystal structure structure determination by the single wavelength of a parallel-stranded G-tetraplex constitutes an exception in anomalous diffraction technique. Besides allowing this respect (14). Since monovalent cations affect the stability of the G-tetrad in a crucial manner, the interpretation that elec- identification of high occupancy binding sites, the tron density peaks in and between the planes formed by four combination of high resolution and anomalous guanines represent sodium ions is unchallenged. diffraction data established here can also pinpoint + + Replacement of Na or K by heavier alkali ions in crystalli- binding sites that feature only partial occupancy. zation of a nucleic acid fragment or soaking crystals in Conversely, high resolution of the data alone does solutions of the heavier alkali ions may be helpful for locating not necessarily allow differentiation between water + + sites occupied by Na or K . For example, crystals of a B-DNA and partially ordered metal ions, as demonstrated dodecamer grown from solutions supplemented with either with the crystal structure of a DNA duplex deter- rubidium or cesium chloride in combination with difference mined to a resolution of 0.6 Å. Fourier synthesis revealed coordination of a single Rb in the minor groove (15). Keeping in mind the differences in ionic INTRODUCTION radius and relative binding affinities of alkali metal ions for + + + + double-stranded DNA (Cs >Rb >K >Na ;16),we may Despite significant advances in the X-ray analysis of 3D structures take this observation as an indication that Na can occupy a of DNA and RNA over the past two decades, reliable detection more or less identical site as Rb . In a similar fashion, binding of light metal ions surrounding nucleic acid molecules remains of K below a so-called AA platform within a domain of group a challenge. Owing to their regular coordination geometry, 2+ 2+ I intron RNA was confirmed by soaking crystals in thallium earth alkali metal ions such as Mg and Ca can often be acetate or cesium hydroxide and examining the corresponding located in Fourier electron density maps. Examples include region of difference electron density maps for changes in crystal structures of small and medium size RNAs determined at both relatively low (1–4) and high (5,6) resolutions. Crystal intensity (17). *To whom correspondence should be addressed. Tel: +1 615 343 8070; Fax: +1 615 343 6707; Email: martin.egli@vanderbilt.edu Correspondence may also be addressed to Muthiah Manoharan. Tel: +1 760 603 2381; Fax: +1 760 929 0036; Email: mmanohar@isisph.com Nucleic Acids Research, 2001, Vol. 29, No. 5 1209 MATERIALS AND METHODS Oligonucleotide synthesis and purification To investigate DNA–ion interactions in crystals we chose A-DNA decamers of sequence GCGTATACGC. This particular DNA sequence was used previously to analyze the conformations of DNA–RNA chimeras (21), DNA hydration (22) and the structural origins of the enhanced RNA affinity (23,24) and exonuclease resistance (25) displayed by 2′-O-modified oligonucleotide analogs. Four DNA decamers with a single 2′-O-aminooxy- ethyl (AOE)-, 2′-O-methyl-3′-methylenephosphonate (MEP)-, 2′-O-fluoroethyl (FET)- or 2′-O-methyl-[tri(oxyethyl)] (TOE)- thymine in place of T6 were included in the crystallization experiments. We found that chemical modification of the T6 sugar moiety in this particular A-DNA decamer leads to crystals of exceptional quality (22). The conformational features of the 2′-O-substituent in TOE were reported before (23) and more detailed accounts of the syntheses, structures and hydration for the other modifications will be described elsewhere. The Z-DNA hexamer CGCGCG was synthesized on the 3 µ mol scale (Pharmacia Expedite), deprotected following established procedures and purified by HPLC. All other oligonucleotides were purified by HPLC and ion exchange chromatography to ≥98%. Figure 1. Idealized curves for the real (f′) and imaginary (f′′) anomalous Crystallizations scattering components of alkali and earth alkali metal ions as a function of X-ray energy (taken from 