Project Summary. Sortase enzymes are cysteine transpeptidases located on the cell surface of Gram-positive bacteria, which attach a variety of proteins to the peptidoglycan cell wall. The first sortase discovered, the Class A sortase from Staphylococcus aureus (saSrtA), is widely used in sortase-mediated ligation (SML) or sortagging protein engineering applications. This is a versatile protein with numerous human health and disease implications; recent examples include the use of an engineered saSrtA to identify and disrupt amyloid-b aggregates in a neurodegeneration model and to construct multi-arm SARS-CoV-2 neutralizing nanobodies. SrtA is also a potential antibiotic target, as it is an essential protein in these pathogenic bacteria. However, recent work from ourselves and others reveals that the stringent target selectivity of saSrtA is not shared amongst other Class A sortases, e.g., those from the Streptococcus or Listeria genera. Furthermore, other classes of sortases are extremely understudied with respect to SrtA proteins, and could provide useful tools for SML applications. Class B sortases (SrtBs), for example, recognize a different motif sequence, which may be exploited to expand the capabilities of SML. Finally, early evidence suggests that the transmembrane region of saSrtA may play an important role in the catalytic efficiency of this enzyme, but this has not been thoroughly investigated. The overall objectives in this application are to (i) dissect selectivity and activity determinants in sortase enzymes from multiple classes, including the transmembrane regions of sortases and/or their substrates, and (ii) use bacterial display to create a high throughput assay to measure sortase selectivity, which would be applicable broadly to all classes of sortases. The central hypothesis is that we can use protein biochemistry, chemical biology, and computational biology to better understand these enzymes. The rationale of this project is that a deep understanding of the general characteristics of sortase biology can be leveraged to develop new SML strategies for protein engineering, and assist in targeted antibiotic design to improve human health. The central hypothesis will be tested by pursuing three specific aims: 1) dissect selectivity and activity determinants in SrtA enzymes by characterizing differences in protein dynamics and elucidating extended motif preferences, as well as develop a high throughout bacterial display assay for sortase selectivity, 2) advance a molecular understanding of SrtB activities in order to improve reaction rates, and 3) characterize the role of the transmembrane regions of sortase and its substrates in catalysis. All aims will utilize protein biochemistry, structural biology, chemical biology, and computational biology. The research proposed in this application is innovative in that we will be developing new assays and applying techniques not previously used with sortase enzymes, continuing our work as a productive collaborative team to investigate aspects of sortase enzymes not currently well understood. The proposed research is significant because our work will inform next-generation SML methods and can be applied to the development of new antibiotics for Gram-positive bacterial pathogens.

Project Narrative. The proposed research is relevant to public health because it defines the selectivity and activity determinants of bacterial sortases, a therapeutic target for antibiotic development against Gram-positive bacteria (e.g., Staphylococcus aureus) and a widely utilized tool in protein engineering. Within these applications, the majority of sortase-mediated ligation efforts utilize a single, highly specific sortase enzyme (from S. aureus), whereas observations for the broader protein family suggest a spectrum of target specificity. A greater understanding of the mechanistic basis of bacterial sortase selectivity and activity can dramatically expand next-generation protein engineering efforts; therefore, the proposed research is directly in line with the mission of the National Institute of General Medical Sciences to “support basic research that… lays the foundation for advances in disease treatment.”