Biophysical Mechanism of Allosteric Regulation of Actin Capping Protein.
Biophysical Mechanism of Allosteric Regulation of Actin Capping Protein.
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肌动蛋白加帽蛋白变构调节的生物物理机制。
DOI：
10.1101/2023.08.16.553570
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Soranno,Andrea
中科院分区：
文献类型：
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作者：
Mooren,OliviaL;Stuchell-Brereton,MelissaD;McConnell,Patrick;Yan,Chenbo;Wilkerson,EmilyM;Goldfarb,Dennis;Cooper,JohnA;Sept,David;Soranno,Andrea
肌动蛋白封盖蛋白（Actin capping protein， CP）可通过位阻和变构机制调节。基于生化功能研究和溶剂可及性实验，生物物理水平上变构调节的分子机制包括三种配体：F-actin、cap - protein - interacting (CPI) motif和V-1/myotrophin结合位点之间的连接。本研究利用单分子Förster共振能量转移（FRET）和分子动力学（MD）研究了CP在原子水平上的变构调节机制，以评估其对链接结合位点配体的构象和结构动力学响应。在没有配体的情况下，单分子FRET和MD在溶液中显示出两种不同的CP构象；以前的晶体学研究只揭示了一种。与cpi基序肽的相互作用诱导CP内的构象，使帽和柄更接近，而与V-1的相互作用使它们彼此远离。比较来自不同蛋白质的CPI基序肽，我们确定了每个CPI基序特异性的CP构象和动力学的变化。单独的CP和带有CPI基序和V-1的复合物的MD模拟揭示了构象变化的原子细节。利用单分子FRET、等温量热法（ITC）和MD模拟分析CP与野生型（wt）和嵌合型CPI-基序肽的相互作用表明，CPI基序c端部分的构象和亲和力差异是内在的。我们得出结论，CP的变构调节涉及到在整个蛋白质中传播的构象变化，以连接不同的结合位点功能。我们的研究结果为CP变构调节的生物物理机制提供了新的见解。
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.