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• W3026695005 abstract "•p53 TAD forms an asymmetric fuzzy complex with S100A4•The crystal structure of the complex was solved by utilizing ANXA2•ANXA2 was found to have better crystal forming ability than wtMBP•ANXA2 chaperon system can be widely used, but works best with smaller targets To fully understand the environmental factors that influence crystallization is an enormous task, therefore crystallographers are still forced to work “blindly” trying as many crystallizing conditions and mutations to improve crystal packing as possible. Numerous times these random attempts simply fail even when using state-of-the-art techniques. As an alternative, crystallization chaperones, having good crystal-forming properties, can be invoked. Today, the almost exclusively used such protein is the maltose-binding protein (MBP) and crystallographers need other widely applicable options. Here, we introduce annexin A2 (ANXA2), which has just as good, if not better, crystal-forming ability than the wild-type MBP. Using ANXA2 as heterologous fusion partner, we were able to solve the atomic resolution structure of a challenging crystallization target, the transactivation domain (TAD) of p53 in complex with the metastasis-associated protein S100A4. p53 TAD forms an asymmetric fuzzy complex with the symmetric S1004 and could interfere with its function. To fully understand the environmental factors that influence crystallization is an enormous task, therefore crystallographers are still forced to work “blindly” trying as many crystallizing conditions and mutations to improve crystal packing as possible. Numerous times these random attempts simply fail even when using state-of-the-art techniques. As an alternative, crystallization chaperones, having good crystal-forming properties, can be invoked. Today, the almost exclusively used such protein is the maltose-binding protein (MBP) and crystallographers need other widely applicable options. Here, we introduce annexin A2 (ANXA2), which has just as good, if not better, crystal-forming ability than the wild-type MBP. Using ANXA2 as heterologous fusion partner, we were able to solve the atomic resolution structure of a challenging crystallization target, the transactivation domain (TAD) of p53 in complex with the metastasis-associated protein S100A4. p53 TAD forms an asymmetric fuzzy complex with the symmetric S1004 and could interfere with its function. Structural biology methods provide powerful tools to understand protein functions and the underlying mechanisms. Protein crystallography is still the most important technique in this field and, as structure solving methods have become more and more advanced, the production of high-quality diffracting crystals remains the largest challenge for crystallographers. The process of crystallization is affected by numerous factors, including the chemical characteristics of the protein, environmental factors, and conformational heterogeneity (Tereshko et al., 2008Tereshko V. Uysal S. Koide A. Margalef K. Koide S. Kossiakoff A.A. Toward chaperone-assisted crystallography: protein engineering enhancement of crystal packing and X-ray phasing capabilities of a camelid single-domain antibody (VHH) scaffold.Protein Sci. 2008; 17: 1175-1187Crossref PubMed Scopus (50) Google Scholar). For crystallization, usually a high amount of purified protein is also required. A popular method to facilitate protein expression and purification is using affinity tags, such as His6-tag (Bornhorst and Falke, 2000Bornhorst J.A. Falke J.J. Purification of proteins using polyhistidine affinity tags.Methods Enzymol. 