Toward an understanding of the conformational plasticity of S100A8 and S100A9 Ca2+-binding proteins
2023; Elsevier BV; Volume: 299; Issue: 4 Linguagem: Inglês
10.1016/j.jbc.2023.102952
ISSN1083-351X
AutoresMagdalena Polakowska, Kamil Steczkiewicz, Roman H. Szczepanowski, Aleksandra Wysłouch‐Cieszyńska,
Tópico(s)Computational Drug Discovery Methods
ResumoS100A8 and S100A9 are small, human, Ca2+-binding proteins with multiple intracellular and extracellular functions in signaling, regulation, and defense. The two proteins are not detected as monomers but form various noncovalent homo- or hetero-oligomers related to specific activities in human physiology. Because of their significant roles in numerous medical conditions, there has been intense research on the conformational properties of various S100A8 and S100A9 proteoforms as essential targets of drug discovery. NMR or crystal structures are currently available only for mutated or truncated protein complexes, mainly with bound metal ions, that may well reflect the proteins' properties outside cells but not in other biological contexts in which they perform. Here, we used structural mass spectrometry methods combined with molecular dynamics simulations to compare the conformations of wildtype full-length S100A8 and S100A9 subunits in biologically relevant homo- and heterodimers and in higher oligomers formed in the presence of calcium or zinc ions. We provide, first, rationales for their functional response to changing environmental conditions, by elucidating differences between proteoforms in flexible protein regions that may provide the plasticity of the binding sites for the multiple targets, and second, the key factors contributing to the variable stability of the oligomers. The described methods and a systematic view of the conformational properties of S100A8 and S100A9 complexes provide a basis for further research to characterize and modulate their functions for basic science and therapies. S100A8 and S100A9 are small, human, Ca2+-binding proteins with multiple intracellular and extracellular functions in signaling, regulation, and defense. The two proteins are not detected as monomers but form various noncovalent homo- or hetero-oligomers related to specific activities in human physiology. Because of their significant roles in numerous medical conditions, there has been intense research on the conformational properties of various S100A8 and S100A9 proteoforms as essential targets of drug discovery. NMR or crystal structures are currently available only for mutated or truncated protein complexes, mainly with bound metal ions, that may well reflect the proteins' properties outside cells but not in other biological contexts in which they perform. Here, we used structural mass spectrometry methods combined with molecular dynamics simulations to compare the conformations of wildtype full-length S100A8 and S100A9 subunits in biologically relevant homo- and heterodimers and in higher oligomers formed in the presence of calcium or zinc ions. We provide, first, rationales for their functional response to changing environmental conditions, by elucidating differences between proteoforms in flexible protein regions that may provide the plasticity of the binding sites for the multiple targets, and second, the key factors contributing to the variable stability of the oligomers. The described methods and a systematic view of the conformational properties of S100A8 and S100A9 complexes provide a basis for further research to characterize and modulate their functions for basic science and therapies. S100A8 and S100A9 are small (10.8 and 13.1 kDa, respectively) α-helical proteins belonging to the S100 family, which is the largest group of calcium-binding proteins in humans (1Jukic A. Bakiri L. Wagner E.F. Tilg H. Adolph T.E. Calprotectin: from biomarker to biological function.Gut. 2021; 70: 1978-1988Crossref PubMed Scopus (105) Google Scholar). They are involved in various functions connected to cell signaling, regulation, and defense (1Jukic