29). Data for elements P, S and the CuKα wavelength Three different ion concentrations were used to grow crystals (1.5418 Å) are included for comparison. The energy E and the corresponding + + + + with Na ,K ,Rb or Cs : 40 (low salt), 230 (medium salt) and wavelength λ of absorption edges are shown together with the color code for 450 mM (high salt) (Table 1). Hanging drops (4 µ l) containing each ion/element. The wavelength of the beam at the Advanced Photon Source 1 mM DNA, 40 mM alkali metal ions (chloride salt), 20 mM ID beamlines can easily be tuned within the 2–0.66 Å (third harmonic) range. Absorption edges below 6 keV correspond to X-ray wavelengths >2 Å, which alkaline cacodylate buffer, pH 6.0, 6 mM spermine and 10 mM present difficulties for use in crystallography. Among alkali metal ions only the MgCl were equilibrated against 1 ml 35% 2-methyl-2,4- + 2+ K absorption edges of Rb and Sr , located near the right-hand side border, are pentanediol (MPD). Low salt crystals appeared within 2 weeks suitable for use in high energy SAD experiments. The anomalous signals of 2+ + 2+ + and were used for data collection [Mg-MEP (22) and Mg-TOE Ba and Cs as well as of Ca and K can be used at low energy. 2+ (23); only Mg and spermine were located]. The phase diagram technique (26,27) was used to grow medium and high To date there are no reports that exploit the anomalous salt crystals. Thus, 1 (medium salt) and 3 µ l(high salt) 1 M scattering contributions of alkali metal ions to determine their alkali chloride solutions, respectively, were added to locations in nucleic acid crystals. All these ions with the untouched droplets left from low salt crystallizations. Initially exception of Na exhibit significant anomalous effects (Fig. 1). crystals in the droplets dissolved, but appeared again within In combination with a tunable X-ray synchrotron source, the 2 weeks after the MPD concentration in the reservoir was 2+ use of anomalous effects for locating metal ions promises to be raised to 40%. Ba -containing crystals of AOE were grown much more sensitive than difference Fourier synthesis along from 4 µ l hanging drops containing 1 mM DNA, 20 mM with monitoring of the R value (18) during the ensuing sodium cacodylate, pH 6.0, 2 mM spermine and 5 mM BaCl , free refinement cycles. Moreover, the presence of anomalously equilibrated against 1 ml 35% MPD. Z-DNA crystals were + + + scattering K ,Rb or Cs ions offers the opportunity to deter- grown from sitting droplets that contained 1.5 mM DNA, mine nucleic acid crystal structures by the single wavelength 20 mM rubidium cacodylate, pH 7.0, and 10 mM spermine and anomalous diffraction (SAD) technique. were equilibrated against a reservoir of 20% MPD. Here we present protocols for the determination of alkali X-ray data collection and processing metal ion-binding sites in nucleic acid crystals and demon- strate that the anomalous diffraction components of Rb and Crystals suitable for data collection were mounted in nylon Cs are suitable for SAD-type structure determination. In the loops and frozen and stored in liquid nitrogen. Data sets with 2+ first application of the SAD technique for crystallographic crystals containing Ba were collected on an in-house rotating structure determination, diffraction data of a protein–peptide anode generator/image plate set-up (Table 1). All other data complex were phased using a single iodine atom (19). The use collections were conducted at the Advanced Photon Source, of SAD to locate sulfur atoms present in almost all proteins using the insertion device beamlines (ID-B) of the DND and shows promise as a general method for solving the phase IMCA collaborative access teams, located at sectors 5 and 17, problem of native crystals of macromolecules (20). Use of the respectively. Both are equipped with 165 mm MARCCD SAD technique successfully demonstrated here for locating detectors. Data to the maximum resolution limits of the alkali metal ions in crystals of oligodeoxynucleotides should individual crystals were collected at wavelengths <1 Å. To be of general