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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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Structure of the S100A4Δ8C/MIIA1908-1923 complex. (A) Ribbon diagram of the S100A4Δ8C/MIIA1908-1923 complex showing the S100A4 subunits in green (subunit A) and brown (subunit B) and the MIIA1908-1923 peptide in blue. (B) Refined model of the S100A4Δ8C/MIIA1908-1923 peptide structure. The myosin-IIA peptide (residues Asp1908-Leu1917) is shown in stick representation superimposed onto the Fo-Fc electron density map, contoured at 3.0σ.
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Figure 4: Structure of the S100A4Δ8C/MIIA1908-1923 complex. (A) Ribbon diagram of the S100A4Δ8C/MIIA1908-1923 complex showing the S100A4 subunits in green (subunit A) and brown (subunit B) and the MIIA1908-1923 peptide in blue. (B) Refined model of the S100A4Δ8C/MIIA1908-1923 peptide structure. The myosin-IIA peptide (residues Asp1908-Leu1917) is shown in stick representation superimposed onto the Fo-Fc electron density map, contoured at 3.0σ.

Mentions: Both the S100A4Δ8C and S100A4Δ8C/MIIA1908-1923 peptide structures were determined by molecular replacement using the native Ca2+-S100A4 structure (2Q91) truncated at Glu91 as the search model [4] (Table 2). For the apo-S100A4Δ8C structure there were four molecules in the asymmetric unit (two S100A4Δ8C dimers), with continuous density from residues Cys3-Gly92 for all molecules. Residues in the favored, allowed and disallowed regions in the Ramachandran plot account for 97.4%, 2.0% and 0.6% of the total residues, respectively. The S100A4Δ8C/MIIA1908-1923 structure contained a single S100A4 dimer in the asymmetric unit with the MIIA1908-1923 peptide bound asymmetrically across the dimer interface. The electron density was continuous from residues Ala2-Gly92 for both molecules in the asymmetric unit, except for residues Gly47-Arg49 (loop region between helix 2 and 3) of subunit A, which were not modeled. Continuous difference density was observed in difference Fourier syntheses (using Fo-Fc coefficients; contoured at 3σ) following the first round of refinement, indicating the presence of a highly ordered peptide, asymmetrically bound to the S100A4 dimer (Figure 4A and 4B). Residues in the favored, allowed and disallowed regions in the Ramachandran plot account for 96.8%, 2.2% and 1.2% of the total residues, respectively. The two residues in the disallowed region, which deviate slightly from expected values, are Arg49 (subunit B) from the disordered region and the C-terminal Gly92.


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)

Structure of the S100A4Δ8C/MIIA1908-1923 complex. (A) Ribbon diagram of the S100A4Δ8C/MIIA1908-1923 complex showing the S100A4 subunits in green (subunit A) and brown (subunit B) and the MIIA1908-1923 peptide in blue. (B) Refined model of the S100A4Δ8C/MIIA1908-1923 peptide structure. The myosin-IIA peptide (residues Asp1908-Leu1917) is shown in stick representation superimposed onto the Fo-Fc electron density map, contoured at 3.0σ.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3924328&req=5

Figure 4: Structure of the S100A4Δ8C/MIIA1908-1923 complex. (A) Ribbon diagram of the S100A4Δ8C/MIIA1908-1923 complex showing the S100A4 subunits in green (subunit A) and brown (subunit B) and the MIIA1908-1923 peptide in blue. (B) Refined model of the S100A4Δ8C/MIIA1908-1923 peptide structure. The myosin-IIA peptide (residues Asp1908-Leu1917) is shown in stick representation superimposed onto the Fo-Fc electron density map, contoured at 3.0σ.
Mentions: Both the S100A4Δ8C and S100A4Δ8C/MIIA1908-1923 peptide structures were determined by molecular replacement using the native Ca2+-S100A4 structure (2Q91) truncated at Glu91 as the search model [4] (Table 2). For the apo-S100A4Δ8C structure there were four molecules in the asymmetric unit (two S100A4Δ8C dimers), with continuous density from residues Cys3-Gly92 for all molecules. Residues in the favored, allowed and disallowed regions in the Ramachandran plot account for 97.4%, 2.0% and 0.6% of the total residues, respectively. The S100A4Δ8C/MIIA1908-1923 structure contained a single S100A4 dimer in the asymmetric unit with the MIIA1908-1923 peptide bound asymmetrically across the dimer interface. The electron density was continuous from residues Ala2-Gly92 for both molecules in the asymmetric unit, except for residues Gly47-Arg49 (loop region between helix 2 and 3) of subunit A, which were not modeled. Continuous difference density was observed in difference Fourier syntheses (using Fo-Fc coefficients; contoured at 3σ) following the first round of refinement, indicating the presence of a highly ordered peptide, asymmetrically bound to the S100A4 dimer (Figure 4A and 4B). Residues in the favored, allowed and disallowed regions in the Ramachandran plot account for 96.8%, 2.2% and 1.2% of the total residues, respectively. The two residues in the disallowed region, which deviate slightly from expected values, are Arg49 (subunit B) from the disordered region and the C-terminal Gly92.

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