A. Bakiri L. Wagner E.F. Tilg H. Adolph T.E. Calprotectin: from biomarker to biological function.Gut. 2021; 70: 1978-1988Crossref PubMed Scopus (105) Google Scholar, 2Donato R. Cannon B.R. Sorci G. Riuzzi F. Hsu K. Weber D.J. et al.Functions of S100 proteins.Curr. Mol. Med. 2013; 13: 24-57Crossref PubMed Scopus (944) Google Scholar). For instance, they are overexpressed and secreted by white blood cells to mediate inflammation by interacting with extracellular receptors, for example, TLR4 and RAGE (3Vogl T. Stratis A. Wixler V. Völler T. Thurainayagam S. Jorch S.K. et al.Autoinhibitory regulation of S100A8/S100A9 alarmin activity locally restricts sterile inflammation.J. Clin. Invest. 2018; 128: 1852-1866Crossref PubMed Scopus (125) Google Scholar, 4Wang S. Song R. Wang Z. Jing Z. Wang S. Ma J. S100A8/A9 in inflammation.Front. Immunol. 2018; 9: 1298Crossref PubMed Scopus (665) Google Scholar, 5Leclerc E. Fritz G. Vetter S.W. Heizmann C.W. Binding of S100 proteins to RAGE: an update.Biochim. Biophys. Acta. 2009; 1793: 993-1007Crossref PubMed Scopus (393) Google Scholar). They also form human "nutritional immunity" complexes that deprive bacteria of transition metal ions essential for their growth (6Zygiel E.M. Nolan E.M. Transition metal sequestration by the host-defense protein calprotectin.Annu. Rev. Biochem. 2018; 87: 621-643Crossref PubMed Scopus (117) Google Scholar, 7Damo S.M. Kehl-Fie T.E. Sugitani N. Holt M.E. Rathi S. 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Chem. 2007; 282: 6126-6135Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Recent affinity-capture mass spectrometry identifications suggest over 150 and 229 interactors for S100A8 and S100A9 proteins, respectively (12Stark C. Breitkreutz B.-J. Reguly T. Boucher L. Breitkreutz A. Tyers M. BioGRID: a general repository for interaction datasets.Nucleic Acids Res. 2006; 34: D535-D539Crossref PubMed Scopus (2873) Google Scholar). Expression levels of S100A8, S100A9, or both proteins change significantly with the development of many human diseases, including rheumatoid arthritis, psoriasis, ulcerative colitis, and Crohn disease, and various human cancers, including breast, prostate, pancreatic, liver, and skin cancer (1Jukic A. Bakiri L. Wagner E.F. Tilg H. Adolph T.E. Calprotectin: from biomarker to biological function.Gut. 2021; 70: 1978-1988Crossref PubMed Scopus (105) Google Scholar, 13Chatziparasidis G. Kantar A. Calprotectin: an ignored biomarker of neutrophilia in pediatric respiratory diseases.Children (Basel). 2021; 8: 428PubMed Google Scholar, 14Allgöwer C. Kretz A.-L. von Karstedt S. Wittau M. Henne-Bruns D. Lemke J. Friend or foe: S100 proteins in cancer.Cancers (Basel). 2020; 12: 2037Crossref PubMed Scopus (71) Google Scholar). The extraordinary diversity of physiological functions of S100A8 and S100A9 has long been the subject of research. However, it is still puzzling, considering that both are small, single-domain proteins, containing only two EF-hand structural motifs connected with a short linker. According to available data, S100A8 and S100A9 have almost identical 3D structures yet perform diverse roles, depending on the biological context (15Korndörfer I.P. Brueckner F. Skerra A. The crystal structure of the human (S100A8/S100A9)2 heterotetramer, calprotectin, illustrates how conformational changes of interacting α-helices can determine specific association of two EF-hand proteins.J. Mol. Biol. 2007; 370: 887-898Crossref PubMed Scopus (219) Google Scholar). The regulation of their function must thus rely not only on their overall static fold but also on more transient factors modulating structural dynamics, thermal stability, or susceptibility to additional factors. Theoretical studies have suggested that intertwined in the conserved S100 protein folds are sequence fragments predicted to have high intrinsic levels of disorder. A higher propensity for disorder was proposed for the sequence of S100A9 than for S100A8 (16Permyakov S.E. Ismailov R.G. Xue B. Denesyuk A.I. Uversky V.N. Permyakov E.A. Intrinsic disorder in S100 proteins.Mol. Biosyst. 