Crystal Structure of the S100A4–Nonmuscle Myosin IIA Tail Fragment Complex Reveals an Asymmetric Target Binding Mechanism
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Crystal structure of the S100A4–nonmuscle myosin IIA tail fragment complex reveals an asymmetric target binding mechanism Bence Kissa,1, Annette Duellib,1, László Radnaia, Katalin A. Kékesic,d, Gergely Katonab,2, and László Nyitraya,2 aDepartment of Biochemistry, Eötvös Loránd University, H-1117 Budapest, Hungary; bDepartment of Chemistry and Molecular Biology, University of Gothenburg, SE-40530 Göteborg, Sweden; cDepartment of Physiology and Neurobiology, and dLaboratory of Proteomics, Institute of Biology, Eötvös Loránd University, H-1117 Budapest, Hungary Edited by* James A. Spudich, Stanford University School of Medicine, Stanford, CA, and approved January 19, 2012 (received for review September 7, 2011) S100A4 is a member of the S100 family of calcium-binding proteins acterized, atomic details of S100A4 binding to any of its partners 2þ that is directly involved in tumor metastasis. It binds to the non- have not been described yet. In two crystal structure of Ca - muscle myosin IIA (NMIIA) tail near the assembly competence bound S100A4 the hydrophobic cleft of each subunit is occupied domain (ACD) promoting filament disassembly, which could be as- by the C terminus of an adjacent dimer (16, 17), which could sociated with increasing metastatic potential of tumor cells. Here, explain the formation of S100A4 oligomers that were observed we investigate the mechanism of S100A4–NMIIA interaction based extracellularly (18). These clefts were also shown to interact with on binding studies and the crystal structure of S100A4 in complex the calmodulin antagonist trifluoperazine (19). Interestingly, the with a 45-residue-long myosin heavy chain fragment. Interestingly, known complex structures of the S100 family do not reveal a we also find that S100A4 binds as strongly to a homologous heavy uniform binding mode as the orientations of the target peptides chain fragment of nonmuscle myosin IIC as to NMIIA. The structure in each complex are considerably different. Nevertheless, all the of the S100A4–NMIIA complex reveals a unique mode of interac- structural models reveal that one S100 dimer binds symmetrically tion in the S100 family: A single, predominantly α-helical myosin to two, predominantly α-helical peptide ligands (20–26). chain is wrapped around the Ca2þ-bound S100A4 dimer occupying One of the most investigated interaction partners of S100A4 both hydrophobic binding pockets. Thermal denaturation experi- is NMIIA. It has been demonstrated that metastasis-associated ments of coiled-coil forming NMIIA fragments indicate that the cellular motility is coupled to S100A4–NMIIA interaction (3, 27). coiled-coil partially unwinds upon S100A4 binding. Based on these The Ca2þ-dependent interaction of S100A4 with NMIIA pre- results, we propose a model for NMIIA filament disassembly: Part vents filament assembly and promotes filament disassembly (8, of the random coil tailpiece and the C-terminal residues of the 10, 28, 29). The increased cytoskeletal dynamics leads to the for- coiled-coil are wrapped around an S100A4 dimer disrupting the mation of few side protrusions and extensive forward protrusions ACD and resulting in filament dissociation. The description of the in S100A4 expressing cells (29). S100A4 binds to the C-terminal complex will facilitate the design of specific drugs that interfere end of the coiled-coil tail of NMIIA (29) overlapping the assem- with the S100A4–NMIIA interaction. bly competence domain (ACD) that is required for filament formation (30–34). Despite several efforts to map the S100A4 Ca2+-binding ∣ myosin filaments ∣ protein–protein interactions ∣ binding site on NMIIA (16, 35, 36), the observations are ambig- cell migration uous, and until now there is no detailed structural model for how the S100A4 dimer dissociates NMIIA filaments. Here, we report 100A4 is a member of the S100 family of small, dimeric, the high-resolution crystal structure of the S100A4–NMIIA frag- SEF-hand Ca2þ-binding proteins. S100 proteins are present ex- ment complex, which reveals a unique target-recognizing me- clusively in vertebrates. They are expressed in a tissue specific chanism in the S100 family. Based on our findings, we propose manner and they regulate specific extra- and intracellular pro- a model for the structural basis of S100A4-induced NMIIA fila- cesses (i.e., cell cycle regulation, cell growth, cell differentiation, ment disassembly. and motility). S100 proteins form mainly homodimers stabilized by noncovalent interactions between two helices from each sub- Results unit (helices 1, 4, 1′,4′) that form an X-type four-helix bundle Affinity of S100A4 to NMII Fragments. Because previous studies on dimerization motif. Each S100 subunit contains two Ca2þ-bind- the NMIIA–S100A4 interaction have not unambiguously deter- ing motifs: an N-terminal pseudo- and a C-terminal canonical mined the myosin sequence requirements for S100A4 