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Structure of the S100A4/myosin-IIA complex.

Ramagopal UA, Dulyaninova NG, Varney KM, Wilder PT, Nallamsetty S, Brenowitz M, Weber DJ, Almo SC, Bresnick AR - BMC Struct. Biol. (2013)

Bottom Line: This asymmetric binding mode was confirmed in NMR studies using a spin-labeled myosin-IIA peptide.These structural studies support the idea that residues 1908-1923 of the myosin-IIA chain heavy represent a core sequence for the S100A4/myosin-IIA complex.In addition, biophysical studies suggest that structural fluctuations within the myosin-IIA coiled-coil may facilitate S100A4 docking onto a single myosin-IIA polypeptide chain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA. anne.bresnick@einstein.yu.edu.

ABSTRACT

Background: S100A4, a member of the S100 family of Ca2+-binding proteins, modulates the motility of both non-transformed and cancer cells by regulating the localization and stability of cellular protrusions. Biochemical studies have demonstrated that S100A4 binds to the C-terminal end of the myosin-IIA heavy chain coiled-coil and disassembles myosin-IIA filaments; however, the mechanism by which S100A4 mediates myosin-IIA depolymerization is not well understood.

Results: We determined the X-ray crystal structure of the S100A4Δ8C/MIIA(1908-1923) peptide complex, which showed an asymmetric binding mode for the myosin-IIA peptide across the S100A4 dimer interface. This asymmetric binding mode was confirmed in NMR studies using a spin-labeled myosin-IIA peptide. In addition, our NMR data indicate that S100A4Δ8C binds the MIIA(1908-1923) peptide in an orientation very similar to that observed for wild-type S100A4. Studies of complex formation using a longer, dimeric myosin-IIA construct demonstrated that S100A4 binding dissociates the two myosin-IIA polypeptide chains to form a complex composed of one S100A4 dimer and a single myosin-IIA polypeptide chain. This interaction is mediated, in part, by the instability of the region of the myosin-IIA coiled-coil encompassing the S100A4 binding site.

Conclusion: The structure of the S100A4/MIIA(1908-1923) peptide complex has revealed the overall architecture of this assembly and the detailed atomic interactions that mediate S100A4 binding to the myosin-IIA heavy chain. These structural studies support the idea that residues 1908-1923 of the myosin-IIA chain heavy represent a core sequence for the S100A4/myosin-IIA complex. In addition, biophysical studies suggest that structural fluctuations within the myosin-IIA coiled-coil may facilitate S100A4 docking onto a single myosin-IIA polypeptide chain.

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S100A4Δ8 exhibits comparable myosin-IIA binding activity to wild-type S100A4. (A) Fluorescence anisotropy measurements of S100A4Δ8 binding to FITC-MIIA1904-1927. Values represent the mean ± standard deviation from two independent experiments. A KD of 0.51 ± 0.05 μM was determined from the fit to a single site saturation binding curve. (B) Representative gel of myosin-IIA disassembly assays performed at a ratio of 1:1 S100A4 dimer:myosin-IIA rod. 1 – myosin-IIA in the absence of S100A4; 2 – myosin-IIA in the presence of S100A4Δ8; 3 – myosin-IIA in the presence of wild-type S100A4 (m = reaction mixture, s = supernatant). (C) Quantification of disassembly assays. Values represent the mean ± standard deviation from two independent experiments.
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Figure 3: S100A4Δ8 exhibits comparable myosin-IIA binding activity to wild-type S100A4. (A) Fluorescence anisotropy measurements of S100A4Δ8 binding to FITC-MIIA1904-1927. Values represent the mean ± standard deviation from two independent experiments. A KD of 0.51 ± 0.05 μM was determined from the fit to a single site saturation binding curve. (B) Representative gel of myosin-IIA disassembly assays performed at a ratio of 1:1 S100A4 dimer:myosin-IIA rod. 1 – myosin-IIA in the absence of S100A4; 2 – myosin-IIA in the presence of S100A4Δ8; 3 – myosin-IIA in the presence of wild-type S100A4 (m = reaction mixture, s = supernatant). (C) Quantification of disassembly assays. Values represent the mean ± standard deviation from two independent experiments.

