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bioRxiv · 10.1101/2023.08.16.553570

Biophysical Mechanism of Allosteric Regulation of Actin Capping Protein

Mooren, O. L.·Stuchell-Brereton, M. D.·McConnell, P.·Yan, C.·Wilkerson, E. M.·Goldfarb, D.·Cooper, J. A.·Sept, D.·Soranno, A.
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Abstract

Actin capping protein (CP) can be regulated by steric and allosteric mechanisms. The molecular mechanism of the allosteric regulation at a biophysical level includes linkage between the binding sites for three ligands: F-actin, Capping-Protein-Interacting (CPI) motifs, and V-1/myotrophin, based on biochemical functional studies and solvent accessibility experiments. Here, we investigated the mechanism of allosteric regulation at the atomic level using single-molecule Forster resonance energy transfer (FRET) and molecular dynamics (MD) to assess the conformational and structural dynamics of CP in response to linked-binding site ligands. In the absence of ligand, both single-molecule FRET and MD revealed two distinct conformations of CP in solution; previous crystallographic studies revealed only one. CPI-motif peptide association induced conformational changes within CP that propagate in one direction, while V-1 association induced conformational changes in the opposite direction. Comparing CPI-motif peptides from different proteins, we identified variations in CP conformations and dynamics that are specific to each CPI motif. MD simulations for CP alone and in complex with a CPI motif and V-1 reveal atomistic details of the conformational changes. Analysis of the interaction of CP with wildtype (wt) and chimeric CPI-motif peptides using single-molecule FRET, isothermal calorimetry (ITC) and MD simulation indicated that conformational and affinity differences are intrinsic to the C-terminal portion of the CPI-motif. We conclude that allosteric regulation of CP involves changes in conformation that disseminate across the protein to link distinct binding-site functions. Our results provide novel insights into the biophysical mechanism of the allosteric regulation of CP.

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Mooren, O. L., Stuchell-Brereton, M. D., McConnell, P., Yan, C., Wilkerson, E. M., Goldfarb, D., Cooper, J. A., Sept, D., Soranno, A.. 2023-08-17. Biophysical Mechanism of Allosteric Regulation of Actin Capping Protein. https://doi.org/10.1101/2023.08.16.553570

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