Cell Surface Protein Anchoring in Gram-positive Bacteria

描述(由申请者提供)：研究将研究用于 表面蛋白共价锚定在革兰氏阳性细菌的细胞壁上。表面蛋白在人类感染的发病过程中起着重要的作用，并由分类酶共价锚定。分类酶介导的附着机制是普遍保守的，负责附着高达30%的表面蛋白，是感染所必需的。本提案中的工作将集中在金黄色葡萄球菌的srtA蛋白上。我们将定位其底物结合部位，确定其活性部位内氨基酸的功能意义，并研究它是如何被钙激活的。这项工作将得到设计的补充，合成和体外测试基于多肽的srtA抑制物，这将使人们能够确定srtA-抑制物复合体的结构，从而深入了解底物识别的分子基础。金黄色葡萄球菌编码第二个类似于排序酶的酶，srtB，它没有已知的功能。我们假设srtB将蛋白质锚定在细胞壁上 通过识别尚未识别的分选信号，我们将使用一种独特的生物扫描方法来确定可被srtA处理的全部氨基酸序列，并寻找srtB分选信号。最后，我们将阐明srtB蛋白的三维结构，以揭示其活性部位的保守结构特征和对其功能的洞察。这项工作的结果将有助于揭示细胞壁锚定的潜在化学，识别靶向细胞壁附着的新的多肽信号，并可能促进新的治疗有用的抗感染药物的开发。

DESCRIPTION (provided by applicant):Research will study the molecular mechanism used to covalently anchor surface proteins to the cell wall of Gram-positive bacteria.Surface proteins play important roles during the pathogenesis of human infections and are covalently anchored by sortase enzymes.The mechanism of sortase-mediated attachment is universally conserved,responsible for attaching up to 30 percent of surface proteins and required for infectivity.Work in this proposal will focus on the SrtA protein from S.aureus.We will localize its substrate-binding site,determine the functional significance of amino acids within its active site,and investigate how it is activated by calcium.This work will be complemented by the design,synthesis and in vitro testing of peptide-based inhibitors of SrtA.This will enable the structure of a SrtA-inhibitor complex to be determined to gain insights in the molecular basis of substrate recognition.S.aureus encodes a second sortase-like enzyme,SrtB,which has no known function.We hypothesize that SrtB anchors proteins to the cell wall by recognizing a sorting signal that has yet to be identified.We will use a unique biopanning method to determine the full range of amino acid sequences that can be processed by SrtA and to search for a SrtB sorting signal.Finally,we will elucidate the three-dimensional structure of the SrtB protein to reveal conserved structural features within its active site and insights into its function.The results of this work will shed light onto the underlying chemistry of cell wall anchoring,identify new peptide signals that target proteins for cell wall attachment,and may facilitate the development of new therapeutically useful anti-infective agents.