2000; 326: 245-254Crossref PubMed Google Scholar), maltose-binding protein (MBP) (Braun et al., 2002Braun P. Hu Y. Shen B. Halleck A. Koundinya M. Harlow E. Labaer J. Proteome-scale purification of human proteins from bacteria.Proc. Natl. Acad. Sci. U S A. 2002; 99: 2654-2659Crossref PubMed Scopus (215) Google Scholar), glutathione S-transferase (GST) (Smith, 2000Smith D.B. Generating fusions to glutathione S-transferase for protein studies.Methods Enzymol. 2000; 326: 254-270Crossref PubMed Google Scholar), thioredoxin (TRX) (LaVallie et al., 2000LaVallie E.R. Lu Z. Diblasio-Smith E.A. Collins-Racie L.A. McCoy J.M. Thioredoxin as a fusion partner for production of soluble recombinant proteins in Escherichia coli.Methods Enzymol. 2000; 326: 322-340Crossref PubMed Google Scholar), or several other short peptides or stable proteins fused to the target proteins (Stevens, 2000Stevens R.C. Design of high-throughput methods of protein production for structural biology.Structure. 2000; 8: R177-R185Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Before crystallization, these affinity tags are usually removed using site-specific proteases, which may lead to the precipitation and/or activity loss of the target molecule (Baneyx, 1999Baneyx F. Recombinant protein expression in Escherichia coli.Curr. Opin. Biotechnol. 1999; 10: 411-421Crossref PubMed Scopus (1010) Google Scholar). A possible way to avoid these problems and increase the solubility of the target protein is to fuse it to the affinity tag via a short, rigid spacer (lacking any protease cleavage site). This way the conformational heterogeneity of the fusion construct is reduced, which might promote crystallizability and the crystallization ability of the chimeric tag can be exploited (Smyth et al., 2003Smyth D.R. Mrozkiewicz M.K. McGrath W.J. Listwan P. Kobe B. Crystal structures of fusion proteins with large-affinity tags.Protein Sci. 2003; 12: 1313-1322Crossref PubMed Scopus (192) Google Scholar). In addition, the known structure of the chimeric tag might be used during data processing to facilitate molecular replacement and structural refinement. For this purpose, MBP, GST, and TRX have been used successfully (Waugh, 2016Waugh D.S. Crystal structures of MBP fusion proteins.Protein Sci. 2016; 25: 559-571Crossref PubMed Scopus (34) Google Scholar, Jin et al., 2017Jin T. Chuenchor W. Jiang J. Cheng J. Li Y. Fang K. Huang M. Smith P. Xiao T.S. Design of an expression system to enhance MBP-mediated crystallization.Sci. Rep. 2017; 7: 40991Crossref PubMed Scopus (28) Google Scholar), together with other well-crystallizing proteins, such as lysozyme (Thorsen et al., 2014Thorsen T.S. Matt R. Weis W.I. Kobilka B.K. Modified T4 lysozyme fusion proteins facilitate G protein-coupled receptor crystallogenesis.Structure. 2014; 22: 1657-1664Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, Rosenbaum et al., 2007Rosenbaum D.M. Cherezov V. Hanson M.A. Rasmussen S.G. Thian F.S. Kobilka T.S. Choi H.J. Yao X.J. Weis W.I. Stevens R.C. Kobilka B.K. GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function.Science. 2007; 318: 1266-1273Crossref PubMed Scopus (1122) Google Scholar, Kobilka, 2013Kobilka B. The structural basis of G-protein-coupled receptor signaling (Nobel Lecture).Angew. Chem. Int. Ed. 2013; 52: 6380-6388Crossref PubMed Scopus (123) Google Scholar) or antibody fragments (Tamura et al., 2019Tamura R. Oi R. Akashi S. Kaneko M.K. Kato Y. Nogi T. Application of the NZ-1 Fab as a crystallization chaperone for PA tag-inserted target proteins.Protein Sci. 2019; 28: 823-836Crossref PubMed Scopus (5) Google Scholar, Lieberman et al., 2011Lieberman R.L. Culver J.A. Entzminger K.C. Pai J.C. Maynard J.A. Crystallization chaperone strategies for membrane proteins.Methods. 