applicability with DNA and RNA. improve completeness and to avoid overloads in the lower 1210 Nucleic Acids Research, 2001, Vol. 29, No. 5 Table 1. Data collection and refinement statistics of A-DNA decamers GCGTATACGC in space group P2 2 2 1 1 1 Compound Ba-AOE Cs-MEP Cs-TOE Rb-FET Rb-TOE K-MEP K-TOE Na-TOE Na-FET Z-DNA Salt form high medium high medium high medium medium medium Unit cell a, Å 24.99 24.82 24.87 24.82 25.16 24.65 25.13 25.13 24.96 18.32 b, Å 44.57 44.33 44.68 45.26 44.83 44.61 44.53 44.53 43.57 30.68 c, Å 45.12 44.82 44.69 43.97 44.82 44.16 45.13 45.13 45.40 42.49 a 2 B factor (Å) DNA 21 8 13 1216 2515 1519 6 Site 1 – 10 15 15 25 30 33 – – – Site2 35(Ba) 2535 3040 3532 2150(Mg) – Site 3 – 60 65 69 70 35 34 25 33 – High resolution X-ray data λ, Å 1.5418 0.8151 0.9500 0.9500 0.9500 0.8151 0.8151 0.8151 0.6630 D, Å 1.70 1.06 1.05 1.05 1.30 1.30 1.30 1.45 0.60 N ref 6913 22 132 19 840 22 802 12 230 12 024 12 024 8695 57 623 % 97.5 97.2 95.1 99.8 99.5 98.9 98.9 99.7 96.0 R 0.068 0.057 0.041 0.046 0.044 0.041 0.041 0.058 0.062 merge R 19.0/21.2 15.0/16.9 16.1/17.8 15.4/17.0 16.5/19.0 20.2/24.6 16.1/17.9 16.1/18.1 15.9/19 16.0/18.5 work/free Anomalous X-ray data λ, Å 1.5418 1.6531 1.7970 0.8151 0.8151 1.6513 D, Å 2.50 2.0 2.0 1.50 1.50 2.0 % 97.5 99.3 98.7 98.8 99.1 98.4 R 0.065 0.082 0.079 0.059 0.044 0.092 merge ∆ F/F 0.0469 0.0784 0.0384 0.0620 0.021 0.019 PP 1.20 1.68 1.35 2.66 – – FOM 0.33 0.41 0.35 0.51 – – Refinement with ion occupancy equal to 1. R factor = Σ |F(hkl) – F(hkl) |/Σ F(hkl) . R = Σ Σ |I(hkl) – <I(hkl)>|/Σ Σ <I(hkl) >. λ, D, the wavelength and resolution of the X-ray data; N ref, %, the hkl o c hkl o merge hkl i i hkl i i number of unique reflections and completeness. R factors based on 2.0 Å anomalous data. ∆ F/F =Σ <|F(+)| – |F(–)|>/<1/2(|F(+)| + |F(–)|>. PP, FOM, phasing power and figure of merit. hkl resolution bins, separate data sets were measured for the low 1I0N (Rb-TOE), 1I0O (K-MEP), 1I0P (K-TOE), 1I0Q (Na- and high resolution ranges in each case. Anomalous data were TOE), 1I0G (Na-FET) and 1I0T (Z-DNA). collected at a wavelength of 0.8151 Å for Rb -containing crystals (third harmonic range) and in the low energy range RESULTS AND DISCUSSION (≥1.54 Å) for all other crystals (Fig. 1). All data were integrated and scaled either in the DENZO/SCALEPACK or HKL2000 Overall strategy suites (28) and selected crystal data and data collection parameters In order to explore the feasibility of using the anomalous are summarized in Table 1. diffraction of alkali metal ions to locate their binding sites in nucleic acid crystals and to provide phase information via SAD, Structure determination, Patterson and electron density we conducted an extensive set of X-ray diffraction experiments at map calculation and refinement the Advanced Photon Source. The study included a variety of Heavy atom searches, Patterson map as well as electron A-form DNA decamer crystals for which multiple data sets at density map calculations and SAD phasing were performed different wavelengths were collected so as to optimize both the with the program CNS (29) and maps were displayed with the magnitude of the anomalous signal and the resolution program TURBO FRODO (30). For all structures initial refine- 2+ (Table 1). The structures of a Ba -form crystal (strong anomalous ments were performed with CNS. To calculate the R values free signal) and of the Z-DNA duplex (resolution 0.6 Å) were used (18) 10% of the data were set aside prior to the refinements. All as ‘anomalous diffraction’ and ‘high resolution’ references. anisotropic refinements with high resolution data were Anomalous data were collected in the low energy range for conducted with the program SHELX-97 (31). Selected refine- + + 2+ + K ,Cs and Ba or, in the case of Rb , at the K absorption edge ment parameters for all structures based on high resolution (Fig. 1). Preliminary diffraction experiments were also conducted data are listed in Table 1. 