Department of Life Sciences, Imperial College London , UK.
PeerJ. 2015 Feb 26;3:e798. doi: 10.7717/peerj.798. eCollection 2015.
Noroviruses are positive-sense single-stranded RNA viruses. They encode an NS6 protease that cleaves a viral polyprotein at specific sites to produce mature viral proteins. In an earlier study we obtained crystals of murine norovirus (MNV) NS6 protease in which crystal contacts were mediated by specific insertion of the C-terminus of one protein (which contains residues P5-P1 of the NS6-7 cleavage junction) into the peptide binding site of an adjacent molecule, forming an adventitious protease-product complex. We sought to reproduce this crystal form to investigate protease-substrate complexes by extending the C-terminus of NS6 construct to include residues on the C-terminal (P') side of the cleavage junction. We report the crystallization and crystal structure determination of inactive mutants of murine norovirus NS6 protease with C-terminal extensions of one, two and four residues from the N-terminus of the adjacent NS7 protein (NS6 1', NS6 2', NS6 4'). We also determined the structure of a chimeric extended NS6 protease in which the P4-P4' sequence of the NS6-7 cleavage site was replaced with the corresponding sequence from the NS2-3 cleavage junction (NS6 4' 2|3).The constructs NS6 1' and NS6 2' yielded crystals that diffracted anisotropically. We found that, although the uncorrected data could be phased by molecular replacement, refinement of the structures stalled unless the data were ellipsoidally truncated and corrected with anisotropic B-factors. These corrections significantly improved phasing by molecular replacement and subsequent refinement.The refined structures of all four extended NS6 proteases are very similar in structure to the mature MNV NS6-and in one case reveal additional details of a surface loop. Although the packing arrangement observed showed some similarities to those observed in the adventitious protease-product crystals reported previously, in no case were specific protease-substrate interactions observed.
诺如病毒是正链单链 RNA 病毒。它们编码 NS6 蛋白酶，该酶在特定位点切割病毒多蛋白，生成成熟的病毒蛋白。在之前的一项研究中，我们获得了鼠诺如病毒 (MNV) NS6 蛋白酶的晶体，其中晶体接触是通过特定插入一个蛋白质的 C 末端（包含 NS6-7 切割连接点的 P5-P1 残基）到相邻分子的肽结合位点来介导的，形成一个偶然的蛋白酶-产物复合物。我们试图复制这种晶体形式，通过延伸 NS6 结构的 C 末端来包含切割连接点的 C 末端（P'）侧的残基，从而研究蛋白酶-底物复合物。我们报告了具有从相邻 NS7 蛋白 N 末端延伸一个、两个和四个残基的 C 末端的鼠诺如病毒 NS6 蛋白酶无活性突变体的结晶和晶体结构测定（NS6 1'、NS6 2'、NS6 4'）。我们还确定了嵌合延伸 NS6 蛋白酶的结构，其中 NS6-7 切割位点的 P4-P4'序列被 NS2-3 切割位点的相应序列取代（NS6 4' 2|3）。构建体 NS6 1'和 NS6 2'产生了各向异性衍射的晶体。我们发现，尽管未校正的数据可以通过分子置换进行相位确定，但如果不将数据椭圆截断并使用各向异性 B 因子进行校正，结构精修就会停滞不前。这些校正显著改善了分子置换和随后的精修的相位。所有四个延伸的 NS6 蛋白酶的结构都非常相似，与成熟的 MNV NS6 相似，在一种情况下还揭示了表面环的额外细节。尽管观察到的包装排列与之前报道的偶然蛋白酶-产物晶体中观察到的排列有一些相似之处，但在任何情况下都没有观察到特定的蛋白酶-底物相互作用。
