The anchoring of the surface proteins to the cell wall in gram-positive bacteria involves a peptide ligation reaction catalyzed by transpeptidase sortase. Most bacterial genomes encode multiple sortases with dedicated functions. Streptococcus pneumoniae (Sp) carries four sortases; a housekeeping sortase (SrtA), and three pilin specific sortases (SrtC1, C2, C3) dedicated to the biosynthesis of covalent pilus. Interestingly, SrtA, meant for performing housekeeping roles, is also implicated in pilus assembly of Sp. The allegiance of SpSrtA to the pathogenic pilus assembly makes it an ideal target for clinical inhibitor development. In this paper, we describe biochemical characterization, crystal structure and peptide substrate preference of SpSrtA. Transpeptidation reaction with a variety of substrates revealed that the enzyme preferred elongated LPXTG sequences and transferred them equally well to both Ala- and Gly-terminated peptides. Curiously, the crystal structure of both wild type and an active site (Cys to Ala) mutant of SpSrtA displayed inter-twined 3D-swapped dimers in which each protomer generated a classic eight-stranded beta-barrel ‘sortase fold'. Size-exclusion chromatography and sedimentation equilibrium measurements revealed the predominant presence of a dimer in equilibrium with its monomer. The crystal structure-based Cys–Cys distance mapping with defined chemical cross-linkers established the existence of 3D-swapped structure in solution. The swapping in SpSrtA, unprecedented for sortase family, may be physiologically relevant and meant to perform regulatory functions.

中文翻译：

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肺炎链球菌的3D交换结构和典型分类酶A的功能属性的询问

在革兰氏阳性细菌中将表面蛋白锚定到细胞壁上涉及由转肽酶分选酶催化的肽连接反应。大多数细菌基因组编码具有特定功能的多种分选酶。肺炎链球菌（Sp）携带4种分选酶。一个管家分选酶（SrtA）和三个专用于共价菌毛生物合成的菌毛蛋白特异性分选酶（SrtC1，C2，C3）。有趣的是，旨在执行家政角色的SrtA也与Sp的菌毛组装有关。SpSrtA对病原菌毛的效忠使它成为临床抑制剂开发的理想靶标。在本文中，我们描述了SpSrtA的生化特性，晶体结构和肽底物偏好。与多种底物的转肽反应表明，该酶更喜欢拉长的LPXTG序列，并将它们同样好地转移到Ala和Gly末端的肽上。奇怪的是，SpSrtA的野生型和活性位点（从Cys到Ala）突变体的晶体结构都显示出相互缠绕的3D交换二聚体，其中每个原发子都产生了经典的八链β-桶“分选折叠”。尺寸排阻色谱法和沉降平衡测量结果表明，二聚体与其单体处于平衡状态。具有定义的化学交联剂的基于晶体结构的Cys-Cys距离图确定了溶液中3D交换结构的存在。SpSrtA中的交换（对于分选酶家族而言是空前的）可能在生理上相关并且意在执行调节功能。