DNA Binding and Phosphorylation Regulate the Core Structure of the NF-κB p50 Transcription Factor

DNA Binding and Phosphorylation Regulate the Core Structure of the NF-κB p50 Transcription Factor B The Author(s), 2018 J. Am. Soc. Mass Spectrom. (2018) DOI: 10.1007/s13361-018-1984-0 FOCUS: HONORING CAROL V. ROBINSON'S ELECTION TO THE NATIONAL ACADEMY OF SCIENCES: RESEARCH ARTICLE DNA Binding and Phosphorylation Regulate the Core Structure of the NF-κB p50 Transcription Factor 1 2 3 2 Matthias Vonderach, Dominic P. Byrne, Perdita E. Barran, Patrick A. Eyers, Claire E. Eyers Centre for Proteome Research, Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool, L69 7ZB, UK Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool, L69 7ZB, UK Michael Barber Centre for Collaborative Mass Spectrometry, Manchester Institute of Biotechnology, The University of Man- chester, 131 Princess Street, Manchester, M1 7DN, UK Abstract. The NF-κB transcription factors are known to be extensively phosphorylated, with dynamic site-specific modification regulating their ability to dimerize and interact with DNA. p50, the proteolytic product of p105 (NF-κB1), forms homodimers that bind DNA but lack intrinsic transactivation function, functioning as repres- sors of transcription from κB promoters. Here, we examine the roles of specific phosphorylation events catalysed by either protein kinase A (PKA ) or Chk1, in regulating the functions of p50 homodimers. LC-MS/MS analysis of proteolysed p50 following in vitro phosphorylation allows us to define Ser328 and Ser337 as PKA - and Chk1-mediated modifications, and pinpoint an additional four Chk1 phosphosites: Ser65, Thr152, Ser242 and Ser248. Native mass spectrometry (MS) reveals Chk1- and PKA -regulated disruption of p50 homodimer formation through Ser337. Additionally, we characterise the Chk1-mediated phosphosite, Ser242, as a regulator of DNA binding, with a S242D p50 phosphomimetic exhibiting a > 10-fold reduction in DNA binding affinity. Conformational dynamics of phosphomimetic p50 variants, including S242D, are further explored using ion-mobility MS (IM-MS). Finally, comparative theoretical modelling with experimentally observed p50 conformers, in the absence and presence of DNA, reveals that the p50 homodimer undergoes conformational contraction during electrospray ionisation that is stabilised by complex formation with κBDNA. Keywords: Native MS, Ion mobility-mass spectrometry, NF-κB, Collision-induced unfolding, Phosphorylation, DNA binding, Molecular modelling Received: 14 March 2018/Revised: 26 April 2018/Accepted: 30 April 2018 such family of ubiquitous transcription factors is NF-kappaB Introduction (NF-κB), and regulated activation of this signal transduction egulated binding of specific transcription factor com- pathway is required for transcriptional control of hundreds of Rplexes to their cognate DNA sequences directly influences genes, under both physiological and pathophysiological condi- the rate at which transcription of individual genes occurs. One tions. This important family of transcription factors is essential for numerous diverse biological functions, including regulation Electronic supplementary material The online version of this article (https:// of inflammation and immune responses, proliferation and apo- doi.org/10.1007/s13361-018-1984-0) contains supplementary material, which ptosis [1]. is available to authorized users. Stable interaction of NF-κB (and other) transcription Correspondence to: Claire Eyers; e-mail: CEyers@liverpool.ac.uk factors with DNA response elements typically requires the M. Vonderach et al.: DNA Binding Stabilises NF-κBDimers formation of either homo- or heterodimers, which permits averaged collision cross section (CCS). Importantly, CCS the recognition of palindromic DNA-sequence motifs by values can be calculated for a given geometry from theoretical adjacent DNA binding domains [2–4]. Specificity of NF- structures derived from density functional theory or molecular κB-mediated transcription is regulated in part by the com- dynamic (MD) simulations using a variety of methods such as binatorial diversity arising from the five related NF-κB projection approximation (PA) [33], exact hard sphere scatter- proteins, with each NF-κB dimer regulating both distinct ing (EHSS) model [34], the trajectory method (TM) [35], and overlapping sets of genes due to subtle differences in projection superposition approximation (PSA) [36]or scatter- their kB consensus DNA binding specificity [5]. Dimeriza- ing on electron density isosurfaces (SEDI) [37]. Such compu- tion of NF-κB proteins, interaction with other transcription- tational strategies allow prediction of putative protein struc- al co-factors and DNA binding, is also regulated by exten- tures by comparison with experimentally derived CCS values sive post-translational modification (PTM), with dynamic derived using a variety of experimental-based structural phosphorylation established as being critical for cellular approaches. function [6–12]. Here, we exploit standard MS-based phosphoproteomics in p105 (NFκB1) is one of the five NF-κB transcription factors combination with native ion mobility-mass spectrometry (IM- and is commonly proteolysed to generate a functional p50 mole- MS) and molecular modelling to define the effects of p50 cule lacking a transactivation domain [1]. The high basal levels of phosphorylation on dimerization and DNA binding. Using nuclear localised p50 homodimers in unstimulated cells are thus travelling wave-IMS (TW-IMS) and comparative molecular thought to act as repressors of transcription from κBpromoters by dynamics (MD) simulations, we demonstrate that the p50 competing for DNA binding with transcriptionally active NF-κB homodimer is stabilised by the presence of its cognate DNA dimers, including the RelA:p50 heterodimer [13, 14]. Regulation oligomer and define specific p50 phosphorylation sites as key of p50 by reversible phosphorylation is much less well understood potential regulators of either DNA binding or homo- than that of the p105 precursor [15–18], but appears to be a critical dimerization. regulator of efficient p50 binding to DNA, and thus transcriptional repression. Phosphorylation of p50 in vitro by the catalytic subunit of protein kinase A (PKA ) has been reported to enhance its Experimental ability to bind DNA in a manner that is independent on its ability Protein Expression and Purification to dimerise [15, 16]. Mutational studies using phosphomimetic variants mapped this critical phosphorylation event to Ser337, and Murine NF-κB p50 (39-364 wild-type; WT) was cloned into Ricciardi and colleagues demonstrated that this site is constitu- the pOPINM vector (OPPF) using the InFusion PCR cloning tively phosphorylated by PKA in unstimulated cells, contributing kit (Clonetech). S65D, S242D, S248D and S337D p50 muta- to κB transcriptional repression under basal (non-stimulated) con- tions were generated by PCR site-directed mutagenesis from ditions [15]. Mutation of two additional p50 Ser residues, Ser65 the WT p50 construct. Appropriate mutations were confirmed and Ser342, to Ala, was also reported to negatively influence the by DNA sequencing. All proteins were produced in BL21 DNA binding ability of p50 [15]. However, regulated phosphor- (DE3) pLysS E. coli cells (Novagen) with expression induced ylation of these residues has not yet been demonstrated. with 0.5 mM IPTG for 3 h at 18 °C and purified with a 3C Mass spectrometry (MS) can be used in a variety of ways to protease cleavable N-terminal His6-MBP-tag. Fusion proteins elucidate information about all levels of protein structure, from were first purified by affinity chromatography using amylose primary to quaternary configurations. Exploitation of ‘native’ resin (NEB), and p50 subunits were cleaved from the MS, where the analyte is transferred from solution into the gas- immobilised affinity medium using 3C protease in 50 mM Tris phase under gentle electrospray ionisation (ESI) conditions (pH 7.4), 100 mM NaCl, 1 mM DTT, 10% (v/v) glycerol and [19–21] from a volatile buffer such as ammonium acetate at 20 mM imidazole. 3C protease (purified as an N-terminal His6- physiological pH, allows non-covalent complexes to be inter- tag fusion protein) was subsequently removed by immobilised rogated. Native MS can thus be used to determine protein metal affinity chromatography. complex stoichiometry [22] or compare the relative dissocia- tion constant (K ) of different ligands [23–25]. Many proteins In Vitro Phosphorylation, Digestion and LC-MS/MS and protein complexes largely retain their solution-phase con- Analysis formation under native ESI conditions [26–28], thus their structure and the effect of protein modification and/or ligand p50 protein (25 μg, 35-381, Enzo Scientific) in 10 mM binding on conformational dynamics and stability can be read- TrisOAc was incubated at 37 °C for 2 h with 10 mM MgCl 2, ily interrogated with gas-phase methods such as ion mobility 250 μM ATP, 1 mM DTT and 1 mM EGTA in the presence of spectrometry (IMS) [29–31] or infrared spectroscopy [32]. In 0.25 μg of either PKA [38] or 4.2 μg Chk1 (MRC PPU IMS, ions are transported by an electric field through a drift cell Reagents and Services, Dundee). Reactions were stopped by filled with an inert gas such as helium or nitrogen, permitting rapid buffer exchange into NH OAc. For digestion, 1 μgof separation of analyte ions based on their charge, mass and protein was denatured prior to digestion by addition of 1% conformation. Consequently, the recorded drift time of ions Waters RapiGest at 80 °C for 10 min. Enzymatic digestion was through the IMS cell can be used to define their rotationally performed at 37 °C overnight using 0.02 μgtrypsinand M. Vonderach et al.: DNA Binding Stabilises NF-κB Dimers stopped by addition of 0.5% TFA