2011; 7: 2164-2180Crossref PubMed Scopus (28) Google Scholar). S100A8 and S100A9 do not exist as monomers. They are isolated from biological samples mostly as noncovalent hetero-oligomers of S100A8 and S100A9 with 1:1 stoichiometry (17Edgeworth J. Gorman M. Bennett R. Freemont P. Hogg N. Identification of p8,14 as a highly abundant heterodimeric calcium binding protein complex of myeloid cells.J. Biol. Chem. 1991; 266: 7706-7713Abstract Full Text PDF PubMed Google Scholar). The S100A8/S100A9 heterodimer (calprotectin) is the preferred assembly if the two recombinant proteins are cofolded in vitro, from denaturing conditions, for example, but it does not form upon the mixing of already folded homodimers, which are the minimal functional units for the individual proteins (18Vogl T. Gharibyan A.L. Morozova-Roche L.A. Pro-inflammatory S100A8 and S100A9 proteins: self-assembly into multifunctional native and amyloid complexes.Int. J. Mol. Sci. 2012; 13: 2893-2917Crossref PubMed Scopus (137) Google Scholar, 19Källberg E. Tahvili S. Ivars F. Leanderson T. Induction of S100A9 homodimer formation in vivo.Biochem. Biophys. Res. Commun. 2018; 500: 564-568Crossref PubMed Scopus (7) Google Scholar, 20Vogl T. Leukert N. Barczyk K. Strupat K. Roth J. Biophysical characterization of S100A8 and S100A9 in the absence and presence of bivalent cations.Biochim. Biophys. Acta. 2006; 1763: 1298-1306Crossref PubMed Scopus (144) Google Scholar). Tissue-specific protein expression profiles and detailed proteomic studies indicate that relative levels of S100A8 and S100A9 in vivo may depart from equimolar, suggesting the significance of other biologically active complexes (21Uhlén M. Fagerberg L. Hallström B.M. Lindskog C. Oksvold P. Mardinoglu A. et al.Tissue-based map of the human proteome.Science. 2015; 347: 1260419Crossref PubMed Scopus (8252) Google Scholar). The proteins can form higher-order oligomers that exhibit specific biological properties depending on environmental conditions (22Tardif M.R. Chapeton-Montes J.A. Posvandzic A. Pagé N. Gilbert C. Tessier P.A. Secretion of S100A8, S100A9, and S100A12 by neutrophils involves reactive oxygen species and potassium efflux.J. Immunol. Res. 2015; 2015: 296149Crossref PubMed Scopus (60) Google Scholar). Oligomerization of S100A8 and S100A9 is related to the binding of divalent metal ions. Each protein binds two Ca2+ ions in noncanonical and canonical loops located in the N- and C-terminal EF-hands (18Vogl T. Gharibyan A.L. Morozova-Roche L.A. Pro-inflammatory S100A8 and S100A9 proteins: self-assembly into multifunctional native and amyloid complexes.Int. J. Mol. Sci. 2012; 13: 2893-2917Crossref PubMed Scopus (137) Google Scholar, 20Vogl T. Leukert N. Barczyk K. Strupat K. Roth J. Biophysical characterization of S100A8 and S100A9 in the absence and presence of bivalent cations.Biochim. Biophys. Acta. 2006; 1763: 1298-1306Crossref PubMed Scopus (144) Google Scholar). Some proteoforms, particularly the heterodimer, also bind transition metal ions, including, Zn2+, Ni2+, Fe2+, and Mn2+, in additional sites (6Zygiel E.M. Nolan E.M. Transition metal sequestration by the host-defense protein calprotectin.Annu. Rev. Biochem. 2018; 87: 621-643Crossref PubMed Scopus (117) Google Scholar, 23Fritz G. Heizmann C. 3D Structures of the Calcium and Zinc Binding S100 Proteins. John Wiley & Sons, Ltd, Hoboken, NJ2006Crossref Google Scholar, 24Gilston B.A. Skaar E.P. Chazin W.J. Binding of transition metals to S100 proteins.Sci. China Life Sci. 2016; 59: 792-801Crossref PubMed Scopus (55) Google Scholar). The oligomers differ in proteolytic stability, ligand/ion specificity, and susceptibility to posttranslational modifications (6Zygiel E.M. Nolan E.M. Transition metal sequestration by the host-defense protein calprotectin.Annu. Rev. Biochem. 2018; 87: 621-643Crossref PubMed Scopus (117) Google Scholar, 25Lim S.Y. Raftery M. Cai H. Hsu K. Yan W.X. Hseih H.-L. et al.S-nitrosylated S100A8: novel anti-inflammatory properties.J. Immunol. 