2011; 55: 293-302Crossref PubMed Scopus (26) Google Scholar). However, so far only MBP appears to be a generally effective crystallization chaperone and at the same time a useful affinity tag (Waugh, 2016Waugh D.S. Crystal structures of MBP fusion proteins.Protein Sci. 2016; 25: 559-571Crossref PubMed Scopus (34) Google Scholar, Clifton et al., 2015Clifton M.C. Dranow D.M. Leed A. Fulroth B. Fairman J.W. Abendroth J. Atkins K.A. Wallace E. Fan D. Xu G. et al.A maltose-binding protein fusion construct yields a robust crystallography platform for MCL1.PLoS One. 2015; 10: e0125010Crossref PubMed Scopus (21) Google Scholar), and little effort was made to find alternatives that might be equally or even more adequate for this dual role. The number of structures in the PDB using crystallization chaperones is increasing, indicating that this approach can be a highly efficient tool for crystallographers solving the structures of hardly crystallizable proteins or protein complexes. A good example for such a difficult target, is the complex between the p53 transactivation domain (TAD) and S100A4. S100A4 is a small, dimeric EF-hand Ca2+-binding protein known by its pathological role in several metastatic tumors and inflammatory diseases (Boye and Maelandsmo, 2010Boye K. Maelandsmo G.M. S100A4 and metastasis: a small actor playing many roles.Am. J. Pathol. 2010; 176: 528-535Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). Its interaction with p53 TAD (1–64) has previously been studied by several groups (Orre et al., 2013Orre L.M. Panizza E. Kaminskyy V.O. Vernet E. Graslund T. Zhivotovsky B. Lehtio J. S100A4 interacts with p53 in the nucleus and promotes p53 degradation.Oncogene. 2013; 32: 5531-5540Crossref PubMed Scopus (61) Google Scholar, Shen et al., 2015Shen W. Chen D. Liu S. Chen L. Yu A. Fu H. Sun X. S100A4 interacts with mutant p53 and affects gastric cancer MKN1 cell autophagy and differentiation.Int. J. Oncol. 2015; 47: 2123-2130Crossref PubMed Scopus (14) Google Scholar, Grigorian et al., 2001Grigorian M. Andresen S. Tulchinsky E. Kriajevska M. Carlberg C. Kruse C. Cohn M. Ambartsumian N. Christensen A. Selivanova G. Lukanidin E. Tumor suppressor p53 protein is a new target for the metastasis-associated Mts1/S100A4 protein: functional consequences of their interaction.J. Biol. Chem. 2001; 276: 22699-22708Crossref PubMed Scopus (258) Google Scholar, Fernandez-Fernandez et al., 2008Fernandez-Fernandez M.R. Rutherford T.J. Fersht A.R. Members of the S100 family bind p53 in two distinct ways.Protein Sci. 2008; 17: 1663-1670Crossref PubMed Scopus (62) Google Scholar, van Dieck et al., 2010van Dieck J. Brandt T. Teufel D.P. Veprintsev D.B. Joerger A.C. Fersht A.R. Molecular basis of S100 proteins interacting with the p53 homologs p63 and p73.Oncogene. 2010; 29: 2024-2035Crossref PubMed Scopus (42) Google Scholar); however, the structure of the complex remains unknown. Note that several other S100 proteins (S100A1, S100A2, S100A4, S100A6, S100A11, and S100B) also bind to p53 TAD (van Dieck et al., 2009bvan Dieck J. Teufel D.P. Jaulent A.M. Fernandez-Fernandez M.R. Rutherford T.J. Wyslouch-Cieszynska A. Fersht A.R. Posttranslational modifications affect the interaction of S100 proteins with tumor suppressor p53.J. Mol. Biol. 2009; 394: 922-930Crossref PubMed Scopus (46) Google Scholar) or to the C-terminal end of p53 containing the tetramerization (TET) (326–356) and the negative regulatory domains (NRD) (364–393) (Fernandez-Fernandez et al., 2005Fernandez-Fernandez M.R. Veprintsev D.B. Fersht A.R. Proteins of the S100 family regulate the oligomerization of p53 tumor suppressor.Proc. Natl. Acad. Sci. U S A. 2005; 102: 4735-4740Crossref PubMed Scopus (147) Google Scholar, Fernandez-Fernandez et al., 2008Fernandez-Fernandez M.R. Rutherford T.J. Fersht A.R. Members of the S100 family bind p53 in two distinct ways.Protein Sci. 2008; 17: 1663-1670Crossref PubMed Scopus (62) Google Scholar). S100A4 is a highly soluble protein, even the name S100 refers to the fact that these proteins remain in solution when saturated ammonium sulfate is used (Moore, 1965Moore B.W. A soluble protein characteristic of the nervous system.Biochem. Biophys. Res. Commun. 