Mentions: Since we were unable to crystallize the wild-type full-length S100A4 with myosin-IIA peptides and our previous structural studies demonstrated that at high protein concentrations residues Glu91-Gln97 mediate interactions between symmetry-related S100A4 dimers in the crystalline state [4], we created a series of S100A4 C-terminal truncations (S100A4Δ3C, S100A4Δ4C, S100A4Δ6C, S100A4Δ7C, S100A4Δ8C, S100A4Δ9C and S100A4Δ13C; where “Δ-number” represents the number of residues deleted from the S100A4 C-terminus) that were expected to reduce S100A4 self-association. The S100A4Δ8C construct was used since it exhibited minimal self-association in the myosin-IIA peptide-bound state as assessed by dynamic light scattering and NMR. The ability of S100A4Δ8C to bind myosin-IIA was assessed in an anisotropy assay using FITC-labeled MIIA1904-1927, which binds wild-type S100A4 with a KD of 0.26 ± 0.03 μM [27]. The measured dissociation constant for S100A4Δ8C (0.51 ± 0.05 μM) was comparable to that observed for the wild-type S100A4 (Figure 3A). In addition, binding was Ca2+-dependent as no binding was observed in the presence of EGTA (data not shown). Using assembly competent myosin-IIA rods (MIIA1339−1960), we monitored the ability of S100A4Δ8C to disassemble preformed myosin-IIA filaments. At a molar stoichiometry of one S100A4Δ8C dimer per myosin-IIA rod, S100A4Δ8C disassembled approximately 85% of the myosin-IIA filaments, which was similar to the disassembly observed in the presence of wild-type S100A4 (Figure 3B and 3C).


Structure of the S100A4/myosin-IIA complex.

Ramagopal UA, Dulyaninova NG, Varney KM, Wilder PT, Nallamsetty S, Brenowitz M, Weber DJ, Almo SC, Bresnick AR - BMC Struct. Biol. (2013)

S100A4Δ8 exhibits comparable myosin-IIA binding activity to wild-type S100A4. (A) Fluorescence anisotropy measurements of S100A4Δ8 binding to FITC-MIIA1904-1927. Values represent the mean ± standard deviation from two independent experiments. A KD of 0.51 ± 0.05 μM was determined from the fit to a single site saturation binding curve. (B) Representative gel of myosin-IIA disassembly assays performed at a ratio of 1:1 S100A4 dimer:myosin-IIA rod. 1 – myosin-IIA in the absence of S100A4; 2 – myosin-IIA in the presence of S100A4Δ8; 3 – myosin-IIA in the presence of wild-type S100A4 (m = reaction mixture, s = supernatant). (C) Quantification of disassembly assays. Values represent the mean ± standard deviation from two independent experiments.
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Figure 3: S100A4Δ8 exhibits comparable myosin-IIA binding activity to wild-type S100A4. (A) Fluorescence anisotropy measurements of S100A4Δ8 binding to FITC-MIIA1904-1927. Values represent the mean ± standard deviation from two independent experiments. A KD of 0.51 ± 0.05 μM was determined from the fit to a single site saturation binding curve. (B) Representative gel of myosin-IIA disassembly assays performed at a ratio of 1:1 S100A4 dimer:myosin-IIA rod. 1 – myosin-IIA in the absence of S100A4; 2 – myosin-IIA in the presence of S100A4Δ8; 3 – myosin-IIA in the presence of wild-type S100A4 (m = reaction mixture, s = supernatant). (C) Quantification of disassembly assays. Values represent the mean ± standard deviation from two independent experiments.
Mentions: Since we were unable to crystallize the wild-type full-length S100A4 with myosin-IIA peptides and our previous structural studies demonstrated that at high protein concentrations residues Glu91-Gln97 mediate interactions between symmetry-related S100A4 dimers in the crystalline state [4], we created a series of S100A4 C-terminal truncations (S100A4Δ3C, S100A4Δ4C, S100A4Δ6C, S100A4Δ7C, S100A4Δ8C, S100A4Δ9C and S100A4Δ13C; where “Δ-number” represents the number of residues deleted from the S100A4 C-terminus) that were expected to reduce S100A4 self-association. The S100A4Δ8C construct was used since it exhibited minimal self-association in the myosin-IIA peptide-bound state as assessed by dynamic light scattering and NMR. The ability of S100A4Δ8C to bind myosin-IIA was assessed in an anisotropy assay using FITC-labeled MIIA1904-1927, which binds wild-type S100A4 with a KD of 0.26 ± 0.03 μM [27]. The measured dissociation constant for S100A4Δ8C (0.51 ± 0.05 μM) was comparable to that observed for the wild-type S100A4 (Figure 3A). In addition, binding was Ca2+-dependent as no binding was observed in the presence of EGTA (data not shown). Using assembly competent myosin-IIA rods (MIIA1339−1960), we monitored the ability of S100A4Δ8C to disassemble preformed myosin-IIA filaments. At a molar stoichiometry of one S100A4Δ8C dimer per myosin-IIA rod, S100A4Δ8C disassembled approximately 85% of the myosin-IIA filaments, which was similar to the disassembly observed in the presence of wild-type S100A4 (Figure 3B and 3C).

