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.
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