and incubation for 45 min at increasing collision energies. Contour plots representing the 37 °C. Phosphopeptides were enriched using TiO spin col- unfolding profile were produced with Origin 9.0. umns (GLSciences) as previously described [39]. LC-MS/MS analysis was performed on an Orbitrap Fusion Tribrid mass K Determination spectrometer (ThermoScientific), attached to an Ultimate 3000 p50 WT or single-point aspartic acid mutants (3 μM) were nano system (Dionex). Peptides were loaded onto the trapping incubated with 0.2–6 μMof κB DNA oligomer 5′-CCCC column (ThermoScientific, PepMap100, C18, 300 μm× CGGGGGCCCCCGGGGG-3′ (Sigma) in 300 mM NH OAc 5 mm), using partial loop injection, for 7 min at a flow rate of for 5 min at room temperature (final volume 10 μL) prior to 9 μL/min with 2% (v/v) MeCN 0.1% (v/v) TFA and then native (IM-)MS analysis. Multi-Gaussian fitting with Origin resolved on an analytical column (Easy-Spray C18 75 μm× 9.0 was used to ascertain the peak areas of all charge states of 500 mm 2 μm bead diameter column) using a 30-min method both the unbound and DNA-bound p50. K values for DNA from 96.2% A (0.1% FA) and 3.8% B (80% MeCN 19.9% binding were determined by nonlinear peak fitting using Eq. 2: −1 H O 0.1% FA) to 100% B at a flow rate of 300 nL min . A full scan mass spectrum was acquired (30K resolution at m/z 200) 0 1 sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi and data-dependent MS/MS analysis performed using a top IPðÞ *L 1 ½ P ½ L ½ L ½ L ½ P 0 0 0 0 0 @ A speed approach (cycle time of 3 s), using HCD and EThcD ¼ −1− þ þ 4 þ − −1 ð2Þ IPðÞ 2 K K K K K D D D D D fragmentation modes, with product ions being detected in the orbitrap (15K resolution). I(PL) and I(P) define the peak area of the DNA-bound Native IM-MS and Collision-Induced Unfolding protein complex and the unbound p50 protein dimer, respec- A commercial TW-IMS instrument (Waters G2-Si) was tively, [P] and [L] are the original protein and DNA concen- 0 0 utilised for native IM-MS. p50 was buffer exchanged into trations [25]. 100 mM NH OAc using 10-kDa molecular cut-off spin filter columns (Amicon) and 1–3 μl of sample (typically 5 μM) was Molecular Modelling and CCS Calculation subjected to electrospray ionisation (ESI) at a voltage of 1.3– ff14SB, OL15 and tip3p force fields implemented in AM- 3 kV using a self-pulled nanospray tip. Sampling cone was set BER16 [43] were used to simulate the effect of protein −2 at 75 V. Trap pressure was adjusted to 5 × 10 mbar, He cell desolvation during ESI on the conformation of the DNA- pressure was 4.53 mbar, IMS pressure was 2.78 mbar and −2 bound and unbound forms of the p50 (39-350) homodimer transfer tube pressure was 5.18 × 10 mbar. IMS was per- (1.NFKB.pdb) [44]. The structure was neutralised by addition formed using a travelling wave height of 29 V and a velocity of Na ions and embedded into a water box containing approx- of 650 m/s. Calibration of the TriWave device was performed imately 35,000 water molecules. Geometry optimization for as previously described [40, 41]using β-lactoglobulin A (Sig- molecular dynamics (MD) simulation was performed utilising ma L7880), avidin (Sigma A9275), transthyretin (Sigma the steepest descent energy minimisation and conjugate gradi- P1742), concanavalin A (Sigma C2010) and serum albumin ent method. Molecular dynamic simulations were performed at (Sigma P7656) as calibrants. Upon removing the time the ions 350 K for 2 ns, with 2 fs integration steps. Langevin dynamics spend in the time of flight mass spectrometer, a logarithmic plot were applied to regulate the temperature. The result of the 2 ns of the ‘corrected drift time t’ versus the by charge q and reduced run was used as input for a further MD simulation in which the mass μ corrected CCS, the so-called reduced CCS was calcu- amount of water was reduced by ~ 10%. Upon completing lated and a straight line extrapolated in order to ascertain the TW 40 × 2 ns runs, a final 10-ns run was performed in the absence slope, m, and intersection, C. The experimental CCS N2→He of solvent, using a charge state of 16+ for the p50 dimer and values (where the TW-IMS-determined drift times in a nitrogen 18+ for the DNA-bound p50 dimer, those being the most atmosphere were converted to helium CCS values [42]) were dominant charge states observed. CCS values of all final MD finally calculated from measured drift times using Eq. 1: structures were computed using Mobcal [34, 35]. RMSD values were calculated utilising CPPTRAJ implemented in AMBER [43]. TW 0 CCS ¼ t � expðÞ C ð1Þ N →He pffiffiffi Results CCSD values are defined as the full width at half maximum Chk1 Induces Extensive Phosphorylation of p50 of the CCS distribution. In Vitro Collision-induced unfolding (CIU) was used to evaluate the transitional unfolding profiles of the protein and protein-DNA Based on mutational analysis and in vitro protein kinase assays, complexes. An individual charge state was isolated with the it was previously reported that phosphorylation of p50 by quadrupole mass filter and subjected to collisional activation in PKA on Ser337 is essential for high-affinity DNA binding. the trap region of the TriWave by application of gradually However, the mechanism whereby pSer337 regulates DNA M. Vonderach et al.: DNA Binding Stabilises NF-κBDimers binding was not defined. Although a S337A p50 mutant ex- Ser240 and Ser246 respectively) both lie in the linker region hibited dramatically reduced DNA binding ability in cells (L3) between the two distinct domains of p50 (Fig. 1b, c). compared to the wild-type p50 protein, this site of modification Phosphorylation of one or both of these residues in this linker is distal from the DNA binding domain, and the ability of region, which adopts a well-defined structure that can fit into S337A p50 to dimerise with p65/RelA was reported to be the major groove of the DNA substrate, is thus likely to have a unaffected [16]. Moreover, the ability of p50 to be phosphor- significant effect on DNA binding ability of p50. In particular, ylated by PKA , or other putative regulatory kinases, at sites Ser242 lies adjacent to a key Lys residue at position 243 distinct from Ser337 was not evaluated in side-by-side (mouse Lys241), which directly interacts with the DNA back- experiments. bone. Consequently, we hypothesised that Ser242 phosphory- Using standard peptide-based tandem MS analysis, we re- lation is likely to disrupt p50 DNA binding. Similarly, Ser65 cently reported PKA -mediated phosphorylation of recombi- (mouse Ser63) lies downstream of a five residue cluster nant p50 (35-381) in the presence of p65/RelA at four sites in (RxRYxCExxS) located in L1, another loop that makes direct addition to Ser337, namely Ser223, Ser226, Ser236 and contacts with the κB DNA. Even though phosphorylation of Thr263 [6]. To assess whether these phosphorylation sites are both Ser328 and Ser337 has been shown to influence the ability dependent on inclusion of RelA in the assay, and thus forma- of p50 to bind DNA, both are localised to the second domain, tion of a RelA:p50 heterodimer, we repeated in vitro distal from the DNA-binding region, suggesting a gross con- phosphosite mapping using PKA with p50 alone. Under these formational change of domain 1 with respect to domain 2 and conditions, only Ser328 and Ser337 were identified as PKA - the DNA-protein interface, rather than a direct effect of phos- regulated phosphosites (Fig. 1, Supp. Figure 1), confirming phorylation of these residues on the ability to bind DNA. previous observations that NF-κB proteins likely adopt differ- ent conformations dependent on their dimerization partners [6]. Phosphorylation of p50 by Chk1 Destabilises MS analysis of the intact phosphorylated p50 identified a single Dimerization phosphate-carrying proteoform (in addition to the non- phosphorylated protein), with no evidence of a doubly phos- To assess the effect of p50 phosphorylation on its ability to phorylated species, suggesting that phosphorylation of p50 on dimerise and bind DNA, we analysed p50 (35-381) by nano- Ser328 and Ser337 is likely to be mutually exclusive (Fig. 1a). electrospray ionisation (nESI)-MS under non-denaturing ‘na- As well as cellular evidence for p50 regulation by PKA tive’ MS conditions, before and after in vitro phosphorylation [18], Chk1 is known to play a major role in phosphorylation- with either PKA or Chk1. As expected, intact non- mediated regulation of this transcription factor, inhibiting DNA phosphorylated p50 was preferentially observed as a dimer binding via phosphorylation at Ser328 [45, 46]. Previous in- with only a small amount of monomer present (Fig. 2). Upon vestigations have focused on phosphorylation of Ser328 by phosphorylation with either protein kinase, there was a small Chk1, even though p50 contains a number of other conserved but consistent increase in the relative abundance of the p50 Chk1 consensus sites. Consistently, we identified a total of six monomer (observed charge states of 11+ to 13+) with respect in vitro Chk1 phosphorylation sites on p50 (35-381) using MS- to the p50 homodimer (observed charge states of 16+ to 19+), based phosphopeptide mapping (Supp. Figure 1), including the demonstrating phosphorylation-mediated destabilisation of the previously reported Ser328 site [46], the overlapping PKA site homodimeric protein. at Ser337, and four novel sites at Ser65, Thr152, Ser242 and Inclusion of a DNA oligomer designed to match the κB Ser248. Chk1 phosphorylation of p50, at least in vitro, is thus consensus sequence for the p50 homodimer (5′-CCCC much more extensive than previously supposed. Analysis of CGGGGGCCCCCCGGGGG-3′) revealed a stabilising effect the intact Chk1-phosphorylated p50 reveals a predominant of DNA binding upon dimer formation. No residual monomer singly phosphorylated species, as well as a doubly