Structural and biochemical analyses of selectivity determinants in chimeric Streptococcus Class A sortase enzymes

Gao, Melody; Johnson, D. Alex; Piper, Isabel M.; Kodama, Hanna M.; Svendsen, Justin E.; Tahti, Elise; Longshore‐Neate, Frederick; Vogel, Brandon; Antos, John M.; Amacher, Jeanine F.

doi:10.1002/pro.4266

Title: Structural and biochemical analyses of selectivity determinants in chimeric Streptococcus Class A sortase enzymes

Journal Article · 03 January 2022 · Protein Science

DOI: https://doi.org/10.1002/pro.4266 · OSTI ID:1838066

^[1];

^[1]; Tahti, Elise ^[1]; Longshore‐Neate, Frederick ^[1];

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^[1]

Department of Chemistry Western Washington University Bellingham Washington USA

Abstract Sequence variation in related proteins is an important characteristic that modulates activity and selectivity. An example of a protein family with a large degree of sequence variation is that of bacterial sortases, which are cysteine transpeptidases on the surface of gram‐positive bacteria. Class A sortases are responsible for attachment of diverse proteins to the cell wall to facilitate environmental adaption and interaction. These enzymes are also used in protein engineering applications for sortase‐mediated ligations (SML) or sortagging of protein targets. We previously investigated SrtA from Streptococcus pneumoniae , identifying a number of putative β7–β8 loop‐mediated interactions that affected in vitro enzyme function. We identified residues that contributed to the ability of S. pneumoniae SrtA to recognize several amino acids at the P1′ position of the substrate motif, underlined in LPXT G , in contrast to the strict P1′ Gly recognition of SrtA from Staphylococcus aureus . However, motivated by the lack of a structural model for the active, monomeric form of S. pneumoniae SrtA, here, we expanded our studies to other Streptococcus SrtA proteins. We solved the first monomeric structure of S. agalactiae SrtA which includes the C‐terminus, and three others of β7–β8 loop chimeras from S. pyogenes and S. agalactiae SrtA. These structures and accompanying biochemical data support our previously identified β7–β8 loop‐mediated interactions and provide additional insight into their role in Class A sortase substrate selectivity. A greater understanding of individual SrtA sequence and structural determinants of target selectivity may also facilitate the design or discovery of improved sortagging tools.

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Sponsoring Organization:: USDOE

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1838066

Journal Information:: Protein Science, Journal Name: Protein Science Journal Issue: 3 Vol. 31; ISSN 0961-8368

Publisher:: Wiley Blackwell (John Wiley & Sons)Copyright Statement

Country of Publication:: United Kingdom

Language:: English

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