Nichols C E, Johnson C, Lockyer M, Charles I G, Lamb H K, Hawkins A R, Stammers D K
Division of Structural Biology, The Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.
Proteins. 2006 Jul 1;64(1):111-23. doi: 10.1002/prot.20982.
The Salmonella typhimurium "yeaZ" gene (StyeaZ) encodes an essential protein of unknown function (StYeaZ), which has previously been annotated as a putative homolog of the Pasteurella haemolytica M22 O-sialoglycoprotein endopeptidase Gcp. YeaZ has also recently been reported as the first example of an RPF from a gram-negative bacterial species. To further characterize the properties of StYeaZ and the widely occurring MK-M22 family, we describe the purification, biochemical analysis, crystallization, and structure determination of StYeaZ. The crystal structure of StYeaZ reveals a classic two-lobed actin-like fold with structural features consistent with nucleotide binding. However, microcalorimetry experiments indicated that StYeaZ neither binds polyphosphates nor a wide range of nucleotides. Additionally, biochemical assays show that YeaZ is not an active O-sialoglycoprotein endopeptidase, consistent with the lack of the critical zinc binding motif. We present a detailed comparison of YeaZ with available structural homologs, the first reported structural analysis of an MK-M22 family member. The analysis indicates that StYeaZ has an unusual orientation of the A and B lobes which may require substantial relative movement or interaction with a partner protein in order to bind ligands. Comparison of the fold of YeaZ with that of a known RPF domain from a gram-positive species shows significant structural differences and therefore potentially distinctive RPF mechanisms for these two bacterial classes.
鼠伤寒沙门氏菌“yeaZ”基因（StyeaZ）编码一种功能未知的必需蛋白（StYeaZ），该蛋白先前被注释为溶血巴斯德氏菌M22 O - 唾液酸糖蛋白内肽酶Gcp的假定同源物。最近，YeaZ也被报道为革兰氏阴性细菌物种中RPF的首个实例。为了进一步表征StYeaZ和广泛存在的MK - M22家族的特性，我们描述了StYeaZ的纯化、生化分析、结晶及结构测定。StYeaZ的晶体结构揭示了一种经典的两叶状肌动蛋白样折叠，其结构特征与核苷酸结合一致。然而，微量热法实验表明，StYeaZ既不结合多磷酸盐，也不结合多种核苷酸。此外，生化分析表明，YeaZ不是一种活性O - 唾液酸糖蛋白内肽酶，这与缺乏关键的锌结合基序一致。我们对YeaZ与现有的结构同源物进行了详细比较，这是首次报道的MK - M22家族成员的结构分析。分析表明，StYeaZ的A叶和B叶具有不寻常的取向，这可能需要大量的相对移动或与伴侣蛋白相互作用才能结合配体。将YeaZ的折叠与革兰氏阳性物种中已知的RPF结构域的折叠进行比较，发现存在显著的结构差异，因此这两类细菌可能具有不同的RPF机制。