phosphory- was observed for the non-phosphorylated p50 in the presence lated form, with relatively low levels of non-phosphorylated of the κB DNA, with stoichiometric formation of the p50 (Fig. 1a). Analogous to the PKA -phosphorylated p50, the p50:p50:DNA complex. A similar stabilising effect was also six sites modified by Chk1 are thus unlikely to be stoichiomet- seen for PKA -phosphorylated p50, with no monomeric p50 rically combinatorial, rather, p50 is modified at specific observed and stoichiometric DNA:protein complex formed. In (discrete) combinations of amino acids. contrast, although the Chk1-phosphorylated p50 homodimer Of the 10 phosphosites that we identified in total on p50 stoichiometrically bound the κB DNA, dimer formation was (with or without RelA), only two, Thr152 and Ser226, are not not enhanced under these conditions (Fig. 2). Chk1 phosphor- completely conserved in model vertebrates (Supp. Figure 2). ylation of p50 thus appears to actively disrupt homo-dimeriza- Thr152 is changed to Ile in Xenopus laevis p50, although it is tion, irrespective of effects on DNA binding. conserved as a Thr in all other species examined. Ser226 was To further evaluate the structural effects of p50 phos- absent in both frog and chicken p50 sequences. Considering phorylation, we used ion mobility-MS (IM-MS) to compare the position of the six PKA and Chk1 phosphosites identified p50 conformation and structural dynamics following treat- in the absence of p65 in the p50 homodimer structure (PDB ment with either PKA or Chk1 (see supplementary infor- entry 1NFK [44]), a number of potential roles for phosphory- mation; Supp. Figure 3). The rotationally averaged collision TW lation might be hypothesised. Ser242 and Ser248 (mouse cross section ( CCS ) of the p50 homodimer was N2→He M. Vonderach et al.: DNA Binding Stabilises NF-κB Dimers Figure 1. Multi-site phosphorylation of p50 by PKA and Chk1 is not combinatorial. (a) Intact mass spectra of p50 before (bottom) and after in vitro phosphorylation with PKA (middle) or Chk1 (top). Depicted are the 30+ (green) and 31+ (blue) charge states of the non-phosphorylated (circles), mono-phosphorylated (triangles) and di-phosphorylated (diamonds) forms of p50. (b) Identified sites of phosphorylation (red) mapped onto the X-ray crystal structure of the mouse p50:p50 homodimer bound to DNA (grey), PDB entry 1NFK. Individual p50 monomers are either in blue or yellow. Sites of phosphorylation are numbered according to the human sequence. Ser328 and Ser337 were identified as PKA phosphosites. All sites were phosphorylated by Chk1. (c) Loops 1 and 3 of p50 with kB DNA show direct interaction of the regions containing Ser65, Ser242 and Ser248 with the DNA determined as 44.3 nm , relatively independent of the cor- charge states, there was no statistically significant change in TW responding charge state (16+ to 18+) (Supp. Figure 3). the absolute CCS value after phosphorylation with N2→He Although subtle differences were observed in the p50 either PKA or Chk1 (Supp. Figure 3). TW TW CCS distribution ( CCSD )uponphos- Interestingly, there was a small but reproducible 1.5% de- N2→He N2→He TW phorylation with PKA , particularly for the 16+ and 17+ crease in the CCS values of the DNA-bound p50 c N2→He Figure 2. Phosphorylation of p50 regulates dimerization. Native mass spectra of p50 before (bottom) or after phosphorylation with either PKA (middle, red) or Chk1 (top, blue), in the absence (left) or presence (right) of the p50 kB DNA oligomer. Charge states are indicated M. Vonderach et al.: DNA Binding Stabilises NF-κBDimers the DNA, and Ser337 is located in the dimerization region and might impart allosteric regulation of dimerization upon phos- phorylation. S65D, S242D, S248D and S337D p50 were analysed by native IM-MS alongside WT p50 (Fig. 3), using multi-Gaussian fitting to evaluate the peak areas and calculate the monomer:dimer ratio for each species. Akin to WT p50, the dominant species of all S→ D p50 protein mass spectra were dimers (Fig. 3). Little, or no, difference was observed in the monomer:dimer ratios for S65D and S242D p50 (Fig. 3,Table 1). However, there was a 2.4-fold relative increase in monomeric S337D p50 com- pared to the non-phosphorylated WT p50. The ability of S337D to disrupt p50 dimerization was further confirmed by size exclusion chromatography (SEC), which revealed a ~ 5-fold relative increase in monomeric S337D p50 based on protein staining and densitometry (Supp. Figure 5). To- gether, these findings support our initial prediction that Figure 3. Ser337 phosphomimetic disrupts dimerization of Ser337 phosphorylation plays a significant role in control- p50. Native mass spectra of p50 (39-364) wild-type (WT, black), ling (disrupting) p50 dimerization. S65D (blue), S242D (red), S248D (brown) and S337D (green) IM-MS analysis of the p50 protein variants revealed a phosphomimetic versions showing p50 monomer and dimer. TW 2 CCS of ~ 42.0–42.5 nm suggest- peak maximum N2→He Non-assigned peaks derive from the cleaved contaminating ing that the dominant dimer conformation is highly similar MBP expression tag. Charge states are indicated for all species analysed (Fig. 4). However, a notable in- crease in conformational flexibility was observed for all mutants compared with WT p50. In particular, the CCSD dimer with decreasing charge state (20+ to 18+), revealing values for S248D and S337D are 4.6 and 5.0 nm ,respec- slight compaction of the complex. Moreover, the asymmetric tively, compared with a CCSD value of just 2.9 nm for CCS profile of the WT p50:p50:DNA complex is indicative of WT p50 (Fig. 4). the presence of two unresolved conformers with charge state TW 2 averaged CCS values of 51.1 and 53.3 nm for the N2→He unmodified p50-DNA dimer. Of note, the relative proportion Phosphomimetic Version of the Chk1-Mediated p50 of the more compact conformer increased with a reduction in Phosphosite Ser242 Destabilises DNA Binding charge state, suggesting gas-phase conformational contraction. Comparable results were also observed following native IM- To evaluate the effect of modification of S248 and S337 on MS analysis of p50 (39-364) (Supp. Figure 4). DNA binding, we employed the titration method and native MS to determine the dissociation constants (K ) for DNA binding for each of the p50 protein variants (Fig. 4; Table 1). Ser337 Phosphomimetic Disrupts p50 Homodimer As expected, the unphosphorylated WT p50 homodimer ex- Formation hibited a relatively high affinity for DNA in this assay, with a To evaluate which of the site-specific, but sub-stoichiometric K value of < 40 nM, similar to that previously reported for PKA - or Chk1-mediated phosphorylation events were respon- both p65 homodimers and p65/p50 heterodimers [47]. All the sible for the observed disruption of dimerization, we expressed aspartic acid mutants analysed exhibited significantly higher Ser→ Asp (potential phosphomimetic) versions of p50 (39- K values than those observed for WT p50, indicating a reduc- 364) for sites predicted to influence either dimer formation or tion in DNA binding affinity. Specifically, S337D p50 exhib- DNA binding: Ser65, Ser242, Ser248 lie in close contact with ited a 3-fold higher dissociation constant than WT p50, with a Table 1. Effect of p50 phosphomimetic variants on protein dimerization and DNA binding. Percentage peak areas of monomers and dimers for p50 WT, S65D, S242D, S248D and S337D and the mutants. DNA binding dissociation constants K , and the relative DNA binding affinity with respect to WT p50, are also presented for each p50 variant Monomer Dimer K value for DNA binding (nM) Relative DNA binding affinity WT 10.5% 89.5% 37 ± 7 100% S65D 9.4% 90.6% 140 ± 16 26.4% S242D 12.4% 87.6% 424 ± 78 8.7% S248D 5.8% 94.2% 112 ± 11 33.0% S337D 25.2% 74.8% 111 ± 15 33.3% M. Vonderach et al.: DNA Binding Stabilises NF-κB Dimers TW Figure 4. Phosphomimetic versions of p50 exhibit increased conformational flexibility compared to WT p50. CCS distri- N2→He butions of wild-type (WT) p50 (39-364) (black) alongside p50 S65D (blue), S242D (red), S248D (brown) and S337D (green) in the absence (left) or presence (right) of DNA measured K of ~ 110 nM, compared with a K of 37 nM for ATDs of S248D and S337D, which lie in the L3 linker region of D D WT p50 under the same conditions. This increase in relative K p50 and the dimerization domain respectively (Fig. 1), suggests is likely attributed to the reduction in the ability of this that both of these phosphomimetic mutations similarly alter the phosphomimetic variant to dimerise (Table 1), a prior require- relative position of the two domains of p50. ment for DNA binding. In agreement with our hypothesis, mimicking the Chk1- mediated p50 phosphorylation site at S242 resulted in an order of magnitude decrease in DNA binding affinity. Replacement of Ser242 with a negatively charged Asp group is predicted to disrupt the direct electrostatic interaction of Lys243 with the phosphate backbone of the DNA. Interestingly, the consistently earlier arrival time distribution (ATD) of S242D when com- pared to that of WT p50 observed in the absence of DNA suggests that this protein can adopt conformations that are likely to be more compact than WT p50 (Fig. 4). In contrast, TW upon DNA binding, the CCS of S242D is consistently N2→He larger than the WT dimeric complex, indicative of a more open conformation. Mutation of the other two identified p50 phosphosites in the DNA binding interface, S65 and S248 (Fig. 1b),resultedina 3.8- and 3.0-fold increase in K values, respectively, compared with the WT protein (Fig. 5), implicating roles for phosphoryla- tion of S65 and S248 in negatively regulating DNA binding of Figure 5. DNA binding of p50 S242D is significantly disrupted. the p50 homodimer. However, ATDs of these two p50 variants Native MS and DNA titration