Bacterial surface proteins and proteinaceous pili extensions play crucial roles in microbial attachment, a critical first step in pathogenesis. The structure and assembly of Chaperon-mediated Gram-negative bacterial pili have been studied in great detail, but very little is known about the assembly and architecture of Gram-positive bacterial pili. In our previous work, we have investigated the structures of enzyme sortases that are responsible for surface protein anchoring and identified a putative Cys-Arg catalytic dyad that is needed for sortase driven transpeptidation. We have investigated the structures of sortase anchored Gram-positive bacterial surface proteins and their complexes with host extracellular matrix proteins, and identified a new paradigm for host pathogen interactions. In our recent work, we identified specific sortases that are involved in Gram-positive pili assembly and also recognized individual subunits of Gram-positive pili that are necessary for bacterial adhesion and pathogenesis. Sortases are unique transpeptidases that recognize C-terminal sorting signals of proteins destined for bacterial surface, either to be attached to the peptidoglycan layer or to be incorporated into the pili of Gram-positive bacteria. Sortases that dictate surface protein destinations exhibit high substrate specificity and very little is known about the structural correlates that are responsible. In this proposal we will address the hypothesis that the structural correlates that dictate sortase substrate specificity extend beyond the substrates C-terminal sorting signals. In addition we suggest, while having similar primary substrate binding pockets, the second substrate binding sites exhibit distinct spatial, polar and structural characteristics between the sortases involved in surface protein anchoring and pilus assembly. Significant progress has been achieved; the work proposed here will build on the preliminary results with the following specific aims: 1) We would characterize sortases of both kind and their active sites with the help of respective substrates, substrate analogues and inhibitors, 2) We would explore the structures of individual major and minor pilus subunits, and finally 3) We would attempt to provide a model for the architecture and assembly of Gram-positive bacterial pili. PUBLIC HEALTH RELEVANCE: Our previous work revealed that sortases belong to a unique class of cysteine transpeptidases that catalyzes the reaction with the help of a novel Cys-Arg catalytic dyad. We have determined the crystal structures of surface proteins anchored by sortases. Our present goal is to understand the structural correlates that dictate sortase substrate specificity and the enzymes' role in Gram-positive pili assembly.