Biophysical Mechanism of Allosteric Regulation of Actin Capping Protein

• Published in: Journal of molecular biology 2023-12, Vol.435 (24), p.168342-168342, Article 168342
• Main Authors: Mooren, Olivia L, Stuchell-Brereton, Melissa D, McConnell, Patrick, Yan, Chenbo, Wilkerson, Emily M, Goldfarb, Dennis, Cooper, John A, Sept, David, Soranno, Andrea
• Format: Article
• Language: English
• Subjects: Actin Capping Proteins - chemistry; Actins - metabolism; Allosteric Regulation; Peptides - chemistry; Protein Binding
• ISSN: 0022-2836; 1089-8638; 1089-8638
• DOI: 10.1016/j.jmb.2023.168342

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 Förster 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. Interaction with CPI-motif peptides induced conformations within CP that bring the cap and stalk closer, while interaction with V-1 moves them away from one another. 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 wild-type (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.