was used to calculate K for DNA are distinct, both in the absence and presence of DNA, indicative binding for each of the p50 homodimers: WT (black), S65D of different effects on gross conformation. The similarity of the (blue), S242D (red), S248D (brown) and S337D (green) M. Vonderach et al.: DNA Binding Stabilises NF-κBDimers the small central cavity which accommodates the helical DNA. In contrast and as previously observed, there was a significant TW difference in the CCS of these two complexes. The N2→He DNA-bound protein dimer exhibited two major conformers of 48.1 and 50.5 nm (Supp. Figure 4). Crucially, the TW EHSS CCS is consistently smaller than the CCS, sug- N2→He gesting ‘contraction’ from the condensed phase structure, con- sistent with many other reports [48–51]. While the difference EHSS TW between the CCS and CCS for the DNA-bound N2→He p50 dimer was between 19 and 27% (conformer-dependent), this increased to 32% for the non DNA-bound complex, indi- cating a more extensive contraction in the absence of DNA in Figure 6. Gas-phase structure of the p50 homodimer is the central core. stabilised in the presence of DNA. Mobcal was used to deter- EHSS To better understand the reasons for this contraction, we mine the theoretical ( CCS) value of the p50 dimer using the exact hard sphere scattering (EHSS) model, in the absence (left) investigated the evaporation process during ESI, monitoring and presence (right) of DNA from the X-ray structure, for com- structural changes during transfer to the gas phase, and parison with experimentally determined cross section values performing a molecular dynamics simulation over 80 ns TW ( CCS ) N2→He (Fig. 7). Root mean square deviation (RMSD) as well as EHSS CCS values were calculated for the final structure of each 2-ns run after removing ~ 10% of the solvent molecules. The Comparison with Theoretical Modelling RMSD values of the unbound WT p50 dimer as a function of the simulation time reveal an increase of up to 20%, implying a To further interrogate the observed differences in p50 con- significant conformational change upon desolvation. formers in the absence and presence of DNA, and to assess Using the projection approximation (PA) and the exact hard whether the condensed phase structure is maintained in the gas- sphere scattering (EHSS) models, the theoretically calculated phase upon ‘native’ ESI, we compared the experimentally TW CCS values of the p50 dimer exhibited conformational con- determined cross sections ( CCS ) with theoretically N2→ He EHSS TW traction, with the CCS fitting between two theoretical calculated ( CCS) values based on the exact hard sphere N2→He values of the final gas-phase simulation. Indeed, the scattering modal (EHSS) implemented in Mobcal (Fig. 6). TW CCS was only 8% smaller than the more exact EHSS Both the unbound and DNA-bound forms of the p50 (WT) N2→He EHSS 2 value (Fig. 7), which is similar to the differences observed in dimer possess very similar CCS of 61.5 and 62.5 nm , other studies [50]. By considering the final modelled gas-phase which is perhaps not surprising given that they only differ by Figure 7. Solvent evaporation during electrospray ionisation results in collapse of the p50 dimer which is partially stabilised in the presence of DNA. Simulation of the evaporation process of the unbound (left) and DNA-bound (right) p50 dimer. Each data point represents the final CCS value calculated using either the projection approximation (PA, blue) or the exact hard sphere scattering (EHSS, red) model of a 2-ns run upon removing 10% of the solvent. Empty symbols correspond to CCS values of the final gas-phase simulation. Root mean square deviation (RMSD) values of each final structure are displayed (top). Green lines exemplify the TW experimentally determined CCS values (CCS ), with two predominant conformers being defined for the DNA-bound p50 N2→He exp dimer M. Vonderach et al.: DNA Binding Stabilises NF-κB Dimers structure, it is apparent that the conformational change in the for Ser337 (phosphorylated by both PKA and Chk1) as a p50 dimer induced upon evaporation is mostly related to the critical regulator of p50 homo-dimerization. These findings removal of the inner cavity. are in contrast to a previous report, which implied a direct The final gas-phase structure of the DNA-bound p50 dimer effect of PKA -mediated phosphorylation at Ser337 on the exhibited a much lower RMSD value (6.7%), implying a more ability of p50:p65 heterodimers to bind DNA, in the absence conservative structural change compared to the unbound di- of an effect on p50 dimerization [15]. mer. Again, the two experimentally determined CCS values are Based on our observations of an order of magnitude de- located between the theoretical PA and EHSS values. Although crease in the relative DNA binding affinity of a S242D p50 we cannot assign structures for the two experimentally ob- mutant, we determined that the Chk1 (but not PKA )-mediated TW served conformations, CCS for the more abundant phosphorylation of Ser242 is a key regulator of p50 homodi- N2→He EHSS ‘open’ conformer is only 5.5% smaller than the CCS mer DNA binding. Ser242 maps to the DNA binding interface value. Consequently, it is likely that the final MD structure is of p50 and lies immediately N-terminal to a critical Lys residue similar to the dominant experimental gas-phase conformer. The that drives a direct interaction with the DNA phosphate back- major changes in this final simulated gas-phase structure occur bone. Phosphorylation of Ser242 is therefore likely to serve as in the outer loop (surface) regions, indicating that the presence a mechanism to directly disrupt this interaction, perhaps upon of DNA in the central cavity of the p50 dimer likely functions Chk1 activation in cells subject to genotoxic stress. to stabilise the structure and prevent collapse of the inner core Finally, comparison of our experimentally derived TW during ESI-IM-MS. CCS values (CCS ) with theoretically calculated N2→He exp Collision-induced unfolding of a single common charge values (CCS ) derived from the p50 dimer X-ray structure the confirmed that although there was little difference in the CCS state (17+) was used to assess the relative stability of these the two complexes in more detail (Supp. Figure 6). A three-step for the p50 dimer upon addition of DNA, the CCS values for exp unfolding profile is observed for the non DNA-bound p50 these complexes were markedly differed. By performing mo- dimer, occurring at collision voltages (CVs) of 40, 48 and lecular dynamics simulations, we uncovered desolvation- 58 V. For the DNA-bound dimer, initial collisional activation mediated structural contraction of the p50 homodimer during induces marginal structural contraction (decreased CCS), be- ESI, which was stabilised by the presence of DNA in the fore collision-mediated elongation. This structural compaction central cavity. The final modelled gas-phase structures, whose event prior to unfolding is similar to that reported previously EHSS values are highly similar to the CCS of the more exp for the dimeric p53 transcription factor [52]. For the DNA- abundant elongated conformer, suggest only minor conforma- bound p50 dimer, the first transition to a more unfolded con- tional changes on the surface of the protein complex when formation takes place at a CV of 59 V, 19 V higher than that compared with the X-ray crystal structure. This stabilising required to start mediating unfolding of the non DNA-bound effect of DNA on the structure of the dimeric p50 transcrip- dimer. These results underpin our findings that DNA binding tion factor was further confirmed by examining the helps to stabilise the structure of the p50 dimer, although collision-induced unfolding conformational profile, and interestingly the unfolded highly activated structures for both again demonstrates how native MS can be used to derive complexes have similar CCS values, suggesting a similar un- structural information for this important class of DNA- folded conformation. binding proteins [52, 53]. Conclusion Funding Information A central goal of this study was to gain insight into the potential This work was supported by the Biotechnology and Biological mechanisms of phosphorylation-mediated transcriptional reg- Sciences Research Council (BBSRC) through grants BB/ ulation through the NF-κB transcription factor p50, by explor- L009501/1 and BB/M012557/1, as well as a research grant ing the effects of specific phosphorylation events on its ability from NorthWest Cancer Research (CR1097). to dimerize and interact with DNA. The two known p50 regulatory protein kinases under investigation, PKA and Chk1, were shown to phosphorylate p50 in vitro on two and six sites respectively by LC-MS. Native MS analysis of p50 in Open Access the absence and presence of the kB DNA oligomer revealed This article is distributed under the terms of the Creative significant differences in protein dimerization after phosphor- Commons Attribution 4.0 International License (http:// ylation with either enzyme, with the change in dimer to mono- creativecommons.org/licenses/by/4.0/), which permits unre- mer ratio suggesting that at least one of the phosphorylation stricted use, distribution, and reproduction in any medium, sites was responsible for regulating p50 dimerization. By ex- provided you give appropriate credit to the original author(s) pressing and evaluating site-specific acidic ‘phosphomimetic’ and the source, provide a link to the Creative Commons versions of p50, where established sites of phosphorylation license, and indicate if changes were made. were mutated to aspartic acid, we were able to define a role M. Vonderach et al.: DNA Binding Stabilises NF-κBDimers 24. 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DNA Binding and Phosphorylation Regulate the Core Structure of the NF-κB p50 Transcription Factor

