Sequence variation in related proteins is an important characteristic that modulates activity and selectivity. An example of a protein family with a large degree of sequence variation is that of bacterial sortases, which are cysteine transpeptidases on the surface of gram-positive bacteria. Class A sortases are responsible for attachment of diverse proteins to the cell wall to facilitate environmental adaption and interaction. These enzymes are also used in protein engineering applications for sortase-mediated ligations (SML) or sortagging of protein targets. We previously investigated SrtA from Streptococcus pneumoniae, identifying a number of putative β7-β8 loop-mediated interactions that affected in vitro enzyme function. We identified residues that contributed to the ability of S. pneumoniae SrtA to recognize several amino acids at the P1’ position of the substrate motif, underlined in LPXTG, in contrast to the strict P1’ Gly recognition of SrtA from Staphylococcus aureus. However, motivated by the lack of a structural model for the active, monomeric form of S. pneumoniae SrtA, here, we expanded our studies to other Streptococcus SrtA proteins. We solved the first monomeric structure of S. agalactiae SrtA which includes the C-terminus, and three others of β7-β8 loop chimeras from S. pyogenes and S. agalactiae SrtA. These structures and accompanying biochemical data support our previously identified β7-β8 loop-mediated interactions and provide additional insight into their role in Class A sortase substrate selectivity. We argue that a greater understanding of individual SrtA sequence and structural determinants of target selectivity can facilitate the design or discovery of improved sortagging tools.

中文翻译：

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嵌合 A 类链球菌分选酶选择性决定簇的结构和生化分析

相关蛋白质的序列变异是调节活性和选择性的重要特征。具有大量序列变异的蛋白质家族的一个例子是细菌分选酶，它是革兰氏阳性细菌表面的半胱氨酸转肽酶。A 类分选酶负责将不同的蛋白质附着到细胞壁上，以促进环境适应和相互作用。这些酶也被在蛋白质工程应用用于分选酶介导的连接反应（SML）或分选标记的蛋白质靶。我们之前研究了来自肺炎链球菌的SrtA ，确定了许多在体外影响的推定的 β7-β8 环介导的相互作用酶功能。我们鉴定了有助于肺炎链球菌SrtA 识别底物基序 P1' 位置几个氨基酸的残基，在 LPXT G 中加下划线，这与来自金黄色葡萄球菌的 SrtA 的严格 P1' Gly 识别形成对比。然而，由于缺乏活性单体形式的肺炎链球菌SrtA的结构模型，在这里，我们将研究扩展到其他链球菌SrtA 蛋白。我们解决了S. agalactiae SrtA的第一个单体结构，其中包括 C 端，以及来自S. pyogenes和S. agalactiae 的其他三个 β7-β8 环嵌合体SrtA。这些结构和伴随的生化数据支持我们之前确定的 β7-β8 环介导的相互作用，并提供了对它们在 A 类分选酶底物选择性中的作用的额外见解。我们认为，更好地了解单个 SrtA 序列和目标选择性的结构决定因素可以促进改进的分拣工具的设计或发现。