1965; 19: 739-744Crossref PubMed Scopus (1302) Google Scholar). This ability makes S100 proteins immensely difficult to crystallize in general, but in the case of p53 the process is even more formidable. In the PDB only NMR structures were published so far containing the whole p53 TAD, which contains both TAD1 and TAD2 subdomains (Harms and Chen, 2006Harms K.L. Chen X. The functional domains in p53 family proteins exhibit both common and distinct properties.Cell Death Differ. 2006; 13: 890-897Crossref PubMed Scopus (59) Google Scholar), in complex with a target protein (Yoon et al., 2018Yoon M.K. Kim B.Y. Lee J.Y. Ha J.H. Kim S.A. Lee D.H. Lee M.S. Lee M.K. Choi J.S. Cho J.H. et al.Cytoplasmic pro-apoptotic function of the tumor suppressor p73 is mediated through a modified mode of recognition of the anti-apoptotic regulator Bcl-XL.J. Biol. Chem. 2018; 293: 19546-19558Crossref PubMed Scopus (11) Google Scholar, Rowell et al., 2012Rowell J.P. Simpson K.L. Stott K. Watson M. Thomas J.O. HMGB1-facilitated p53 DNA binding occurs via HMG-Box/p53 transactivation domain interaction, regulated by the acidic tail.Structure. 2012; 20: 2014-2024Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, Feng et al., 2009Feng H. Jenkins L.M. Durell S.R. Hayashi R. Mazur S.J. Cherry S. Tropea J.E. Miller M. Wlodawer A. Appella E. Bai Y. Structural basis for p300 Taz2-p53 TAD1 binding and modulation by phosphorylation.Structure. 2009; 17: 202-210Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, Okuda and Nishimura, 2014Okuda M. Nishimura Y. Extended string binding mode of the phosphorylated transactivation domain of tumor suppressor p53.J. Am. Chem. Soc. 2014; 136: 14143-14152Crossref PubMed Scopus (32) Google Scholar, Krauskopf et al., 2018Krauskopf K. Gebel J. Kazemi S. Tuppi M. Lohr F. Schafer B. Koch J. Guntert P. Dotsch V. Kehrloesser S. Regulation of the activity in the p53 family depends on the organization of the transactivation domain.Structure. 2018; 26: 1091-1100.e4Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar, Krois et al., 2016Krois A.S. Ferreon J.C. Martinez-Yamout M.A. Dyson H.J. Wright P.E. Recognition of the disordered p53 transactivation domain by the transcriptional adapter zinc finger domains of CREB-binding protein.Proc. Natl. Acad. Sci. U S A. 2016; 113: E1853-E1862Crossref PubMed Scopus (41) Google Scholar, Lee et al., 2010Lee C.W. Martinez-Yamout M.A. Dyson H.J. Wright P.E. Structure of the p53 transactivation domain in complex with the nuclear receptor coactivator binding domain of CREB binding protein.Biochemistry. 2010; 49: 9964-9971Crossref PubMed Scopus (118) Google Scholar). The absence of X-ray structures, including the long TAD, despite the large number of studies, shows that the crystallization of this domain in any protein complex is an inherently difficult task. Remarkably, here using annexin A2 (ANXA2) as a crystallization chaperon we were able to solve the atomic resolution structure of this p53 TAD-S100A4 complex. ANXA2 is a non-EF-hand Ca2+-binding protein (Gerke and Moss, 2002Gerke V. Moss S.E. Annexins: from structure to function.Physiol. Rev. 2002; 82: 331-371Crossref PubMed Scopus (1537) Google Scholar). Its ability to aggregate (“annex”) phospholipid membranes in a Ca2+-dependent manner underlies its biological functions, such as vesicular transport, and exo- and endocytosis (Gerke and Moss, 2002Gerke V. Moss S.E. Annexins: from structure to function.Physiol. Rev. 2002; 82: 331-371Crossref PubMed Scopus (1537) Google Scholar, Drust and Creutz, 1988Drust D.S. Creutz C.E. Aggregation of chromaffin granules by calpactin at micromolar levels of calcium.Nature. 