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Abstract

B The Author(s), 2018 J. Am. Soc. Mass Spectrom. (2018) DOI: 10.1007/s13361-018-1984-0 FOCUS: HONORING CAROL V. ROBINSON'S ELECTION TO THE NATIONAL ACADEMY OF SCIENCES: RESEARCH ARTICLE DNA Binding and Phosphorylation Regulate the Core Structure of the NF-κB p50 Transcription Factor 1 2 3 2 Matthias Vonderach, Dominic P. Byrne, Perdita E. Barran, Patrick A. Eyers, Claire E. Eyers Centre for Proteome Research, Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool, L69 7ZB, UK Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool, L69 7ZB, UK Michael Barber Centre for Collaborative Mass Spectrometry, Manchester Institute of Biotechnology, The University of Man- chester, 131 Princess Street, Manchester, M1 7DN, UK Abstract. The NF-κB transcription factors are known to be extensively phosphorylated, with dynamic site-specific modification regulating their ability to dimerize and interact with DNA. p50, the proteolytic product of p105 (NF-κB1), forms homodimers that bind DNA but lack intrinsic transactivation function, functioning as repres- sors of transcription from κB promoters. Here, we examine the roles of specific phosphorylation events catalysed by either protein kinase A (PKA ) or Chk1, in regulating the functions of p50 homodimers. LC-MS/MS analysis of proteolysed p50 following in vitro phosphorylation allows us to define Ser328 and Ser337 as PKA - and Chk1-mediated modifications, and pinpoint an additional four Chk1 phosphosites: Ser65, Thr152, Ser242 and Ser248. Native mass spectrometry (MS) reveals Chk1- and PKA -regulated disruption of p50 homodimer formation through Ser337. Additionally, we characterise the Chk1-mediated phosphosite, Ser242, as a regulator of DNA binding, with a S242D p50 phosphomimetic exhibiting a > 10-fold reduction in DNA binding affinity. Conformational dynamics of phosphomimetic p50 variants, including S242D, are further explored using ion-mobility MS (IM-MS). Finally, comparative theoretical modelling with experimentally observed p50 conformers, in the absence and presence of DNA, reveals that the p50 homodimer undergoes conformational contraction during electrospray ionisation that is stabilised by complex formation with κBDNA. Keywords: Native MS, Ion mobility-mass spectrometry, NF-κB, Collision-induced unfolding, Phosphorylation, DNA binding, Molecular modelling Received: 14 March 2018/Revised: 26 April 2018/Accepted: 30 April 2018 such family of ubiquitous transcription factors is NF-kappaB Introduction (NF-κB), and regulated activation of this signal transduction egulated binding of specific transcription factor com- pathway is required for transcriptional control of hundreds of Rplexes to their cognate DNA sequences directly influences genes, under both physiological and pathophysiological condi- the rate at which transcription of individual genes occurs. One tions. This important family of transcription factors is essential for numerous diverse biological functions, including regulation Electronic supplementary material The online version of this article (https:// of inflammation and immune responses, proliferation and apo- doi.org/10.1007/s13361-018-1984-0) contains supplementary material, which ptosis [1]. is available to authorized users. Stable interaction of NF-κB (and other) transcription Correspondence to: Claire Eyers; e-mail: CEyers@liverpool.ac.uk factors with DNA response elements typically requires the M. Vonderach et al.: DNA Binding Stabilises NF-κBDimers formation of either homo- or heterodimers, which permits averaged collision cross section (CCS). Importantly, CCS the recognition of palindromic DNA-sequence motifs by values can be calculated for a given geometry from theoretical adjacent DNA binding domains [2–4]. Specificity of NF- structures derived from density functional theory or molecular κB-mediated transcription is regulated in part by the com- dynamic (MD) simulations using a variety of methods such as binatorial diversity arising from the five related NF-κB projection approximation (PA) [33], exact hard sphere scatter- proteins, with each NF-κB dimer regulating both distinct ing (EHSS) model [34], the trajectory method (TM) [35], and overlapping sets of genes due to subtle differences in projection superposition approximation (PSA) [36]or scatter- their kB consensus DNA binding specificity [5]. Dimeriza- ing on electron density isosurfaces (SEDI) [37]. Such compu- tion of NF-κB proteins, interaction with other transcription- tational strategies allow prediction of putative protein struc- al co-factors and DNA binding, is also regulated by exten- tures by comparison with experimentally derived CCS values sive post-translational modification (PTM), with dynamic derived using a variety of experimental-based structural phosphorylation established as being critical for cellular approaches. function [6–12]. Here, we exploit standard MS-based phosphoproteomics in p105 (NFκB1) is one of the five NF-κB transcription factors combination with native ion mobility-mass spectrometry (IM- and is commonly proteolysed to generate a functional p50 mole- MS) and molecular modelling to define the effects of p50 cule lacking a transactivation domain [1]. The high basal levels of phosphorylation on dimerization and DNA binding. Using nuclear localised p50 homodimers in unstimulated cells are thus travelling wave-IMS (TW-IMS) and comparative molecular thought to act as repressors of transcription from κBpromoters by dynamics (MD) simulations, we demonstrate that the p50 competing for DNA binding with transcriptionally active NF-κB homodimer is stabilised by the presence of its cognate DNA dimers, including the RelA:p50 heterodimer [13, 14]. Regulation oligomer and define specific p50 phosphorylation sites as key of p50 by reversible phosphorylation is much less well understood potential regulators of either DNA binding or homo- than that of the p105 precursor [15–18], but appears to be a critical dimerization. regulator of efficient p50 binding to DNA, and thus transcriptional repression. Phosphorylation of p50 in vitro by the catalytic subunit of protein kinase A (PKA ) has been reported to enhance its Experimental ability to bind DNA in a manner that is independent on its ability Protein Expression and Purification to dimerise [15, 16]. Mutational studies using phosphomimetic variants mapped this critical phosphorylation event to Ser337, and Murine NF-κB p50 (39-364 wild-type; WT) was cloned into Ricciardi and colleagues demonstrated that this site is constitu- the pOPINM vector (OPPF) using the InFusion PCR cloning tively phosphorylated by PKA in unstimulated cells, contributing kit (Clonetech). S65D, S242D, S248D and S337D p50 muta- to κB transcriptional repression under basal (non-stimulated) con- tions were generated by PCR site-directed mutagenesis from ditions [15]. Mutation of two additional p50 Ser residues, Ser65 the WT p50 construct. Appropriate mutations were confirmed and Ser342, to Ala, was also reported to negatively influence the by DNA sequencing. All proteins were produced in BL21 DNA binding ability of p50 [15]. However, regulated phosphor- (DE3) pLysS E. coli cells (Novagen) with expression induced ylation of these residues has not yet been demonstrated. with 0.5 mM IPTG for 3 h at 18 °C and purified with a 3C Mass spectrometry (MS) can be used in a variety of ways to protease cleavable N-terminal His6-MBP-tag. Fusion proteins elucidate information about all levels of protein structure, from were first purified by affinity chromatography using amylose primary to quaternary configurations. Exploitation of ‘native’ resin (NEB), and p50 subunits were cleaved from the MS, where the analyte is transferred from solution into the gas- immobilised affinity medium using 3C protease in 50 mM Tris phase under gentle electrospray ionisation (ESI) conditions (pH 7.4), 100 mM NaCl, 1 mM DTT, 10% (v/v) glycerol and [19–21] from a volatile buffer such as ammonium acetate at 20 mM imidazole. 3C protease (purified as an N-terminal His6- physiological pH, allows non-covalent complexes to be inter- tag fusion protein) was subsequently removed by immobilised rogated. Native MS can thus be used to determine protein metal affinity chromatography. complex stoichiometry [22] or compare the relative dissocia- tion constant (K ) of different ligands [23–25]. Many proteins In Vitro Phosphorylation, Digestion and LC-MS/MS and protein complexes largely retain their solution-phase con- Analysis formation under native ESI conditions [26–28], thus their structure and the effect of protein modification and/or ligand p50 protein (25 μg, 35-381, Enzo Scientific) in 10 mM binding on conformational dynamics and stability can be read- TrisOAc was incubated at 37 °C for 2 h with 10 mM MgCl 2, ily interrogated with gas-phase methods such as ion mobility 250 μM ATP, 1 mM DTT and 1 mM EGTA in the presence of spectrometry (IMS) [29–31] or infrared spectroscopy [32]. In 0.25 μg of either PKA [38] or 4.2 μg Chk1 (MRC PPU IMS, ions are transported by an electric field through a drift cell Reagents and Services, Dundee). Reactions were stopped by filled with an inert gas such as helium or nitrogen, permitting rapid buffer exchange into NH OAc. For digestion, 1 μgof separation of analyte ions based on their charge, mass and protein was denatured prior to digestion by addition of 1% conformation. Consequently, the recorded drift time of ions Waters RapiGest at 80 °C for 10 min. Enzymatic digestion was through the IMS cell can be used to define their rotationally performed at 37 °C overnight using 0.02 μgtrypsinand M. Vonderach et al.: DNA Binding Stabilises NF-κB Dimers stopped by addition of 0.5% TFA and incubation for 45 min at increasing collision energies. Contour plots