Limits...
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.

Show MeSH

Related in: MedlinePlus

NMR assignments for MIIA1893-1923-bound Ca2+-S100A4. (A) A 1H-15N heteronuclear single quantum coherence spectrum (HSQC) of MIIA1893-1923-bound S100A4 shown together with the resonance assignments. (B) Insert of the HSQC spectrum illustrating the assignments for the most crowded region of the HSQC spectrum. The two weak contours labeled with an asterisk (*) were not assigned since they did not have observable correlations to any other residues.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3924328&req=5

Figure 7: NMR assignments for MIIA1893-1923-bound Ca2+-S100A4. (A) A 1H-15N heteronuclear single quantum coherence spectrum (HSQC) of MIIA1893-1923-bound S100A4 shown together with the resonance assignments. (B) Insert of the HSQC spectrum illustrating the assignments for the most crowded region of the HSQC spectrum. The two weak contours labeled with an asterisk (*) were not assigned since they did not have observable correlations to any other residues.

Mentions: Since MIIA1893-1923 bound S100A4 with a higher affinity than MIIA1908-1923[4], and exhibited better spectral properties than MIIA1908-1923, we examined this longer peptide in NMR studies to further investigate the solution properties of the asymmetric binding mode observed for myosin-IIA in the S100A4Δ8C/MIIA1908-1923 complex. As expected for a complex with a dissociation constant in the nanomolar range, MIIA1893-1923-bound S100A4 exhibited resonances with slow exchange on the chemical shift timescale. This property resulted in spectra exhibiting narrow linewidths, which is consistent with little, if any, broadening contributions from intermediate exchange. The 1H-15N HSQC of the Ca2+-S100A4/MIIA1893-1923 complex showed that peak doubling occurred for approximately two-thirds of the observable 1H-15N correlations throughout all regions of the protein (Figure 7). The resonance doubling complicated the assignment of backbone and side chain resonances, but it was still possible to sequence-specifically assign all of the observed correlations using standard NMR techniques. The observed peak doubling is consistent with the binding mode of the shorter MIIA1908-1923 peptide in our X-ray structure, in which peptide binding breaks the symmetry of the S100A4 dimer interface that is observed in the Ca2+-bound S100A4, resulting in two distinct electronic shielding environments for several residues.


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)

NMR assignments for MIIA1893-1923-bound Ca2+-S100A4. (A) A 1H-15N heteronuclear single quantum coherence spectrum (HSQC) of MIIA1893-1923-bound S100A4 shown together with the resonance assignments. (B) Insert of the HSQC spectrum illustrating the assignments for the most crowded region of the HSQC spectrum. The two weak contours labeled with an asterisk (*) were not assigned since they did not have observable correlations to any other residues.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: NMR assignments for MIIA1893-1923-bound Ca2+-S100A4. (A) A 1H-15N heteronuclear single quantum coherence spectrum (HSQC) of MIIA1893-1923-bound S100A4 shown together with the resonance assignments. (B) Insert of the HSQC spectrum illustrating the assignments for the most crowded region of the HSQC spectrum. The two weak contours labeled with an asterisk (*) were not assigned since they did not have observable correlations to any other residues.
Mentions: Since MIIA1893-1923 bound S100A4 with a higher affinity than MIIA1908-1923[4], and exhibited better spectral properties than MIIA1908-1923, we examined this longer peptide in NMR studies to further investigate the solution properties of the asymmetric binding mode observed for myosin-IIA in the S100A4Δ8C/MIIA1908-1923 complex. As expected for a complex with a dissociation constant in the nanomolar range, MIIA1893-1923-bound S100A4 exhibited resonances with slow exchange on the chemical shift timescale. This property resulted in spectra exhibiting narrow linewidths, which is consistent with little, if any, broadening contributions from intermediate exchange. The 1H-15N HSQC of the Ca2+-S100A4/MIIA1893-1923 complex showed that peak doubling occurred for approximately two-thirds of the observable 1H-15N correlations throughout all regions of the protein (Figure 7). The resonance doubling complicated the assignment of backbone and side chain resonances, but it was still possible to sequence-specifically assign all of the observed correlations using standard NMR techniques. The observed peak doubling is consistent with the binding mode of the shorter MIIA1908-1923 peptide in our X-ray structure, in which peptide binding breaks the symmetry of the S100A4 dimer interface that is observed in the Ca2+-bound S100A4, resulting in two distinct electronic shielding environments for several residues.

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