1988; 331: 88-91Crossref PubMed Scopus (330) Google Scholar). ANXA2 consists of a disordered N-terminal (NTD) (2–33) and a highly conserved, rigid C-terminal “core” domains (CTD) (34–339). The convex side of the CTD is responsible for Ca2+-dependent membrane binding while the concave side directs membrane aggregation and anchors the NTD via a highly conserved G-[TS]-[VI] motif localized in the C terminus of the NTD (C-NTD) (23–33) (Ecsédi et al., 2017Ecsédi P. Kiss B. Gogl G. Radnai L. Buday L. Koprivanacz K. Liliom K. Leveles I. Vertessy B. Jeszenoi N. et al.Regulation of the equilibrium between closed and open conformations of annexin A2 by N-terminal phosphorylation and S100A4-binding.Structure. 2017; 25: 1195-1207.e5Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). ANXA2 is a highly soluble and stable protein and its ability to easily form crystals has recently been observed in our laboratory (Ecsédi et al., 2017Ecsédi P. Kiss B. Gogl G. Radnai L. Buday L. Koprivanacz K. Liliom K. Leveles I. Vertessy B. Jeszenoi N. et al.Regulation of the equilibrium between closed and open conformations of annexin A2 by N-terminal phosphorylation and S100A4-binding.Structure. 2017; 25: 1195-1207.e5Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Moreover, beside the case of the p53 TAD-S100A4 complex, studied here, we have already used ANXA2 previously to determine the structure of a PDZ domain with a bound peptide ligand (Gogl et al., 2018Gogl G. Biri-Kovacs B. Poti A.L. Vadaszi H. Szeder B. Bodor A. Schlosser G. Acs A. Turiak L. Buday L. et al.Dynamic control of RSK complexes by phosphoswitch-based regulation.FEBS J. 2018; 285: 46-71Crossref PubMed Scopus (10) Google Scholar). Results presented in this paper suggest that ANXA2 could be another promising crystallization helper molecule similarly to MBP and it is likely applicable to determine the structure of other difficult protein complexes. A previous NMR study suggested that only parts of TAD1 (residues 17–40) and TAD2 (residues 41–57) are involved in complex formation with S100A4 (van Dieck et al., 2009bvan Dieck J. Teufel D.P. Jaulent A.M. Fernandez-Fernandez M.R. Rutherford T.J. Wyslouch-Cieszynska A. Fersht A.R. Posttranslational modifications affect the interaction of S100 proteins with tumor suppressor p53.J. Mol. Biol. 2009; 394: 922-930Crossref PubMed Scopus (46) Google Scholar). However, several prolines, the contribution of which to the interaction could not be predicted by NMR, are located in N- and C-terminal directions (P4, P8, P12, P13, P58, and P60), thus the first 60 residues of p53 was arbitrarily chosen to express as a recombinant peptide (TAD1−60) and used in the binding experiments. It was found, using isothermal titration calorimetry measurements, that the interaction between p53 TAD1−60 and S100A4 is asymmetric (a single peptide binds to one S100A4 dimer) and its dissociation constant is in the micromolar range (Figure 1A). According to our circular dichroism (CD) measurements, analyzed with the BesStSel secondary structure prediction program (Micsonai et al., 2015Micsonai A. Wien F. Kernya L. Lee Y.H. Goto Y. Refregiers M. Kardos J. Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy.Proc. Natl. Acad. Sci. U S A. 2015; 112: E3095-E3103Crossref PubMed Scopus (693) Google Scholar), the structure of the originally disordered TAD1−60 peptide changes to a more helical structure upon complex formation with S100A4 (α helix content increases from ∼4.6% to ∼29.4%) (Figure 1B). Note here that by correcting our data with the CD spectrum of the blank buffer ruled out its contribution during the α helix content estimations of peptides. A similar effect was observed with other S100 ligands (Fernandez-Fernandez et al., 2008Fernandez-Fernandez M.R. Rutherford T.J. Fersht A.R. Members of the S100 family bind p53 in two distinct ways.Protein Sci. 2008; 17: 1663-1670Crossref PubMed Scopus (62) Google Scholar, Kiss et al., 2012Kiss B. Duelli A. Radnai L. Kekesi K.A. Katona G. Nyitray L. Crystal structure of the S100A4-nonmuscle myosin IIA tail fragment complex reveals an asymmetric target binding mechanism.Proc. Natl. Acad. Sci. U S A. 2012; 109: 6048-6053Crossref PubMed Scopus (45) Google Scholar). Previously, Lee et al., 2000Lee H. Mok K.H. Muhandiram R. Park K.H. Suk J.E. Kim D.H. Chang J. Sung Y.C. Choi K.Y. Han K.H. Local structural elements in the mostly unstructured transcriptional activation domain of human p53.J. Biol. Chem. 2000; 275: 29426-29432Crossref PubMed Scopus (268) Google Scholar have found several regions in the apo p53 TAD peptide with relatively high secondary structure propensity (residues 18–26, 40–44, and 48–53) using NMR spectroscopy (Figure 1C). The number of residues in these transiently formed nascent structural elements are in good agreement with the CD data presented here. Disordered binding regions (IDRs) of proteins usually fold upon binding (Dunker et al., 2001Dunker A.K. Lawson J.D. Brown C.J. Williams R.M. Romero P. Oh J.S. Oldfield C.J. Campen A.M. Ratliff C.M. Hipps K.W. et al.Intrinsically disordered protein.J. Mol. Graph Model. 2001; 19: 26-59Crossref PubMed Scopus (1688) Google Scholar), therefore one can assume that those short-term observed folds of TAD could be key elements in complex formation (Kim and Han, 2018Kim D.H. Han K.H. Transient secondary structures as general target-binding motifs in intrinsically disordered proteins.Int. J. Mol. Sci. 2018; 19https://doi.org/10.3390/ijms19113614Crossref Scopus (17) Google Scholar). Nevertheless, the low overall helix propensity of the peptide even in the bound form predicts that TAD probably retains partial flexibility and forms a dynamic, partially folded so-called fuzzy complex (Tompa and Fuxreiter, 2008Tompa P. Fuxreiter M. Fuzzy complexes: polymorphism and structural disorder in protein-protein interactions.Trends Biochem. Sci. 2008; 33: 2-8Abstract Full Text Full Text PDF PubMed Scopus (707) Google Scholar) with S100A4. These results together with other findings (van Dieck et al., 2009bvan Dieck J. Teufel D.P. Jaulent A.M. Fernandez-Fernandez M.R. Rutherford T.J. Wyslouch-Cieszynska A. Fersht A.R. Posttranslational modifications affect the interaction of S100 proteins with tumor suppressor p53.J. Mol. Biol. 2009; 394: 922-930Crossref PubMed Scopus (46) Google Scholar, van Dieck et al., 2010van Dieck J. Brandt T. Teufel D.P. Veprintsev D.B. Joerger A.C. Fersht A.R. Molecular basis of S100 proteins interacting with the p53 homologs p63 and p73.Oncogene. 2010; 29: 2024-2035Crossref PubMed Scopus (42) Google Scholar) now ensures the previous prediction that the first 16 and the last 8 residues do not participate in the interaction. Therefore, a truncated TAD peptide (residues 17–56, TAD17−56) was produced and its binding to S100A4 was evaluated using a competitive fluorescence polarization assay. It revealed that the truncated TAD17−56 has the same affinity to S100A4 as the longer TAD1−60 peptide (Kd values ∼0.7 and ∼0.9 μM, respectively) (Figure 1D). Thus the subsequent experiments were conducted using this “minimal” binding sequence of p53 to ease crystallization. We have made several attempts to crystallize the p53 TAD17−56-S100A4 complex alone or by using GST or wild-type MBP (wtMBP) tags as crystallization chaperones (connected by an “SGSGG” short linker). If either p53 TAD17−56 or S100A4 was fused to GST or MBP, respectively, then mixed with the other