representing the 37 °C. Phosphopeptides were enriched using TiO spin col- unfolding profile were produced with Origin 9.0. umns (GLSciences) as previously described [39]. LC-MS/MS analysis was performed on an Orbitrap Fusion Tribrid mass K Determination spectrometer (ThermoScientific), attached to an Ultimate 3000 p50 WT or single-point aspartic acid mutants (3 μM) were nano system (Dionex). Peptides were loaded onto the trapping incubated with 0.2–6 μMof κB DNA oligomer 5′-CCCC column (ThermoScientific, PepMap100, C18, 300 μm× CGGGGGCCCCCGGGGG-3′ (Sigma) in 300 mM NH OAc 5 mm), using partial loop injection, for 7 min at a flow rate of for 5 min at room temperature (final volume 10 μL) prior to 9 μL/min with 2% (v/v) MeCN 0.1% (v/v) TFA and then native (IM-)MS analysis. Multi-Gaussian fitting with Origin resolved on an analytical column (Easy-Spray C18 75 μm× 9.0 was used to ascertain the peak areas of all charge states of 500 mm 2 μm bead diameter column) using a 30-min method both the unbound and DNA-bound p50. K values for DNA from 96.2% A (0.1% FA) and 3.8% B (80% MeCN 19.9% binding were determined by nonlinear peak fitting using Eq. 2: −1 H O 0.1% FA) to 100% B at a flow rate of 300 nL min . A full scan mass spectrum was acquired (30K resolution at m/z 200) 0 1 sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi and data-dependent MS/MS analysis performed using a top IPðÞ *L 1 ½ P ½ L ½ L ½ L ½ P 0 0 0 0 0 @ A speed approach (cycle time of 3 s), using HCD and EThcD ¼ −1− þ þ 4 þ − −1 ð2Þ IPðÞ 2 K K K K K D D D D D fragmentation modes, with product ions being detected in the orbitrap (15K resolution). I(PL) and I(P) define the peak area of the DNA-bound Native IM-MS and Collision-Induced Unfolding protein complex and the unbound p50 protein dimer, respec- A commercial TW-IMS instrument (Waters G2-Si) was tively, [P] and [L] are the original protein and DNA concen- 0 0 utilised for native IM-MS. p50 was buffer exchanged into trations [25]. 100 mM NH OAc using 10-kDa molecular cut-off spin filter columns (Amicon) and 1–3 μl of sample (typically 5 μM) was Molecular Modelling and CCS Calculation subjected to electrospray ionisation (ESI) at a voltage of 1.3– ff14SB, OL15 and tip3p force fields implemented in AM- 3 kV using a self-pulled nanospray tip. Sampling cone was set BER16 [43] were used to simulate the effect of protein −2 at 75 V. Trap pressure was adjusted to 5 × 10 mbar, He cell desolvation during ESI on the conformation of the DNA- pressure was 4.53 mbar, IMS pressure was 2.78 mbar and −2 bound and unbound forms of the p50 (39-350) homodimer transfer tube pressure was 5.18 × 10 mbar. IMS was per- (1.NFKB.pdb) [44]. The structure was neutralised by addition formed using a travelling wave height of 29 V and a velocity of Na ions and embedded into a water box containing approx- of 650 m/s. Calibration of the TriWave device was performed imately 35,000 water molecules. Geometry optimization for as previously described [40, 41]using β-lactoglobulin A (Sig- molecular dynamics (MD) simulation was performed utilising ma L7880), avidin (Sigma A9275), transthyretin (Sigma the steepest descent energy minimisation and conjugate gradi- P1742), concanavalin A (Sigma C2010) and serum albumin ent method. Molecular dynamic simulations were performed at (Sigma P7656) as calibrants. Upon removing the time the ions 350 K for 2 ns, with 2 fs integration steps. Langevin dynamics spend in the time of flight mass spectrometer, a logarithmic plot were applied to regulate the temperature. The result of the 2 ns of the ‘corrected drift time t’ versus the by charge q and reduced run was used as input for a further MD simulation in which the mass μ corrected CCS, the so-called reduced CCS was calcu- amount of water was reduced by ~ 10%. Upon completing lated and a straight line extrapolated in order to ascertain the TW 40 × 2 ns runs, a final 10-ns run was performed in the absence slope, m, and intersection, C. The experimental CCS N2→He of solvent, using a charge state of 16+ for the p50 dimer and values (where the TW-IMS-determined drift times in a nitrogen 18+ for the DNA-bound p50 dimer, those being the most atmosphere were converted to helium CCS values [42]) were dominant charge states observed. CCS values of all final MD finally calculated from measured drift times using Eq. 1: structures were computed using Mobcal [34, 35]. RMSD values were calculated utilising CPPTRAJ implemented in AMBER [43]. TW 0 CCS ¼ t � expðÞ C ð1Þ N →He pffiffiffi Results CCSD values are defined as the full width at half maximum Chk1 Induces Extensive Phosphorylation of p50 of the CCS distribution. In Vitro Collision-induced unfolding (CIU) was used to evaluate the transitional unfolding profiles of the protein and protein-DNA Based on mutational analysis and in vitro protein kinase assays, complexes. An individual charge state was isolated with the it was previously reported that phosphorylation of p50 by quadrupole mass filter and subjected to collisional activation in PKA on Ser337 is essential for high-affinity DNA binding. the trap region of the TriWave by application of gradually However, the mechanism whereby pSer337 regulates DNA M. Vonderach et al.: DNA Binding Stabilises NF-κBDimers binding was not defined. Although a S337A p50 mutant ex- Ser240 and Ser246 respectively) both lie in the linker region hibited dramatically reduced DNA binding ability in cells (L3) between the two distinct domains of p50 (Fig. 1b, c). compared to the wild-type p50 protein, this site of modification Phosphorylation of one or both of these residues in this linker is distal from the DNA binding domain, and the ability of region, which adopts a well-defined structure that can fit into S337A p50 to dimerise with p65/RelA was reported to be the major groove of the DNA substrate, is thus likely to have a unaffected [16]. Moreover, the ability of p50 to be phosphor- significant effect on DNA binding ability of p50. In particular, ylated by PKA , or other putative regulatory kinases, at sites Ser242 lies adjacent to a key Lys residue at position 243 distinct from Ser337 was not evaluated in side-by-side (mouse Lys241), which directly interacts with the DNA back- experiments. bone. Consequently, we hypothesised that Ser242 phosphory- Using standard peptide-based tandem MS analysis, we re- lation is likely to disrupt p50 DNA binding. Similarly, Ser65 cently reported PKA -mediated phosphorylation of recombi- (mouse Ser63) lies downstream of a five residue cluster nant p50 (35-381) in the presence of p65/RelA at four sites in (RxRYxCExxS) located in L1, another loop that makes direct addition to Ser337, namely Ser223, Ser226, Ser236 and contacts with the κB DNA. Even though phosphorylation of Thr263 [6]. To assess whether these phosphorylation sites are both Ser328 and Ser337 has been shown to influence the ability dependent on inclusion of RelA in the assay, and thus forma- of p50 to bind DNA, both are localised to the second domain, tion of a RelA:p50 heterodimer, we repeated in vitro distal from the DNA-binding region, suggesting a gross con- phosphosite mapping using PKA with p50 alone. Under these formational change of domain 1 with respect to domain 2 and conditions, only Ser328 and Ser337 were identified as PKA - the DNA-protein interface, rather than a direct effect of phos- regulated phosphosites (Fig. 1, Supp. Figure 1), confirming phorylation of these residues on the ability to bind DNA. previous observations that NF-κB proteins likely adopt differ- ent conformations dependent on their dimerization partners [6]. Phosphorylation of p50 by Chk1 Destabilises MS analysis of the intact phosphorylated p50 identified a single Dimerization phosphate-carrying proteoform (in addition to the non- phosphorylated protein), with no evidence of a doubly phos- To assess the effect of p50 phosphorylation on its ability to phorylated species, suggesting that phosphorylation of p50 on dimerise and bind DNA, we analysed p50 (35-381) by nano- Ser328 and Ser337 is likely to be mutually exclusive (Fig. 1a). electrospray ionisation (nESI)-MS under non-denaturing ‘na- As well as cellular evidence for p50 regulation by PKA tive’ MS conditions, before and after in vitro phosphorylation [18], Chk1 is known to play a major role in phosphorylation- with either PKA or Chk1. As expected, intact non- mediated regulation of this transcription factor, inhibiting DNA phosphorylated p50 was preferentially observed as a dimer binding via phosphorylation at Ser328 [45, 46]. Previous in- with only a small amount of monomer present (Fig. 2). Upon vestigations have focused on phosphorylation of Ser328 by phosphorylation with either protein kinase, there was a small Chk1, even though p50 contains a number of other conserved but consistent increase in the relative abundance of the p50 Chk1 consensus sites. Consistently, we identified a total of six monomer (observed charge states of 11+ to 13+) with respect in vitro Chk1 phosphorylation sites on p50 (35-381) using MS- to the p50 homodimer (observed charge states of 16+ to 19+), based phosphopeptide mapping (Supp. Figure 1), including the demonstrating phosphorylation-mediated destabilisation of the previously reported Ser328 site [46], the overlapping PKA site homodimeric protein. at Ser337, and four novel sites at Ser65, Thr152, Ser242 and Inclusion of a DNA oligomer designed to match the κB Ser248. Chk1 phosphorylation of p50, at least in vitro, is thus consensus sequence for the p50 homodimer (5′-CCCC much more extensive than previously supposed. Analysis of CGGGGGCCCCCCGGGGG-3′) revealed a stabilising effect the intact Chk1-phosphorylated p50 reveals a predominant of DNA binding upon dimer formation. No residual monomer singly phosphorylated species, as well as a doubly phosphory- was observed for the non-phosphorylated p50 in the presence lated