2+ with decamer crystals containing Sr and confirmed the useful- ness of this ion for SAD-based structure determination (data Coordinates not shown). While anomalous data were typically collected to Final coordinates and structure factors for all structures were resolutions of ∼2 Å, data to high resolution (up to 1.05 Å) were deposited in the Protein Data Bank (PDB). PDB codes: 1I0F also collected at a shorter wavelength for each crystal (Table (Ba-AOE), 1I0J (Cs-MEP), 1I0K (Cs-TOE), 1I0M (Rb-FET), 1). The benefits of atomic resolution data for analyzing ion and Nucleic Acids Research, 2001, Vol. 29, No. 5 1211 solvent interactions in crystals are obvious and an approach that combines the precision of high resolution with the sensi- tivity of anomalous diffraction can be expected to be superior to all previous crystallographic methods to locate alkali metal ions. In addition, to potentially gain an insight into both low and high occupancy DNA binding sites for these metal ions their concentrations in the crystallization solutions were varied (see Materials and Methods). SAD phasing Data collection at the appropriate wavelengths with DNA crystals containing alkali or earth alkali metal ions demonstrates the merits of some of these ions for phasing (Table 1). The heavy atom search procedure in the program CNS was applied to anomalous difference Patterson maps calculated at 2.5, 2.0 and 2+ + 1.5 Å based on data sets for DNA crystals containing Ba ,Cs and Rb , respectively. An example of an anomalous Patterson map is depicted in Figure S1 (Supplementary Material avail- able online). One ion-binding site was located both in Cs-TOE (medium salt) and Ba-AOE crystals (sites 1 and 2, respec- tively, Fig. 2A). Ions at both sites 1 and 2 were located in Cs- MEP and Rb-FET crystals grown under high salt conditions. In the Harker sections peak 1 was twice as high as peak 2. Based on the sites determined in the Patterson maps, SAD phasing was then performed using data to 2.5, 2.0 and 1.8 Å resolution 2+ + + for Ba ,Cs and Rb , respectively. Values for phasing power and figure of merit are listed in Table 1. However, the heavy atom search procedure was not successful in the case of the Rb-TOE (medium salt) and K-MEP (high salt) structures. In order to find additional binding sites the above SAD phases were used in combination with the anomalous differ- ence structure factors to compute anomalous difference Fourier and double-difference (or log likelihood gradient) maps. Examples of anomalous difference Fourier and double- difference maps for the Rb-FET structure are depicted in Figure 2A. In this manner a third ion-binding site was retrieved in the Rb-FET and Cs-MEP structures (site 3). Moreover, a second site (site 2) previously absent in the Cs-TOE structure was also found in this fashion. The resulting phases were subsequently improved by density modification. 2+ As expected, Ba present in the reference structure proved Figure 2. Maps calculated at 1.8 Å resolution with SAD phases for the Rb-FET structure and superimposed on the final structure (selected DNA residues are very potent for phasing and a single site allowed structure numbered). (A) The anomalous difference Fourier (blue) and double-difference determination via SAD. Similarly, Cs - (high and medium salt (red) maps contoured at the 4σ level. Ion-binding sites 1 and 2 (shown with forms) and Rb -form (high salt form) crystals were used for their symmetry mates, designated by lower case letters) were located in the SAD-type phasing. Electron density for two thirds of the anomalous difference Patterson map and used for phasing. Ion-binding site 3 was found at this stage and is characterized by a significantly greater peak duplex was clearly visible with the Ba-AOE, Cs-MEP and Rb- height than the remaining peaks in the double-difference density map. (B)Stereo FET structures and a portion of the Rb-FET experimental diagram of the density-modified electron density map contoured at the 1σ level electron density map is depicted in Figure 2B. and superimposed on the central TAT portion of the structure. DNA atoms are colored yellow, red, cyan, orange and green for carbon, oxygen, nitrogen, Ion-binding sites and coordination modes