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Title: The “Lid” in the Streptococcus pneumoniae SrtC1 Sortase Adopts a Rigid Structure that Regulates Substrate Access to the Active Site

Many species of Gram-positive bacteria use sortase enzymes to assemble long, proteinaceous pili structures that project from the cell surface to mediate microbial adhesion. Sortases construct highly stable structures by catalyzing a transpeptidation reaction that covalently links pilin subunits together via isopeptide bonds. Most Gram-positive pili are assembled by class C sortases that contain a “lid”, a structurally unique N-terminal extension that occludes the active site. It has been hypothesized that the “lid” in many sortases is mobile and thus capable of readily being displaced from the enzyme to facilitate substrate binding. Here in this work, we show using NMR dynamics measurements, in vitro assays, and molecular dynamics simulations that the lid in the class C sortase from Streptococcus pneumoniae (SrtC1) adopts a rigid conformation in solution that is devoid of large magnitude conformational excursions that occur on mechanistically relevant time scales. Additionally, we show that point mutations in the lid induce dynamic behavior that correlates with increased hydrolytic activity and sorting signal substrate access to the active site cysteine residue. In conclusion, these results suggest that the lid of the S. pneumoniae SrtC1 enzyme has a negative regulatory function and imply that a significant energetic barrier must be surmountedmore » by currently unidentified factors to dislodge it from the active site to initiate pilus biogenesis.« less
Authors:
 [1] ;  [2] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [2]
  1. Univ. of California, Los Angeles, CA (United States). Department of Chemistry and Biochemistry and the UCLA-DOE Institute of Genomics and Proteomics
  2. Illinois Institute of Technology, Chicago, IL (United States). Department of Physics and Center for Molecular Study of Condensed Soft Matter
Publication Date:
Grant/Contract Number:
FC02-02ER63421
Type:
Accepted Manuscript
Journal Name:
Journal of Physical Chemistry. B, Condensed Matter, Materials, Surfaces, Interfaces and Biophysical Chemistry
Additional Journal Information:
Journal Volume: 120; Journal Issue: 33; Journal ID: ISSN 1520-6106
Publisher:
American Chemical Society
Research Org:
Univ. of California, Los Angeles, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
OSTI Identifier:
1466769

Jacobitz, Alex W., Naziga, Emmanuel B., Yi, Sung Wook, McConnell, Scott A., Peterson, Robert, Jung, Michael E., Clubb, Robert T., and Wereszczynski, Jeff. The “Lid” in the Streptococcus pneumoniae SrtC1 Sortase Adopts a Rigid Structure that Regulates Substrate Access to the Active Site. United States: N. p., Web. doi:10.1021/acs.jpcb.6b01930.
Jacobitz, Alex W., Naziga, Emmanuel B., Yi, Sung Wook, McConnell, Scott A., Peterson, Robert, Jung, Michael E., Clubb, Robert T., & Wereszczynski, Jeff. The “Lid” in the Streptococcus pneumoniae SrtC1 Sortase Adopts a Rigid Structure that Regulates Substrate Access to the Active Site. United States. doi:10.1021/acs.jpcb.6b01930.
Jacobitz, Alex W., Naziga, Emmanuel B., Yi, Sung Wook, McConnell, Scott A., Peterson, Robert, Jung, Michael E., Clubb, Robert T., and Wereszczynski, Jeff. 2016. "The “Lid” in the Streptococcus pneumoniae SrtC1 Sortase Adopts a Rigid Structure that Regulates Substrate Access to the Active Site". United States. doi:10.1021/acs.jpcb.6b01930. https://www.osti.gov/servlets/purl/1466769.
@article{osti_1466769,
title = {The “Lid” in the Streptococcus pneumoniae SrtC1 Sortase Adopts a Rigid Structure that Regulates Substrate Access to the Active Site},
author = {Jacobitz, Alex W. and Naziga, Emmanuel B. and Yi, Sung Wook and McConnell, Scott A. and Peterson, Robert and Jung, Michael E. and Clubb, Robert T. and Wereszczynski, Jeff},
abstractNote = {Many species of Gram-positive bacteria use sortase enzymes to assemble long, proteinaceous pili structures that project from the cell surface to mediate microbial adhesion. Sortases construct highly stable structures by catalyzing a transpeptidation reaction that covalently links pilin subunits together via isopeptide bonds. Most Gram-positive pili are assembled by class C sortases that contain a “lid”, a structurally unique N-terminal extension that occludes the active site. It has been hypothesized that the “lid” in many sortases is mobile and thus capable of readily being displaced from the enzyme to facilitate substrate binding. Here in this work, we show using NMR dynamics measurements, in vitro assays, and molecular dynamics simulations that the lid in the class C sortase from Streptococcus pneumoniae (SrtC1) adopts a rigid conformation in solution that is devoid of large magnitude conformational excursions that occur on mechanistically relevant time scales. Additionally, we show that point mutations in the lid induce dynamic behavior that correlates with increased hydrolytic activity and sorting signal substrate access to the active site cysteine residue. In conclusion, these results suggest that the lid of the S. pneumoniae SrtC1 enzyme has a negative regulatory function and imply that a significant energetic barrier must be surmounted by currently unidentified factors to dislodge it from the active site to initiate pilus biogenesis.},
doi = {10.1021/acs.jpcb.6b01930},
journal = {Journal of Physical Chemistry. B, Condensed Matter, Materials, Surfaces, Interfaces and Biophysical Chemistry},
number = 33,
volume = 120,
place = {United States},
year = {2016},
month = {4}
}