binding EF-hand (1, 2). The elevated concentration of S100A4 is ob- (16, 35, 36), isothermal titration calorimetric (ITC) measure- served in a number of human cancers such as breast, colorectal, ments were carried out with peptides and fragments illustrated in ovarian, pancreatic, and others (3, 4). Overexpression of S100A4 Fig. 2A. Our results support the findings of Badyal et al. (36) that K ≈ 30 μ enhanced the metastatic capability of mammary tumors (5, 6). the 16-mer peptide MP0 shows lower affinity ( d M) to Extracellularly, binding to annexin A2 (ANXA2) S100A4 acti- vates matrix metalloproteinases through plasminogen activation Author contributions: B.K., G.K., and L.N. designed research; B.K., A.D., and K.A.K. (7), whereas in the cytoplasm it interacts with cytoskeletal ele- performed research; B.K., A.D., L.R., G.K., and L.N. analyzed data; and B.K., A.D., G.K., and ments like F-actin (8), tropomyosin (9), and nonmuscle myosin L.N. wrote the paper. IIA (NMIIA) (10), and it has a role in cell shape remodeling. The authors declare no conflict of interest. S100A4 has also been shown to interact with several other pro- *This Direct Submission article had a prearranged editor. β teins: p53, CCN3, liprin 1, methionine aminopeptidase 2, and Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, Smad3 (11–15). www.pdb.org (PDB ID code 3ZWH). 2þ Upon Ca binding, S100A4 undergoes a large conforma- 1B.K. and A.D. contributed equally to this work. tional shift in the canonical EF-hand resulting in the exposure 2To whom correspondence should be addressed. E-mail: [email protected] or of a hydrophobic binding pocket formed by helices 3, 4, the hinge [email protected]. region (loop 2), and the C-terminal coil region (3) (Fig. 1). This article contains supporting information online at www.pnas.org/lookup/suppl/ 2þ Although the structural transition on binding Ca is well char- doi:10.1073/pnas.1114732109/-/DCSupplemental. 6048–6053 ∣ PNAS ∣ April 17, 2012 ∣ vol. 109 ∣ no. 16 www.pnas.org/cgi/doi/10.1073/pnas.1114732109 Downloaded by guest on September 27, 2021 one myosin heavy chain peptide to one S100A4 dimer (Fig. 2C). Because ITC measurements with the longer myosin fragment K ≈ 6 MF2 suggested a considerably tighter interaction ( d nM) (Fig. S1B), we added N-terminal residues to MPC, resulting in a 45-residue-long NMIIA fragment (MPT). Peptides MPT and MPN bind to S100A4 with the same stoichiometry as MPC, but K ≈ 8 B–D with higher affinities ( d nM, Fig. 2 ). Although the affinities are similar, enthalpy changes are higher for MPN, whereas the entropic cost of MPT binding to S100A4 is lower, indicating different binding mechanisms for the coiled-coil and random coil regions of the myosin heavy chain (Table S1). In order to investigate the isoform specificity of S100A4 binding to the three NMII paralogs, we also measured the affinity of the homologous MPTb and MPTc fragments (Fig. 2A) to S100A4 by K ≈ ITC. MPTb shows reduced affinity compared to MPT ( d 125 nM). On the contrary, MPTc binds to S100A4 as strongly Fig. 1. Overview of S100A4 in complex with the 45-residue-long NMIIA tail as the NMIIA peptide (Fig. S1 E and F and Table S1). fragment along the twofold symmetry axis of the dimer. S100A4 subunit A and B are shown in green and blue, respectively, while NMIIA peptide in 2þ Overall Description of the S100A4-MPT Complex. yellow (residues 1893–1913) and orange (residues 1914–1935), and Ca ions Our efforts to crys- are gray. The main secondary structural elements are indicated. tallize wild-type S100A4 in complex with NMIIA peptides failed. Therefore, we created a triple mutant S100A4 (Cys3Ser/Cys81Ser/ S100A4 than it was previously reported (16) (Fig. S1A and Cys86Ser), where the solvent exposed thiol groups were elimi- nated in order to avoid disulfide formation, leading to the loss Table S1). The C-terminal extension of this peptide (MPC) binds K ≈ 2 μ in binding capability of S100A4 to myosin IIA fragments. These with micromolar affinity ( d M) and with a stoichiometry of mutations were introduced not only to the wild-type, but to the Phe45Trp mutant S100A4, which was originally designed for fa- cilitating spectroscopic measurements. The construct with the four mutations was successfully crystallized and the crystal struc- ture was solved by molecular replacement to 1.9 Å resolution (Table S2). According to ITC measurement, MPT binds to the mutant S100A4 with about two orders of magnitude weaker af- K ≈ 1 5 μ finity than to the wild-type protein ( d . M), but the stoi- chiometry of the interaction did not change (Fig. S1D). Fig. 1 shows how a single chain of MPT (NMIIA heavy chain residues 1894–1935) wraps around the dimeric S100A4 protein in the crys- tal structure. Symmetry Breakage in the S100A4 Homodimer upon MPT Binding. Whereas MPT binds both S100A4 subunits with approximately 2 2 the same area of interaction surface (1;308 Å and 1;251 Å for subunit A and B, respectively), each subunit of the symmetric homodimer has to adapt its conformation to the different char- acter of the two termini of MPT.