partner, no crystal was observed in any case. The poor crystal-forming ability of the complex presumably comes from the high solubility of S100A4 and the dynamic, fuzzy binding mode of p53. Following these unavailing attempts, two fusion constructs of ANXA2 were designed, based on its 3D structures (Ecsédi et al., 2017Ecsédi P. Kiss B. Gogl G. Radnai L. Buday L. Koprivanacz K. Liliom K. Leveles I. Vertessy B. Jeszenoi N. et al.Regulation of the equilibrium between closed and open conformations of annexin A2 by N-terminal phosphorylation and S100A4-binding.Structure. 2017; 25: 1195-1207.e5Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, Gogl et al., 2018Gogl G. Biri-Kovacs B. Poti A.L. Vadaszi H. Szeder B. Bodor A. Schlosser G. Acs A. Turiak L. Buday L. et al.Dynamic control of RSK complexes by phosphoswitch-based regulation.FEBS J. 2018; 285: 46-71Crossref PubMed Scopus (10) Google Scholar), and cloned into a modified pET15 vector together with an N-terminal short multi-cloning site (for inserting the target proteins) followed by a cleavable His6-tag with the aim of using it as a crystallization chaperone similarly to MBP/GST. One construct (ANXA223−339) contains the so-called C-NTD of ANXA2 that transiently binds to the core domain (Figure 2A). Its presence considerably stabilizes ANXA2 (Ecsédi et al., 2017Ecsédi P. Kiss B. Gogl G. Radnai L. Buday L. Koprivanacz K. Liliom K. Leveles I. Vertessy B. Jeszenoi N. et al.Regulation of the equilibrium between closed and open conformations of annexin A2 by N-terminal phosphorylation and S100A4-binding.Structure. 2017; 25: 1195-1207.e5Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), thus facilitating crystal formation; however, its structural plasticity may result in a less compact fold with a long disordered linker between the target protein and ANXA2. The other construct (ANXA229−339) does not contain the C-NTD (Figure 2B), which precludes the formation of such a long spacer but results in a less stable core domain (DNA sequences and ANXA2 containing plasmids were uploaded and sent to Addgene with IDs of 136543, 136544, 136545, and 136546). In a previously published paper, where we have solved the crystal structure of the second PDZ domain of the membrane-associated guanylate kinase (MAGI-1), the former ANXA2 construct was used (Gogl et al., 2018Gogl G. Biri-Kovacs B. Poti A.L. Vadaszi H. Szeder B. Bodor A. Schlosser G. Acs A. Turiak L. Buday L. et al.Dynamic control of RSK complexes by phosphoswitch-based regulation.FEBS J. 2018; 285: 46-71Crossref PubMed Scopus (10) Google Scholar). In the case of p53 TAD17−56 we had to apply the latter strategy since S100A4 interacts with the C-NTD of ANXA2 (Ecsédi et al., 2017Ecsédi P. Kiss B. Gogl G. Radnai L. Buday L. Koprivanacz K. Liliom K. Leveles I. Vertessy B. Jeszenoi N. et al.Regulation of the equilibrium between closed and open conformations of annexin A2 by N-terminal phosphorylation and S100A4-binding.Structure. 2017; 25: 1195-1207.e5Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). To increase the chance of crystal formation, we have produced a chimera where a single-chain S100A4Δ8 dimer (scS100A4Δ8) was fused to ANXA229−339 using the previously designed vectors. In case of scS100A4Δ8 the last eight residues forming a short disordered region, thus presumably negatively affecting crystal formation, were deleted and the two subunits were covalently joined into a single-chain construct (a short linker with the sequence of “SAGSAG” was used between the subunits). Using this chimera, ANXA2 could drive crystal packing instead of the highly soluble S100A4. In our first attempts, where this scS100A4Δ8-ANXA229−339 construct was complexed with p53 TAD17−56, the p53 peptide ligand dissociated from the crystals and only the