Bottom Line: This asymmetric binding mode was confirmed in NMR studies using a spin-labeled myosin-IIA peptide.These structural studies support the idea that residues 1908-1923 of the myosin-IIA chain heavy represent a core sequence for the S100A4/myosin-IIA complex.In addition, biophysical studies suggest that structural fluctuations within the myosin-IIA coiled-coil may facilitate S100A4 docking onto a single myosin-IIA polypeptide chain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA. anne.bresnick@einstein.yu.edu.

ABSTRACT

Background: S100A4, a member of the S100 family of Ca2+-binding proteins, modulates the motility of both non-transformed and cancer cells by regulating the localization and stability of cellular protrusions. Biochemical studies have demonstrated that S100A4 binds to the C-terminal end of the myosin-IIA heavy chain coiled-coil and disassembles myosin-IIA filaments; however, the mechanism by which S100A4 mediates myosin-IIA depolymerization is not well understood.

Results: We determined the X-ray crystal structure of the S100A4Δ8C/MIIA(1908-1923) peptide complex, which showed an asymmetric binding mode for the myosin-IIA peptide across the S100A4 dimer interface. This asymmetric binding mode was confirmed in NMR studies using a spin-labeled myosin-IIA peptide. In addition, our NMR data indicate that S100A4Δ8C binds the MIIA(1908-1923) peptide in an orientation very similar to that observed for wild-type S100A4. Studies of complex formation using a longer, dimeric myosin-IIA construct demonstrated that S100A4 binding dissociates the two myosin-IIA polypeptide chains to form a complex composed of one S100A4 dimer and a single myosin-IIA polypeptide chain. This interaction is mediated, in part, by the instability of the region of the myosin-IIA coiled-coil encompassing the S100A4 binding site.

Conclusion: The structure of the S100A4/MIIA(1908-1923) peptide complex has revealed the overall architecture of this assembly and the detailed atomic interactions that mediate S100A4 binding to the myosin-IIA heavy chain. These structural studies support the idea that residues 1908-1923 of the myosin-IIA chain heavy represent a core sequence for the S100A4/myosin-IIA complex. In addition, biophysical studies suggest that structural fluctuations within the myosin-IIA coiled-coil may facilitate S100A4 docking onto a single myosin-IIA polypeptide chain.

Show MeSH
Related in: MedlinePlus