2008; 181: 5627-5636Crossref PubMed Scopus (91) Google Scholar, 26Lim S.Y. Raftery M.J. Goyette J. Geczy C.L. S-glutathionylation regulates inflammatory activities of S100A9.J. Biol. Chem. 2010; 285: 14377-14388Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 27Hoskin T.S. Crowther J.M. Cheung J. Epton M.J. Sly P.D. Elder P.A. et al.Oxidative cross-linking of calprotectin occurs in vivo, altering its structure and susceptibility to proteolysis.Redox Biol. 2019; 24: 101202Crossref PubMed Scopus (26) Google Scholar). For instance, S100A8/S100A9 heterodimers, released from the low-Ca2+ interior of cells, form tetramers in the Ca2+-rich intercellular space. In psoriasis, calprotectin dimers bind to TLR4 receptors to induce inflammation signals. Tetrameric calprotectin loses this capability because the TLR4-binding epitope hides at the interface between the dimers. Thus, the localization of its dimeric form capable of TLR4 binding is focused, allowing for precise control of the spread of the inflammation signal (3Vogl T. Stratis A. Wixler V. Völler T. Thurainayagam S. Jorch S.K. et al.Autoinhibitory regulation of S100A8/S100A9 alarmin activity locally restricts sterile inflammation.J. Clin. Invest. 2018; 128: 1852-1866Crossref PubMed Scopus (125) Google Scholar). The Ca2+-bound tetramer acts instead as an antibacterial agent through a "nutritional immunity" mechanism by depriving bacteria of essential transition metal ions. Strong binding of Zn2+, Mn2+, Ni2+, and Fe2+ is provided by two sites formed at the heterodimer interface. The first is a classical H3D site built of the side chains of H83A8, H87A8, H20A9, and D30A9. The second is a unique H6 site involving the two imidazoles of H17A8 and H27A8 from S100A8 and the four imidazoles of the H91A9, H95A9, H103A9, and H105A9 residues from the S100A9 tail. Transition ion affinity drops significantly in the absence of Ca2+ (6Zygiel E.M. Nolan E.M. Transition metal sequestration by the host-defense protein calprotectin.Annu. Rev. Biochem. 2018; 87: 621-643Crossref PubMed Scopus (117) Google Scholar, 7Damo S.M. Kehl-Fie T.E. Sugitani N. Holt M.E. Rathi S. Murphy W.J. et al.Molecular basis for manganese sequestration by calprotectin and roles in the innate immune response to invading bacterial pathogens.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 3841-3846Crossref PubMed Scopus (285) Google Scholar, 28Nakashige T.G. Zygiel E.M. Drennan C.L. Nolan E.M. Nickel sequestration by the host-defense protein human calprotectin.J. Am. Chem. Soc. 2017; 139: 8828-8836Crossref PubMed Scopus (85) Google Scholar, 29Lin H. Andersen G.R. Yatime L. Crystal structure of human S100A8 in complex with zinc and calcium.BMC Struct. Biol. 2016; 16: 8Crossref PubMed Scopus (21) Google Scholar). In an excess of Zn2+ ions, calprotectin assembles into higher, noncanonical oligomers, leading to harmful amyloid deposits, as observed in Zn2+-rich prostate cancer cells (30Yanamandra K. Alexeyev O. Zamotin V. Srivastava V. Shchukarev A. Brorsson A.-C. et al.Amyloid formation by the pro-inflammatory S100A8/A9 proteins in the ageing prostate.PLoS One. 2009; 4: e5562Crossref PubMed Scopus (86) Google Scholar). The mechanistic rationales underlying effects like heterodimerization and metal ion–dependent tetramerization are still under debate. Available structures represent primarily static data and describe only the limited, successfully crystallized conformers determined with flexible regions excluded, mainly in a metal ion–loaded form (Table S1). Remarkably, there is no structure of calprotectin in the apo form. All published structures have a similar main-chain conformation and do not clearly explain observed functional differences between oligomeric assemblies under varying conditions. Also, most crystallized proteins have their reactive cysteines (C42A8 and C3A9) mutated or removed, which is a disadvantage because S100A8 and S100A9 proteins are particularly susceptible to oxidative modifications in vivo, and the posttranslational redox modifications of methionine and cysteine residues regulate some of their biological activities (25Lim S.Y. Raftery M. Cai H. Hsu K. Yan W.X. Hseih H.