form, with relatively low levels of non-phosphorylated of the κB DNA, with stoichiometric formation of the p50 (Fig. 1a). Analogous to the PKA -phosphorylated p50, the p50:p50:DNA complex. A similar stabilising effect was also six sites modified by Chk1 are thus unlikely to be stoichiomet- seen for PKA -phosphorylated p50, with no monomeric p50 rically combinatorial, rather, p50 is modified at specific observed and stoichiometric DNA:protein complex formed. In (discrete) combinations of amino acids. contrast, although the Chk1-phosphorylated p50 homodimer Of the 10 phosphosites that we identified in total on p50 stoichiometrically bound the κB DNA, dimer formation was (with or without RelA), only two, Thr152 and Ser226, are not not enhanced under these conditions (Fig. 2). Chk1 phosphor- completely conserved in model vertebrates (Supp. Figure 2). ylation of p50 thus appears to actively disrupt homo-dimeriza- Thr152 is changed to Ile in Xenopus laevis p50, although it is tion, irrespective of effects on DNA binding. conserved as a Thr in all other species examined. Ser226 was To further evaluate the structural effects of p50 phos- absent in both frog and chicken p50 sequences. Considering phorylation, we used ion mobility-MS (IM-MS) to compare the position of the six PKA and Chk1 phosphosites identified p50 conformation and structural dynamics following treat- in the absence of p65 in the p50 homodimer structure (PDB ment with either PKA or Chk1 (see supplementary infor- entry 1NFK [44]), a number of potential roles for phosphory- mation; Supp. Figure 3). The rotationally averaged collision TW lation might be hypothesised. Ser242 and Ser248 (mouse cross section ( CCS ) of the p50 homodimer was N2→He M. Vonderach et al.: DNA Binding Stabilises NF-κB Dimers Figure 1. Multi-site phosphorylation of p50 by PKA and Chk1 is not combinatorial. (a) Intact mass spectra of p50 before (bottom) and after in vitro phosphorylation with PKA (middle) or Chk1 (top). Depicted are the 30+ (green) and 31+ (blue) charge states of the non-phosphorylated (circles), mono-phosphorylated (triangles) and di-phosphorylated (diamonds) forms of p50. (b) Identified sites of phosphorylation (red) mapped onto the X-ray crystal structure of the mouse p50:p50 homodimer bound to DNA (grey), PDB entry 1NFK. Individual p50 monomers are either in blue or yellow. Sites of phosphorylation are numbered according to the human sequence. Ser328 and Ser337 were identified as PKA phosphosites. All sites were phosphorylated by Chk1. (c) Loops 1 and 3 of p50 with kB DNA show direct interaction of the regions containing Ser65, Ser242 and Ser248 with the DNA determined as 44.3 nm , relatively independent of the cor- charge states, there was no statistically significant change in TW responding charge state (16+ to 18+) (Supp. Figure 3). the absolute CCS value after phosphorylation with N2→He Although subtle differences were observed in the p50 either PKA or Chk1 (Supp. Figure 3). TW TW CCS distribution ( CCSD )uponphos- Interestingly, there was a small but reproducible 1.5% de- N2→He N2→He TW phorylation with PKA , particularly for the 16+ and 17+ crease in the CCS values of the DNA-bound p50 c N2→He Figure 2. Phosphorylation of p50 regulates dimerization. Native mass spectra of p50 before (bottom) or after phosphorylation with either PKA (middle, red) or Chk1 (top, blue), in the absence (left) or presence (right) of the p50 kB DNA oligomer. Charge states are indicated M. Vonderach et al.: DNA Binding Stabilises NF-κBDimers the DNA, and Ser337 is located in the dimerization region and might impart allosteric regulation of dimerization upon phos- phorylation. S65D, S242D, S248D and S337D p50 were analysed by native IM-MS alongside WT p50 (Fig. 3), using multi-Gaussian fitting to evaluate the peak areas and calculate the monomer:dimer ratio for each species. Akin to WT p50, the dominant species of all S→ D p50 protein mass spectra were dimers (Fig. 3). Little, or no, difference was observed in the monomer:dimer ratios for S65D and S242D p50 (Fig. 3,Table 1). However, there was a 2.4-fold relative increase in monomeric S337D p50 com- pared to the non-phosphorylated WT p50. The ability of S337D to disrupt p50 dimerization was further confirmed by size exclusion chromatography (SEC), which revealed a ~ 5-fold relative increase in monomeric S337D p50 based on protein staining and densitometry (Supp. Figure 5). To- gether, these findings support our initial prediction that Figure 3. Ser337 phosphomimetic disrupts dimerization of Ser337 phosphorylation plays a significant role in control- p50. Native mass spectra of p50 (39-364) wild-type (WT, black), ling (disrupting) p50 dimerization. S65D (blue), S242D (red), S248D (brown) and S337D (green) IM-MS analysis of the p50 protein variants revealed a phosphomimetic versions showing p50 monomer and dimer. TW 2 CCS of ~ 42.0–42.5 nm suggest- peak maximum N2→He Non-assigned peaks derive from the cleaved contaminating ing that the dominant dimer conformation is highly similar MBP expression tag. Charge states are indicated for all species analysed (Fig. 4). However, a notable in- crease in conformational flexibility was observed for all mutants compared with WT p50. In particular, the CCSD dimer with decreasing charge state (20+ to 18+), revealing values for S248D and S337D are 4.6 and 5.0 nm ,respec- slight compaction of the complex. Moreover, the asymmetric tively, compared with a CCSD value of just 2.9 nm for CCS profile of the WT p50:p50:DNA complex is indicative of WT p50 (Fig. 4). the presence of two unresolved conformers with charge state TW 2 averaged CCS values of 51.1 and 53.3 nm for the N2→He unmodified p50-DNA dimer. Of note, the relative proportion Phosphomimetic Version of the Chk1-Mediated p50 of the more compact conformer increased with a reduction in Phosphosite Ser242 Destabilises DNA Binding charge state, suggesting gas-phase conformational contraction. Comparable results were also observed following native IM- To evaluate the effect of modification of S248 and S337 on MS analysis of p50 (39-364) (Supp. Figure 4). DNA binding, we employed the titration method and native MS to determine the dissociation constants (K ) for DNA binding for each of the p50 protein variants (Fig. 4; Table 1). Ser337 Phosphomimetic Disrupts p50 Homodimer As expected, the unphosphorylated WT p50 homodimer ex- Formation hibited a relatively high affinity for DNA in this assay, with a To evaluate which of the site-specific, but sub-stoichiometric K value of < 40 nM, similar to that previously reported for PKA - or Chk1-mediated phosphorylation events were respon- both p65 homodimers and p65/p50 heterodimers [47]. All the sible for the observed disruption of dimerization, we expressed aspartic acid mutants analysed exhibited significantly higher Ser→ Asp (potential phosphomimetic) versions of p50 (39- K values than those observed for WT p50, indicating a reduc- 364) for sites predicted to influence either dimer formation or tion in DNA binding affinity. Specifically, S337D p50 exhib- DNA binding: Ser65, Ser242, Ser248 lie in close contact with ited a 3-fold higher dissociation constant than WT p50, with a Table 1. Effect of p50 phosphomimetic variants on protein dimerization and DNA binding. Percentage peak areas of monomers and dimers for p50 WT, S65D, S242D, S248D and S337D and the mutants. DNA binding dissociation constants K , and the relative DNA binding affinity with respect to WT p50, are also presented for each p50 variant Monomer Dimer K value for DNA binding (nM) Relative DNA binding affinity WT 10.5% 89.5% 37 ± 7 100% S65D 9.4% 90.6% 140 ± 16 26.4% S242D 12.4% 87.6% 424 ± 78 8.7% S248D 5.8% 94.2% 112 ± 11 33.0% S337D 25.2% 74.8% 111 ± 15 33.3% M. Vonderach et al.: DNA Binding Stabilises NF-κB Dimers TW Figure 4. Phosphomimetic versions of p50 exhibit increased conformational flexibility compared to WT p50. CCS distri- N2→He butions of wild-type (WT) p50 (39-364) (black) alongside p50 S65D (blue), S242D (red), S248D (brown) and S337D (green) in the absence (left) or presence (right) of DNA measured K of ~ 110 nM, compared with a K of 37 nM for ATDs of S248D and S337D, which lie in the L3 linker region of D D WT p50 under the same conditions. This increase in relative K p50 and the dimerization domain respectively (Fig. 1), suggests is likely attributed to the reduction in the ability of this that both of these phosphomimetic mutations similarly alter the phosphomimetic variant to dimerise (Table 1), a prior require- relative position of the two domains of p50. ment for DNA binding. In agreement with our hypothesis, mimicking the Chk1- mediated p50 phosphorylation site at S242 resulted in an order of magnitude decrease in DNA binding affinity. Replacement of Ser242 with a negatively charged Asp group is predicted to disrupt the direct electrostatic interaction of Lys243 with the phosphate backbone of the DNA. Interestingly, the consistently earlier arrival time distribution (ATD) of S242D when com- pared to that of WT p50 observed in the absence of DNA suggests that this protein can adopt conformations that are likely to be more compact than WT p50 (Fig. 4). In contrast, TW upon DNA binding, the CCS of S242D is consistently N2→He larger than the WT dimeric complex, indicative of a more open conformation. Mutation of the other two identified p50 phosphosites in the DNA binding interface, S65 and S248 (Fig. 1b),resultedina 3.8- and 3.0-fold increase in K values, respectively, compared with the WT protein (Fig. 5), implicating roles for phosphoryla- tion of S65 and S248 in negatively regulating DNA binding of Figure 5. DNA binding of p50 S242D is significantly disrupted. the p50 homodimer. However, ATDs of these two p50 variants Native MS and DNA titration was used to calculate K for DNA are distinct, both in the absence and presence