Cell Surface Protein Anchoring in Gram-positive Bacteria

描述（由申请人提供）：研究将研究用于 将表面蛋白共价锚在革兰氏阳性菌的细胞壁上。表面蛋白在人类感染的发病过程中起着重要作用，并通过分选酶共价锚定。分选酶介导的附着机制是普遍保守的，负责附着高达30%的表面蛋白质，并为感染性所需。这项建议的工作将集中在金黄色葡萄球菌的SrtA蛋白。我们将定位其底物结合位点，确定其活性位点内氨基酸的功能意义，并研究其如何被钙激活。这项工作将通过设计，这将使得能够确定SrtA-抑制剂复合物的结构，以获得底物识别的分子基础的见解。金黄色葡萄球菌编码第二种分选酶，我们假设SrtB将蛋白质锚定在细胞壁上， 通过识别尚未鉴定的分选信号。我们将使用独特的生物淘选方法来确定可以被SrtA处理的全部氨基酸序列，并搜索SrtB分选信号。最后，我们将阐明这三点-SrtB蛋白的三维结构，揭示其活性位点内的保守结构特征，并深入了解其功能。这项工作的结果将有助于阐明细胞壁锚定的基础化学，识别靶向蛋白质用于细胞壁附着的新肽信号，并且可以促进新的治疗上有用的抗感染剂的开发。

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.