-L. et al.S-nitrosylated S100A8: novel anti-inflammatory properties.J. Immunol. 2008; 181: 5627-5636Crossref PubMed Scopus (91) Google Scholar, 26Lim S.Y. Raftery M.J. Goyette J. Geczy C.L. S-glutathionylation regulates inflammatory activities of S100A9.J. Biol. Chem. 2010; 285: 14377-14388Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 27Hoskin T.S. Crowther J.M. Cheung J. Epton M.J. Sly P.D. Elder P.A. et al.Oxidative cross-linking of calprotectin occurs in vivo, altering its structure and susceptibility to proteolysis.Redox Biol. 2019; 24: 101202Crossref PubMed Scopus (26) Google Scholar, 31Stephan J.R. Yu F. Costello R.M. Bleier B.S. Nolan E.M. Oxidative post-translational modifications accelerate proteolytic degradation of calprotectin.J. Am. Chem. Soc. 2018; 140: 17444-17455Crossref PubMed Scopus (18) Google Scholar). A promising direction is to combine biophysical methods with computer simulations, as has been already done to study the conformational dynamics of S100A11 (32Xiao Y. Shaw G.S. Konermann L. Calcium-mediated control of S100 proteins: allosteric communication via an agitator/signal blocking mechanism.J. Am. Chem. Soc. 2017; 139: 11460-11470Crossref PubMed Scopus (15) Google Scholar). In this work, we applied hydrogen–deuterium exchange mass spectrometry (HDx-MS) combined with molecular dynamics (MD) simulations as a complementary technique for structural studies. We aimed to provide the missing conformational comparison of the full-length, nonmutated S100A8 and S100A9 proteins arranged in different oligomeric structures. HDx-MS has already been helpful, in our hands, in studying conformational consequences of posttranslational modifications in proteins that escape classical structure elucidation (33Bajor M. Zaręba-Kozioł M. Zhukova L. Goryca K. Poznański J. Wysłouch-Cieszyńska A. An interplay of S-nitrosylation and metal ion binding for astrocytic S100B protein.PLoS One. 2016; 11e0154822Crossref Scopus (13) Google Scholar). A significant advantage of the method is that proteins can be analyzed without labeling and under widely varying conditions. Most importantly, HDx-MS results reflect the structural flexibility and dynamics of the investigated systems (34Narang D. Lento C. J Wilson D. HDX-MS: an analytical tool to capture protein motion in action.Biomedicines. 2020; 8: 224Crossref PubMed Google Scholar, 35Giladi M. Khananshvili D. Hydrogen-deuterium exchange mass-spectrometry of secondary active transporters: from structural dynamics to molecular mechanisms.Front. Pharmacol. 2020; 11: 70Crossref PubMed Scopus (18) Google Scholar, 36Masson G.R. Burke J.E. Ahn N.G. Anand G.S. Borchers C. Brier S. et al.Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments.Nat. Methods. 2019; 16: 595-602Crossref PubMed Scopus (322) Google Scholar). By superimposing HDx profiles with molecular dynamic simulations, we tried to infer how the conformational characteristics of S100A8 and S100A9 subunits evolve when switching from homodimers to heterodimers, and from apo forms to Ca2+- or Zn2+-loaded oligomers. Eventually, we demonstrate that their properties are modulated at the level of conformational plasticity, tuned at particular protein regions in different proteoforms. Wildtype human S100A8 and S100A9 proteins were individually overexpressed in E. coli and purified to homogeneity under denaturing conditions using HPLC (Fig. S1A). S100A8 and S100A9 complexes were obtained by refolding denatured proteins back to their native structures by dialysis from pH 2.5 to pH 7.5, as described (20Vogl T. Leukert N. Barczyk K. Strupat K. Roth J. Biophysical characterization of S100A8 and S100A9 in the absence and presence of bivalent cations.Biochim. Biophys. Acta. 2006; 1763: 1298-1306Crossref PubMed Scopus (144) Google Scholar). Refolded proteins assembled into either a homodimer when using pure S100A8 or S100A9 or heterodimers for an equimolar mixture