Vega-Rocha Susana, Gronenborn Bruno, Gronenborn Angela M, Campos-Olivas Ramón
Structural and Computational Biology Program, Spanish National Cancer Center (CNIO), Madrid 28029, Spain.
Biochemistry. 2007 May 29;46(21):6201-12. doi: 10.1021/bi700159q. Epub 2007 May 2.
Nanoviruses are a family of plant viruses that possess a genome of multiple circular single-stranded DNA (ssDNA) components and are strikingly similar in their replication mode to the plant geminiviruses and to the circoviruses that infect birds or mammals. These viruses multiply by rolling circle replication using virus-encoded multifunctional replication initiator proteins (Rep proteins) that catalyze the initiation of replication on a double-stranded DNA (dsDNA) intermediate and the resolution of the ssDNA into circles. Here we report the solution NMR three-dimensional structure of the endonuclease domain from the master Rep (M-Rep) protein of faba bean necrotic yellows virus (FBNYV), a representative of the nanoviruses. The domain comprises amino acids 2-95 (M-Rep2-95), and its global fold is similar to those previously described for the gemini- and circovirus Rep endonuclease domains, consisting of a central 5-stranded antiparallel beta-sheet covered on one side by an alpha-helix and irregular loops and on the other, more open side of the domain, by an alpha-helix containing the catalytic tyrosine residue (the catalytic helix). Longer domain constructs extending to amino acids 117 and 124 were also characterized. They contain an additional alpha-helix, are monomeric, and exhibit catalytic activity indistinguishable from that of M-Rep2-95. The binding site for the catalytic metal was identified by paramagnetic broadening and maps to residues on the exposed face of the central beta-sheet. A comparison with the previously determined Rep endonuclease domain structures of tomato yellow leaf curl Sardinia virus (TYLCSV), a geminivirus, and that of porcine circovirus type 2 (PCV2) Rep allows the identification of a positively charged surface that is most likely involved in dsDNA binding, and reveals common features shared by all endonuclease domains of nanovirus, geminivirus, and circovirus Rep proteins.
纳米病毒是一类植物病毒，其基因组由多个环状单链DNA（ssDNA）组分构成，在复制模式上与植物双生病毒以及感染鸟类或哺乳动物的圆环病毒极为相似。这些病毒通过滚环复制进行增殖，利用病毒编码的多功能复制起始蛋白（Rep蛋白），该蛋白催化双链DNA（dsDNA）中间体上的复制起始以及ssDNA环化的解析。在此，我们报道了蚕豆坏死黄化病毒（FBNYV）（纳米病毒的代表）主Rep（M-Rep）蛋白的核酸内切酶结构域的溶液核磁共振三维结构。该结构域包含氨基酸2 - 95（M-Rep2 - 95），其整体折叠结构与先前描述的双生病毒和圆环病毒Rep核酸内切酶结构域相似，由一个中央的5股反平行β折叠组成，一侧被一个α螺旋和不规则环覆盖，另一侧在结构域更开放的一侧被一个包含催化酪氨酸残基的α螺旋（催化螺旋）覆盖。还对延伸至氨基酸117和124的更长结构域构建体进行了表征。它们包含一个额外的α螺旋，呈单体形式，并且表现出与M-Rep2 - 95无法区分的催化活性。通过顺磁加宽鉴定了催化金属的结合位点，其映射到中央β折叠暴露面上的残基。与先前确定的双生病毒番茄黄化曲叶撒丁岛病毒（TYLCSV）和猪圆环病毒2型（PCV2）Rep的Rep核酸内切酶结构域结构进行比较，能够确定一个最有可能参与dsDNA结合的带正电表面，并揭示了纳米病毒、双生病毒和圆环病毒Rep蛋白所有核酸内切酶结构域共有的特征。