scS100A4Δ8-ANXA229−339 chimera was found in apo form (not deposited in the PDB). We believe that the presence of the fuzzy interaction is unfavorable for crystal formation, thus crystals lacking p53, caused by the temporary dissociation of the peptide, could form more easily causing the elimination of the peptide from its binding site in those structures. To solve this problem we covalently bound p53 TAD17−56 to the N terminus of scS100A4Δ8-ANXA229−339 via a GS linker (“GGSG” plus “HM” as cloning artifact) (Figure 2C) to form the ternary chimeric construct of p53 TAD17−56-scS100A4Δ8-ANXA229−339. Note here that the artificially produced scS100A4Δ8 allowed the presence of only one covalently bound ANXA229−339 and p53 TAD17−56 in the chimera. We have performed model buildings, based on known S100 complexes, to estimate the optimal linker length with the assumption that the flexible residues, located at both the N and C termini of each protein, could act as potential extenders in case the designed linker would turn out to be short and would constrain the interaction itself. Using these optimal linkers the chimeras could be prevented to become overly flexible, which usually hampers crystal formation. Using the p53 TAD17−56-scS100A4Δ8-ANXA229−339 construct we could solve the structure of the complex at 3.1 Å resolution (Figure 3A) (Table 1). Two chimeric molecules were found in the asymmetric unit. In both chains, ANXA229−339 was visible, but in chain B the scS100A4Δ8 was presumably very flexible and the electron density map of the complex was very weak. In the case of chain A, however, both scS100A4Δ8 and the p53 fragment could be built into the electron density map. The electron density of the linkers between p53 TAD17−56 and scS100A4Δ8, as well as between the S100A4 subunits, were not visible indicating that those additional residues did not affect the binding mode of p53 TAD17−56 to scS100A4Δ8. Three short segments of p53 were observed to be in α-helical conformation in the structure. The residues of these observed helices nicely overlap with the residues of the nascent secondary elements and form helices previously found by others in apo (Lee et al., 2000Lee H. Mok K.H. Muhandiram R. Park K.H. Suk J.E. Kim D.H. Chang J. Sung Y.C. Choi K.Y. Han K.H. Local structural elements in the mostly unstructured transcriptional activation domain of human p53.J. Biol. Chem. 2000; 275: 29426-29432Crossref PubMed Scopus (268) Google Scholar) or in complexed p53 (Lee et al., 2010Lee C.W. Martinez-Yamout M.A. Dyson H.J. Wright P.E. Structure of the p53 transactivation domain in complex with the nuclear receptor coactivator binding domain of CREB binding protein.Biochemistry. 2010; 49: 9964-9971Crossref PubMed Scopus (118) Google Scholar). The N-terminal α helix (18–25), part of TAD1, makes exclusively hydrophobic interactions (F19, L22, W23, and L25 of p53) with one of the binding pocket of S100A4 dimer. Note that S20/D21 and K24 of p53 form an intramolecular salt bridge stabilizing this α helix. At the C-terminal end of the TAD segment, two more α helices (residues 37–42 and 47–53) are visible in the complex. S37 and S46 N-terminally cap the evolved helices respectively, further stabilizing their structure. Hydrophobi" @default.
• W3026695005 created "2020-05-29" @default.
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• W3026695005 date "2020-08-01" @default.
• W3026695005 modified "2023-10-17" @default.
• W3026695005 title "Structure Determination of the Transactivation Domain of p53 in Complex with S100A4 Using Annexin A2 as a Crystallization Chaperone" @default.
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• W3026695005 doi "https://doi.org/10.1016/j.str.2020.05.001" @default.
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