of purified S100A8 and S100A9. Anion exchange chromatography and gel filtration showed that the dimers were noncovalent, and HPLC, SDS-PAGE, and liquid chromatography (LC)–electrospray ionization (ESI)–MS of whole proteins confirmed the composition of appropriate proteoforms (Fig. S1, A–C). Circular dichroism (CD) spectroscopy demonstrated the formation of the expected α-helical structures observed in these proteins previously (20Vogl T. Leukert N. Barczyk K. Strupat K. Roth J. Biophysical characterization of S100A8 and S100A9 in the absence and presence of bivalent cations.Biochim. Biophys. Acta. 2006; 1763: 1298-1306Crossref PubMed Scopus (144) Google Scholar, 37NematiNiko F. Chegini K.G. Asghari H. Amini A. Gheibi N. Modifying effects of carboxyl group on the interaction of recombinant S100A8/A9 complex with tyrosinase.Biochim. Biophys. Acta. 2017; 1865: 370-379Crossref Scopus (4) Google Scholar, 38Imani M. Bahrami Y. Jaliani H.Z. Ardestani S.K. In solution cation-induced secondary and tertiary structure alterations of human calprotectin.Protein J. 2014; 33: 465-473Crossref PubMed Scopus (5) Google Scholar) (Fig. S1, D and E). Size-exclusion chromatography (SEC) (Fig. 1, A–C) and analytical ultracentrifugation (Fig. 1D) indicated that pure S100A8 and S100A9 proteins formed only homodimers regardless of the presence of Ca2+/Zn2+ ions. Heterodimers assembled into tetramers upon the addition of Ca2+ or into even higher oligomers with Zn2+. According to native MS results, the predominant proteoform for S100A8/S100A9 in the presence of excess Ca2+/Zn2+ ions remained the tetramer (Fig. 1, E and F). Since human S100A8 and S100A9 proteins possess reduced cysteine residues, we paid special attention throughout the purification process to prevent the formation of a covalent disulfide or other oxidation product. Before each experiment, we confirmed the reduced state of cysteine thiols by HPLC and MS analysis. The redox stability of cysteines differed markedly among the various purified proteoforms. Calprotectin and (S100A8)2 did not form covalent disulfide bonds in solution even after several days of storage at 4 °C without a thiol-reducing agent, while (S100A9)2 had to be used immediately after purification, as it formed covalent dimers more efficiently. Proper folding of all analyzed proteoforms was also assessed by thermal stability measurements using nanodifferential fluorescence based on tryptophan fluorescence analysis. S100A8 and S100A9 proteins have one tryptophan residue each. In homodimers at room temperature, W88A9 is more exposed to the solvent (fluorescence at 350 nm/330 nm [F] = 0.94) than is W54A8 (F = 0.74). The F value for Trp fluorescence in the heterodimer is an arithmetic mean of the values obtained for the homodimers (F = 0.84). Upon Ca2+-induced tetramerization, the Trp fluorescence drops (F = 0.71), indicating an additional burial of Trp side chains. With increasing temperature, the tryptophans in (S100A8)2 become buried (red shifted in fluorescence), while in (S100A9)2 and calprotectin they become exposed (blue shifted). The observed unfolding transitions were sudden and occurred at quite different temperatures. Calprotectin showed much higher stability (Tm[A8A9apo] = 70.2 °C, Tm[A8A9Ca2+] ≈ 75 °C) compared with homodimers (Tm[A8apo] = Tm[A8Ca2+] ≈ 60.5 °C, Tm[A9apo] = Tm[A9Ca2+] ≈ 61.7 °C) (Fig. S1F). We performed HDx-MS experiments to gain insight into the conformational dynamics of the homogenous S100A8 and S100A9 oligomers. Using tandem mass spectrometry, we observed that digestion of each studied proteoform by pepsin at pH 2.5 yielded, among others, 39 peptides from the S100A8 subunit and 49 from S100A9, altogether covering 93% of the proteins (Fig. 2 and Table S2). Only two fragments from the N termini of helices IV (A869–74 and A979–83) could not be detected. Despite minor differences between our protocols (see Experimental procedures) and those previously reported in the literature for a Cys/Ser mutant of calprotectin (39Adhikari J. Stephan J.R. Rempel D.L. Nolan