phosphorus and fluorine, respectively. Electron density for the FET substituent of T6 is clearly visible in this map. Our experiments reveal up to three alkali metal ion-binding sites in the A-DNA decamer crystals (Fig. 2A). Sites 1 and 3 are exclusively occupied by alkali ions, whereas site 2 can accommodate both alkali and earth alkali metal ions (Fig. 3). the minor groove–terminal base pair interface formed by three Site 1 involves phosphate groups from three adjacent duplexes neighboring duplexes (Fig. 6). Summaries of the distances to and the coordination modes for individual alkali metal ions are DNA atoms and water molecules for individual alkali ions at depicted in Figure 4. For a superposition of this binding site sites 1, 2 and 3 are given in Tables S2–S4, respectively. from seven different structures see Figure S2. Site 2 is located All analyzed duplexes exhibit a kink of between 12° and 15° in the major groove and comprises three adjacent base pairs (Fig. 5). Sites 3 and 1 are fairly closely spaced and at the into the major groove at the T4pA5 step and ions bound at former ions are engaged in direct contact to backbone atoms at site 2 stabilize this kink (Fig. 3). Numerical details for helical 1212 Nucleic Acids Research, 2001, Vol. 29, No. 5 Figure 3. (A) Superposition of A-DNA decamer duplexes crystallized in the presence of different alkali or earth alkali metal ions. The bottom halves of duplexes were used in the superposition (r.m.s.d. <0.5 Å). The view depicts binding site 2 in the major groove common for all ions and demonstrates similar kinks for duplexes at the T4pA5 base-pair steps. Binding site 1 involving phosphate groups is exclusively occupied by alkali metal ions and is shown along with symmetry- related sites (1a and 1b). DNA duplexes listed in Table 1 are drawn as stick models with color codes indicated. The low salt Mg-MEP (21) and Mg-TOE (22) forms 2+ 2+ were also included in the superposition and are colored red and magenta, respectively. Mg and Ba coordinated in the major groove are shown as spheres with larger radius. Alkali metal ions are drawn as smaller spheres. (B) Schematic of the major groove of the A-DNA decamer with sequence GCGTATACGC. A green circle indicates ions binding at the G3pT4 step. No ion was found at the chemically identical G13pA14 step. Asterisks indicate 2′-O-modified nucleotides. Figure 5. Major groove solvation around GpTpA portions. (A) G3pT4pA5: Mg-MEP and Mg-TOE (low salt). (B) G3pT4pA5: Ba-AOE. (C) G3pT4pA5: consensus geometry of site 2 in the Cs-MEP and Rb-FET (high salt) as well as Figure 4. Coordination geometry of alkali metal ions bound at site 1, involving in Cs-, Rb-, K- and Na-TOE (medium salt) crystals. The water drawn with an phosphate groups from three adjacent duplexes. (A) Cs-MEP, Rb-FET (high open circle is absent in all medium salt forms. (D) Hydration of the chemically salt), Cs-TOE and Rb-TOE (medium salt) structures. (B) K-MEP (high salt) equivalent G13pT14pA15 portion. Residues from strands one and two are and K-TOE (medium salt) structures. Ordered ions (green) are arranged in the drawn with solid and open bonds, respectively, C1′ atoms are black and ion–DNA plane defined by P10, P15 and P20 (A and B) and ion–DNA (green) and ion–water (green) and ion–water contacts (red) are represented by dashed lines. contacts (red) are represented by dashed lines. (C)Na-TOE and Na-FET structures (medium salt). (D) Mg-MEP (low salt). No ordered ions were found in the structures depicted in (C) and (D). P P distances are in Å and correspond to average values with standard deviations in parentheses (A and C). Water the nature of the bound ion. The packing mode of decamers in molecules are red and open circles in (B) are waters absent in K-MEP due to the orthorhombic lattice involves stacking of terminal base limited resolution. pairs from two duplexes into the minor groove of a third. The observed metal ions either stabilize lattice interactions (sites 1 parameters of all duplexes are depicted in Figure S3. Interest- and 3) or the kink of duplexes (site 2), the latter