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Directed evolution provides insight into conformational substrate sampling by SrtA. PLoS One 2017;12(8):e0184271

Date

09/01/2017

Pubmed ID

28859178

Pubmed Central ID

PMC5578623

DOI

10.1371/journal.pone.0184271

Scopus ID

2-s2.0-85028825732 (requires institutional sign-in at Scopus site)   8 Citations

Abstract

The Sortase family of transpeptidases are found in numerous gram-positive bacteria and involved in divergent physiological processes including anchoring of surface proteins to the cell wall as well as pili assembly. As essential proteins, sortase enzymes have been the focus of considerable interest for the development of novel anti-microbials, however, more recently their function as unique transpeptidases has been exploited for the synthesis of novel bio-conjugates. Yet, for synthetic purposes, SrtA-mediated conjugation suffers from the enzyme's inherently poor catalytic efficiency. Therefore, to identify SrtA variants with improved catalytic efficiency, we used directed evolution to select a catalytically enhanced SrtA enzyme. An analysis of improved SrtA variants in the context of sequence conservation, NMR and x-ray crystal structures, and kinetic data suggests a novel mechanism for catalysis involving large conformational changes that delivers substrate to the active site pocket. Indeed, using DEER-EPR spectroscopy, we reveal that upon substrate binding, SrtA undergoes a large scissors-like conformational change that simultaneously translates the sort-tag substrate to the active site in addition to repositioning key catalytic residues for esterification. A better understanding of Sortase dynamics will significantly enhance future engineering and drug discovery efforts.

Author List

Suliman M, Santosh V, Seegar TCM, Dalton AC, Schultz KM, Klug CS, Barton WA

Authors

Candice S. Klug PhD Professor in the Biophysics department at Medical College of Wisconsin
Kathryn M. Schultz Research Scientist I in the Biophysics department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Aminoacyltransferases
Bacterial Proteins
Catalysis
Catalytic Domain
Crystallography, X-Ray
Cysteine Endopeptidases
Directed Molecular Evolution
Electron Spin Resonance Spectroscopy
Kinetics
Protein Conformation
Staphylococcus aureus
Substrate Specificity