Bravo A, Serrano-Heras G, Salas M
Centro de Biologia Molecular Severo Ochoa (Consejo Superior de Investigaciones Cientificas-Universidad Autónoma de Madrid (SIC-UAM)), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
J Biol Chem. 2001 Jun 15;276(24):21250-6. doi: 10.1074/jbc.M011296200. Epub 2001 Mar 29.
The bacteriophage phi29 replication protein p1 self-interacts in vitro, generating highly ordered structures. Specifically, the 53-amino acid protein p1DeltaN33, which retains the sequence of p1 spanning amino acids Met(34) to Lys(85), assembles into two-dimensional protofilament sheets. The region of protein p1 located between residues Glu(38) and Asn(65) presumably forms an alpha-helical coiled-coil structure. Here we have examined the role of this coiled-coil sequence in the formation of protofilament sheets. Using sedimentation assays and negative-stain electron microscopy analysis, we demonstrate that residues Leu(46), Met(53), and Leu(60), but not Leu(39), are essential for p1DeltaN33 assembly into sheets. Remarkably, replacement of Leu(46) by Val shifts the pathway of molecular assembly, leading to the formation of filamentous polymers approximately 10 nm in diameter. These results show, for the first time, that a short coiled-coil motif can mediate protein assembly into protofilament sheet structures.
噬菌体phi29复制蛋白p1在体外发生自我相互作用，形成高度有序的结构。具体而言，保留了p1从甲硫氨酸（34）到赖氨酸（85）序列的53个氨基酸的蛋白p1DeltaN33组装成二维原丝片层。蛋白p1位于谷氨酸（38）和天冬酰胺（65）残基之间的区域大概形成一个α-螺旋卷曲螺旋结构。在此，我们研究了这个卷曲螺旋序列在原丝片层形成中的作用。通过沉降分析和负染电子显微镜分析，我们证明亮氨酸（46）、甲硫氨酸（53）和亮氨酸（6）残基对于p1DeltaN33组装成片层是必需的，而亮氨酸（39）则不是。值得注意的是，用缬氨酸取代亮氨酸（46）会改变分子组装途径，导致形成直径约10纳米的丝状聚合物。这些结果首次表明，一个短的卷曲螺旋基序可以介导蛋白质组装成原丝片层结构。