likely being ingly, the magnitude of the kink appears to be independent of caused by the particular packing mode. Thus, each duplex is in Nucleic Acids Research, 2001, Vol. 29, No. 5 1213 + + Figure 6. Coordination geometry of Cs and Rb at binding site 3 in the Cs-MEP and Rb-FET (high salt) structures. Residues from three adjacent duplexes at the minor groove–terminal base pair interface are drawn with solid, gray and open bonds and are numbered. Ion–DNA and ion–water contacts are drawn with green and red dashed lines, respectively. An ion bound at site 1 and the water molecule (W) corresponding to the peak indicated by red arrows and lines in Figures 7 and 8, respectively, are included. In the Rb-, K- and Na-TOE (medium salt) structures, the peak at site 3 was found to be shifted from its posi- tion in the Cs-MEP and Rb-FET structures (see Table S4).. Figure 8. Dependence of ‘solvent’ peak height in electron density maps of selected DNA decamers as a function of X-ray data resolution, nature of alkali metal ion and ion concentration. Blue, cyan and green arrows represent ions coordinated at sites 1, 2 and 3, respectively, and red arrows represent hydration site W (Fig. 6). + + lower the occupancies for K and Rb . On the other hand, site 2 appears to be ideal for Cs , independent of the ion concentration, while the geometric constraints of the binding site may not be + + ideal for Rb and K . Based on an analysis of ion–ligand distances, site 3 appears to be well suited for coordination by + + Cs and Rb . However, the high B factors of these ions (Table 1) plus the fact that they were not visible in Patterson maps suggest relatively low occupancies. The reduced occupancies at site 3comparedwithsite 1are probably duetothe different numbers of phosphates present at the two sites (three versus one at sites 1 and 3, respectively). These data are in line with the observation that sites 1 and 3 are exclusively occupied by Figure 7. Average distances between alkali metal ions and oxygen atoms alkali metal ions and that site 2 is optimal for the larger alkali (DNA or water) plotted as a function of ion type for high and medium salt concentrations. The black solid line indicates ideal coordination distances for and all earth alkali metal ions. Taken together, it appears that individual alkali metal ions. Open circles indicate coordination spheres that analyses of ion–ligand distances in DNA crystals can give lack water molecules compared with fully occupied sites (high salt Cs-MEP qualitative information about ion occupancy. and Rb-FET; see Tables S2–S4). Anomalous diffraction versus high resolution data Do resolution-dependent Fourier electron density maps and contact with seven ions; three symmetry-related ions each at anomalous maps provide a consistent picture concerning the the type 1 and 3 sites and one ion at site 2. locations of alkali metal ions in the DNA crystals? We examined this question by comparing the putative identities of Occupancies of binding sites peaks in both types of maps (Fig. 8). Accordingly, we found In addition to B-factor refinements (values for individual ions only one case (Cs-MEP, high salt form) where the top three are listed in Table 1), analyses of ion–ligand distances in peaks in the electron density map coincide with the three crystals may furnish insight into the occupancy of a particular site. strongest peaks in the anomalous map, independent of the For example, comparisons of alkali metal ion–ligand distances at resolution of the former. In all other cases the locations of at sites 1–3 as a function of the metal ion concentration demonstrate least two of the peaks identified as ions based on the anoma- that site 1 can contract to accommodate the smaller ions (Fig. 7). lous data deviate from those hinted at as ions based on electron + + + For the high salt Cs ,Rb and K crystal forms the site appears density peak lists (Fig. 8). 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Nucleic Acids Research – Oxford University Press

Published: Mar 1, 2001

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