E.M. Gross M.L. Calcium binding to the innate immune protein human calprotectin revealed by integrated mass spectrometry.J. Am. Chem. Soc. 2020; 142: 13372-13383Crossref PubMed Scopus (10) Google Scholar), the digestion results remained consistent, with only a slightly decreased number of obtained peptides (39 versus 40 for S100A8, and 49 versus 55 for S100A9, Table S2). We measured the deuterium uptake levels for the same sets of peptides in each proteoform at five exchange time points: immediately after D2O addition and at 10 s, 1 min, 5 min, and 24 h of HDx. HDx-MS for apo homodimers demonstrated substantial differences in deuteration levels between (S100A8)2 and (S100A9)2. The HDx profile for apo (S100A8)2 consists of alternating regions characterized by lower and higher deuterium uptake that correlate well with the localization of α-helices (Figs. 3A and S2). Less protected fragments correspond to the loose parts, such as the protein's termini, Ca2+-binding loops, and linker between the two EF-hand motifs. While the exchange levels eventually saturated after 5 min for most of the protein sequence, the peptides from helix I (i.e., A85–15) remained barely accessible to the solvent. For (S100A9)2, HDx levels were much less differentiated (Figs. 3B and S3), and the regions within α-helices not as pronounced. The only peptide with low (<25%) deuterium uptake after 10 s derives from helix I (A914–19) and corresponds to the most protected fragment in (S100A8)2. Yet, in contrast to (S100A8)2, after 1 and 5 min of exchange, the exchange rates for this peptide reached 60% and 90%, respectively. Also, helices II and IV were more protected in (S100A8)2 than in (S100A9)2 (∼30% versus ∼55%, and 45% versus 80% at the 10-s time point, respectively). Conversely, the Ca2+-binding loops are more dynamic in (S100A8)2 (80%–100% after 10 s) as compared with the loops in (S100A9)2 (∼70%). In both homodimers, the binding of calcium stabilizes the Ca2+-binding loops only slightly, with the most pronounced effect on the canonical loop in (S100A9)2. There are, however, also substantial differences. (S100A8)2 becomes less dynamic mainly within helix II, and this is clearly pronounced for the peptide A831–39 at all measured times (Figs. 4, A and C and S2). In (S100A9)2, on the other hand, the HDx profile deviated substantially, becoming differentiated between the more protected helices I, II, and III and the less protected linker and helix IV (Figs. 4B and S3). After 5 min, both profiles converged back to resemble their apo counterparts. Only helices I and II remained more protected, especially in (S100A8)2 (Fig 4, C and D), and the C terminus of (S100A9)2 remained less protected. Heterodimerization had an even more profound effect on HDx dynamics in each protein compared with either apo or Ca2+-loaded homodimers. In the S100A8 subunit, helices I and II and the linker became further protected, increasing the contrast with the accessible Ca2+-binding loops and helix III. Despite the overall protein stabilization, helix IV experienced increased exchange rates relative to the homodimer (Figs. S2 and S3). S100A9 displayed an overall significant decrease in HDx levels, especially for helices I, II, and III within the linker; helix IV; and the Ca2+-binding loops. In contrast to the homodimers, the HDx profiles of the subunits in heterodimers retained definition even after 5 min of exchange, and their HDx profiles became more alike. Notably, heterodimerization in the absence of metal ions decreased deuterium uptake by both Ca2+-binding loops in S100A9. The loop protection was higher than after Ca2+ addition to the homodimers (Fig. 4B), especially after 5 min of exchange (Fig. 4D). Loading calprotectin with Ca2+ ions promoted further decline in HDx ratios in both subunits. Now the last core structural element, helix III, became protected (Fig. 5, A and B). This effect was already pronounced for S100A9 in the apo heterodimer, but here it became apparent for b
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