of DNA, indicative binding for each of the p50 homodimers: WT (black), S65D of different effects on gross conformation. The similarity of the (blue), S242D (red), S248D (brown) and S337D (green) M. Vonderach et al.: DNA Binding Stabilises NF-κBDimers the small central cavity which accommodates the helical DNA. In contrast and as previously observed, there was a significant TW difference in the CCS of these two complexes. The N2→He DNA-bound protein dimer exhibited two major conformers of 48.1 and 50.5 nm (Supp. Figure 4). Crucially, the TW EHSS CCS is consistently smaller than the CCS, sug- N2→He gesting ‘contraction’ from the condensed phase structure, con- sistent with many other reports [48–51]. While the difference EHSS TW between the CCS and CCS for the DNA-bound N2→He p50 dimer was between 19 and 27% (conformer-dependent), this increased to 32% for the non DNA-bound complex, indi- cating a more extensive contraction in the absence of DNA in Figure 6. Gas-phase structure of the p50 homodimer is the central core. stabilised in the presence of DNA. Mobcal was used to deter- EHSS To better understand the reasons for this contraction, we mine the theoretical ( CCS) value of the p50 dimer using the exact hard sphere scattering (EHSS) model, in the absence (left) investigated the evaporation process during ESI, monitoring and presence (right) of DNA from the X-ray structure, for com- structural changes during transfer to the gas phase, and parison with experimentally determined cross section values performing a molecular dynamics simulation over 80 ns TW ( CCS ) N2→He (Fig. 7). Root mean square deviation (RMSD) as well as EHSS CCS values were calculated for the final structure of each 2-ns run after removing ~ 10% of the solvent molecules. The Comparison with Theoretical Modelling RMSD values of the unbound WT p50 dimer as a function of the simulation time reveal an increase of up to 20%, implying a To further interrogate the observed differences in p50 con- significant conformational change upon desolvation. formers in the absence and presence of DNA, and to assess Using the projection approximation (PA) and the exact hard whether the condensed phase structure is maintained in the gas- sphere scattering (EHSS) models, the theoretically calculated phase upon ‘native’ ESI, we compared the experimentally TW CCS values of the p50 dimer exhibited conformational con- determined cross sections ( CCS ) with theoretically N2→ He EHSS TW traction, with the CCS fitting between two theoretical calculated ( CCS) values based on the exact hard sphere N2→He values of the final gas-phase simulation. Indeed, the scattering modal (EHSS) implemented in Mobcal (Fig. 6). TW CCS was only 8% smaller than the more exact EHSS Both the unbound and DNA-bound forms of the p50 (WT) N2→He EHSS 2 value (Fig. 7), which is similar to the differences observed in dimer possess very similar CCS of 61.5 and 62.5 nm , other studies [50]. By considering the final modelled gas-phase which is perhaps not surprising given that they only differ by Figure 7. Solvent evaporation during electrospray ionisation results in collapse of the p50 dimer which is partially stabilised in the presence of DNA. Simulation of the evaporation process of the unbound (left) and DNA-bound (right) p50 dimer. Each data point represents the final CCS value calculated using either the projection approximation (PA, blue) or the exact hard sphere scattering (EHSS, red) model of a 2-ns run upon removing 10% of the solvent. Empty symbols correspond to CCS values of the final gas-phase simulation. Root mean square deviation (RMSD) values of each final structure are displayed (top). Green lines exemplify the TW experimentally determined CCS values (CCS ), with two predominant conformers being defined for the DNA-bound p50 N2→He exp dimer M. Vonderach et al.: DNA Binding Stabilises NF-κB Dimers structure, it is apparent that the conformational change in the for Ser337 (phosphorylated by both PKA and Chk1) as a p50 dimer induced upon evaporation is mostly related to the critical regulator of p50 homo-dimerization. These findings removal of the inner cavity. are in contrast to a previous report, which implied a direct The final gas-phase structure of the DNA-bound p50 dimer effect of PKA -mediated phosphorylation at Ser337 on the exhibited a much lower RMSD value (6.7%), implying a more ability of p50:p65 heterodimers to bind DNA, in the absence conservative structural change compared to the unbound di- of an effect on p50 dimerization [15]. mer. Again, the two experimentally determined CCS values are Based on our observations of an order of magnitude de- located between the theoretical PA and EHSS values. Although crease in the relative DNA binding affinity of a S242D p50 we cannot assign structures for the two experimentally ob- mutant, we determined that the Chk1 (but not PKA )-mediated TW served conformations, CCS for the more abundant phosphorylation of Ser242 is a key regulator of p50 homodi- N2→He EHSS ‘open’ conformer is only 5.5% smaller than the CCS mer DNA binding. Ser242 maps to the DNA binding interface value. Consequently, it is likely that the final MD structure is of p50 and lies immediately N-terminal to a critical Lys residue similar to the dominant experimental gas-phase conformer. The that drives a direct interaction with the DNA phosphate back- major changes in this final simulated gas-phase structure occur bone. Phosphorylation of Ser242 is therefore likely to serve as in the outer loop (surface) regions, indicating that the presence a mechanism to directly disrupt this interaction, perhaps upon of DNA in the central cavity of the p50 dimer likely functions Chk1 activation in cells subject to genotoxic stress. to stabilise the structure and prevent collapse of the inner core Finally, comparison of our experimentally derived TW during ESI-IM-MS. CCS values (CCS ) with theoretically calculated N2→He exp Collision-induced unfolding of a single common charge values (CCS ) derived from the p50 dimer X-ray structure the confirmed that although there was little difference in the CCS state (17+) was used to assess the relative stability of these the two complexes in more detail (Supp. Figure 6). A three-step for the p50 dimer upon addition of DNA, the CCS values for exp unfolding profile is observed for the non DNA-bound p50 these complexes were markedly differed. By performing mo- dimer, occurring at collision voltages (CVs) of 40, 48 and lecular dynamics simulations, we uncovered desolvation- 58 V. For the DNA-bound dimer, initial collisional activation mediated structural contraction of the p50 homodimer during induces marginal structural contraction (decreased CCS), be- ESI, which was stabilised by the presence of DNA in the fore collision-mediated elongation. This structural compaction central cavity. The final modelled gas-phase structures, whose event prior to unfolding is similar to that reported previously EHSS values are highly similar to the CCS of the more exp for the dimeric p53 transcription factor [52]. For the DNA- abundant elongated conformer, suggest only minor conforma- bound p50 dimer, the first transition to a more unfolded con- tional changes on the surface of the protein complex when formation takes place at a CV of 59 V, 19 V higher than that compared with the X-ray crystal structure. This stabilising required to start mediating unfolding of the non DNA-bound effect of DNA on the structure of the dimeric p50 transcrip- dimer. These results underpin our findings that DNA binding tion factor was further confirmed by examining the helps to stabilise the structure of the p50 dimer, although collision-induced unfolding conformational profile, and interestingly the unfolded highly activated structures for both again demonstrates how native MS can be used to derive complexes have similar CCS values, suggesting a similar un- structural information for this important class of DNA- folded conformation. binding proteins [52, 53]. Conclusion Funding Information A central goal of this study was to gain insight into the potential This work was supported by the Biotechnology and Biological mechanisms of phosphorylation-mediated transcriptional reg- Sciences Research Council (BBSRC) through grants BB/ ulation through the NF-κB transcription factor p50, by explor- L009501/1 and BB/M012557/1, as well as a research grant ing the effects of specific phosphorylation events on its ability from NorthWest Cancer Research (CR1097). to dimerize and interact with DNA. The two known p50 regulatory protein kinases under investigation, PKA and Chk1, were shown to phosphorylate p50 in vitro on two and six sites respectively by LC-MS. Native MS analysis of p50 in Open Access the absence and presence of the kB DNA oligomer revealed This article is distributed under the terms of the Creative significant differences in protein dimerization after phosphor- Commons Attribution 4.0 International License (http:// ylation with either enzyme, with the change in dimer to mono- creativecommons.org/licenses/by/4.0/), which permits unre- mer ratio suggesting that at least one of the phosphorylation stricted use, distribution, and reproduction in any medium, sites was responsible for regulating p50 dimerization. By ex- provided you give appropriate credit to the original author(s) pressing and evaluating site-specific acidic ‘phosphomimetic’ and the source, provide a link to the Creative Commons versions of p50, where established sites of phosphorylation license, and indicate if changes were made. were mutated to aspartic acid, we were able to define a role M. Vonderach et al.: DNA Binding Stabilises NF-κBDimers 24. 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Journal of The American Society for Mass SpectrometrySpringer Journals

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