Structural and biochemical analyses of selectivity determinants in chimeric Streptococcus Class A sortase enzymes
Structural and biochemical analyses of selectivity determinants in chimeric Streptococcus Class A sortase enzymes
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DOI：
10.1002/pro.4266
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发表时间：
2022-01-03
期刊：
影响因子：
8
通讯作者：
Amacher, Jeanine F.
中科院分区：
文献类型：
--
作者：
Gao, Melody;Johnson, D. Alex;Amacher, Jeanine F.
相关蛋白质的序列变异是调节活性和选择性的重要特征。具有大程度序列变异的蛋白质家族的实例是细菌分选酶，其是革兰氏阳性细菌表面上的半胱氨酸转肽酶。A类分选酶负责将不同蛋白质附着到细胞壁以促进环境适应和相互作用。这些酶还用于蛋白质工程应用中，用于分选酶介导的连接（SML）或蛋白质靶的分选标记。我们以前研究了肺炎链球菌的SrtA，确定了一些推定的β 7-β 8环介导的相互作用，影响体外酶功能。我们确定了有助于S.与来自金黄色葡萄球菌的SrtA的严格P1 ' Gly识别相反，肺炎链球菌SrtA识别底物基序的P1 '位置处的几个氨基酸，在LPXTG中加下划线。然而，由于缺乏活性单体形式S的结构模型，在此，我们将我们的研究扩展到其他链球菌SrtA蛋白。我们解决了S的第一个单体结构。无乳链球菌SrtA，包括C-末端，和来自S.化脓性链球菌和S.无乳菌SrtA.这些结构和伴随的生物化学数据支持我们先前鉴定的β 7-β 8环介导的相互作用，并提供了对其在A类分选酶底物选择性中的作用的额外见解。对单个SrtA序列和靶选择性的结构决定因素的更好理解也可以促进改进的分选工具的设计或发现。
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 beta 7-beta 8 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 beta 7-beta 8 loop chimeras from S. pyogenes and S. agalactiae SrtA. These structures and accompanying biochemical data support our previously identified beta 7-beta 8 loop-mediated interactions and provide additional insight into their role in Class A sortase substrate selectivity. A greater understanding of individual SrtA sequence and structural determinants of target selectivity may also facilitate the design or discovery of improved sortagging tools.