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Structures of Streptococcus pyogenes class A sortase in complex with substrate and product mimics provide key details of target recognition

Johnson, D. Alex and Piper, Isabel M. and Vogel, Brandon A. and Jackson, Sophie N. and Svendsen, Justin E. and Kodama, Hanna M. and Lee, Darren E. and Lindblom, Katy M. and McCarty, James and Antos, John M. and Amacher, Jeanine F. (2022) Structures of Streptococcus pyogenes class A sortase in complex with substrate and product mimics provide key details of target recognition. Journal of Biological Chemistry, 298 (10). Art. No. 102446. ISSN 0021-9258. doi:10.1016/j.jbc.2022.102446.

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The cell wall is a critical extracellular barrier for bacteria and many other organisms. In bacteria, this structural layer consists of peptidoglycan, which maintains cell shape and structural integrity and provides a scaffold for displaying various protein factors. To attach proteins to the cell wall, Gram-positive bacteria utilize sortase enzymes, which are cysteine transpeptidases that recognize and cleave a specific sorting signal, followed by ligation of the sorting signal–containing protein to the peptidoglycan precursor lipid II (LII). This mechanism is the subject of considerable interest as a target for therapeutic intervention and as a tool for protein engineering, where sortases have enabled sortase-mediated ligation or sortagging strategies. Despite these uses, there remains an incomplete understanding of the stereochemistry of substrate recognition and ligation product formation. Here, we solved the first structures of sortase A from Streptococcus pyogenes bound to two substrate sequences, LPATA and LPATS. In addition, we synthesized a mimetic of the product of sortase-mediated ligation involving LII (LPAT-LII) and solved the complex structure in two ligand conformations. These structures were further used as the basis for molecular dynamics simulations to probe sortase A-ligand dynamics and to construct a model of the acyl–enzyme intermediate, thus providing a structural view of multiple key states in the catalytic mechanism. Overall, this structural information provides new insights into the recognition of the sortase substrate motif and LII ligation partner and will support the continued development of sortases for protein engineering applications.

Item Type:Article
Related URLs:
URLURL TypeDescription
Piper, Isabel M.0000-0001-9347-2645
Vogel, Brandon A.0000-0003-2563-0677
Jackson, Sophie N.0000-0001-9133-6906
Svendsen, Justin E.0000-0002-5873-5339
Kodama, Hanna M.0000-0003-2872-2948
Lee, Darren E.0000-0002-5053-863X
McCarty, James0000-0002-3838-6004
Antos, John M.0000-0002-2555-1875
Amacher, Jeanine F.0000-0002-3646-7521
Additional Information:We thank the other members of the Amacher and Antos labs for helpful discussions and assistance. We also thank the Berkeley Center for Structural Biology for being an excellent resource for the crystallography community as well as the incredible staff at the PDB for their assistance in defining the isopeptide-linked LPAT-LII ligand in the spySrtA complex structures. The Berkeley Center for Structural Biology is supported in part by the National Institutes of Health, National Institute of General Medical Sciences, and the Howard Hughes Medical Institute. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the US Department of Energy under contract no. DE-AC02-05CH11231. We thank Sarina Kiesser for assistance and expertise with the QTOF system. This work used the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by the National Science Foundation (NSF) grant number ACI-1548562. This work used the SDSC Expanse through allocation TG-BIO210091.
Funding AgencyGrant Number
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Department of Energy (DOE)DE-AC02-05CH11231
Issue or Number:10
Record Number:CaltechAUTHORS:20221130-645685700.5
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:118173
Deposited By: Research Services Depository
Deposited On:23 Dec 2022 19:06
Last Modified:23 Dec 2022 19:06

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