Kodera Noriyuki, Abe Hiroshi, Nguyen Phuong Doan N, Ono Shoichiro
WPI-Nano Life Science Institute, Kanazawa University, Kanazawa, Ishikawa, Japan.
Department of Biology, Graduate School of Science, Chiba University, Chiba, Japan.
J Biol Chem. 2021 Jan-Jun;296:100649. doi: 10.1016/j.jbc.2021.100649. Epub 2021 Apr 9.
Cyclase-associated protein (CAP) is a conserved actin-binding protein that regulates multiple aspects of actin dynamics, including polymerization, depolymerization, filament severing, and nucleotide exchange. CAP has been isolated from different cells and tissues in an equimolar complex with actin, and previous studies have shown that a CAP-actin complex contains six molecules each of CAP and actin. Intriguingly, here, we successfully isolated a complex of Xenopus cyclase-associated protein 1 (XCAP1) with actin from oocyte extracts, which contained only four molecules each of XCAP1 and actin. This XCAP1-actin complex remained stable as a single population of 340 kDa species during hydrodynamic analyses using gel filtration or analytical ultracentrifugation. Examination of the XCAP1-actin complex by high-speed atomic force microscopy revealed a tripartite structure: one middle globular domain and two globular arms. The two arms were observed in high and low states. The arms at the high state were spontaneously converted to the low state by dissociation of actin from the complex. However, when extra G-actin was added, the arms at the low state were converted to the high state. Based on the known structures of the N-terminal helical-folded domain and C-terminal CARP domain, we hypothesize that the middle globular domain corresponds to a tetramer of the N-terminal helical-folded domain of XCAP1 and that each arm in the high state corresponds to a heterotetramer containing a dimer of the C-terminal CARP domain of XCAP1 and two G-actin molecules. This novel configuration of a CAP-actin complex should help to understand how CAP promotes actin filament disassembly.
环化酶相关蛋白（CAP）是一种保守的肌动蛋白结合蛋白，可调节肌动蛋白动力学的多个方面，包括聚合、解聚、丝切断和核苷酸交换。CAP已从不同细胞和组织中以与肌动蛋白等摩尔复合物的形式分离出来，先前的研究表明，CAP-肌动蛋白复合物包含六个分子的CAP和肌动蛋白。有趣的是，在这里，我们成功地从卵母细胞提取物中分离出非洲爪蟾环化酶相关蛋白1（XCAP1）与肌动蛋白的复合物，该复合物仅包含四个分子的XCAP1和肌动蛋白。在使用凝胶过滤或分析超速离心进行的流体动力学分析中，这种XCAP1-肌动蛋白复合物作为单一的340 kDa物种群体保持稳定。通过高速原子力显微镜检查XCAP1-肌动蛋白复合物，发现了一种三方结构：一个中间球状结构域和两个球状臂。观察到两个臂处于高态和低态。高态的臂通过肌动蛋白从复合物中解离而自发转变为低态。然而，当添加额外的G-肌动蛋白时，低态的臂转变为高态。基于已知的N端螺旋折叠结构域和C端CARP结构域的结构，我们假设中间球状结构域对应于XCAP1的N端螺旋折叠结构域的四聚体，并且高态的每个臂对应于一个异源四聚体，该异源四聚体包含XCAP1的C端CARP结构域的二聚体和两个G-肌动蛋白分子。这种CAP-肌动蛋白复合物新的结构有助于理解CAP如何促进肌动蛋白丝的解聚。
