Molecular basis for the activation of a catalytic asparagine residue in a self-cleaving bacterial autotransporter

Travis J. Barnard, James Gumbart, Janine H. Peterson, Nicholas Noinaj, Nicole C. Easley, Nathalie Dautin, Adam J. Kuszak, Emad Tajkhorshid, Harris D. Bernstein, Susan K. Buchanan

Research output: Contribution to journalArticle

Abstract

Autotransporters are secreted proteins produced by pathogenic Gram-negative bacteria. They consist of a membrane-embedded β-domain and an extracellular passenger domain that is sometimes cleaved and released from the cell surface. We solved the structures of three noncleavable mutants of the autotransporter EspP to examine how it promotes asparagine cyclization to cleave its passenger. We found that cyclization is facilitated by multiple factors. The active-site asparagine is sterically constrained to conformations favorable for cyclization, while electrostatic interactions correctly orient the carboxamide group for nucleophilic attack. During molecular dynamics simulations, water molecules were observed to enter the active site and to form hydrogen bonds favorable for increasing the nucleophilicity of the active-site asparagine. When the activated asparagine attacks its main-chain carbonyl carbon, the resulting oxyanion is stabilized by a protonated glutamate. Upon cleavage, this proton could be transferred to the leaving amine group, helping overcome a significant energy barrier. Together, these findings provide insight into factors important for asparagine cyclization, a mechanism broadly used for protein cleavage.

Original languageEnglish (US)
Pages (from-to)128-142
Number of pages15
JournalJournal of Molecular Biology
Volume415
Issue number1
DOIs
StatePublished - Jan 6 2012

Fingerprint

Asparagine
Cyclization
Catalytic Domain
Molecular Dynamics Simulation
Gram-Negative Bacteria
Static Electricity
Amines
Protons
Glutamic Acid
Hydrogen
Proteins
Carbon
Type V Secretion Systems
Membranes
Water

Keywords

  • EspP
  • asparagine cyclization
  • autocleavage
  • crystal structure
  • outer membrane protein

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

Cite this

Barnard, T. J., Gumbart, J., Peterson, J. H., Noinaj, N., Easley, N. C., Dautin, N., ... Buchanan, S. K. (2012). Molecular basis for the activation of a catalytic asparagine residue in a self-cleaving bacterial autotransporter. Journal of Molecular Biology, 415(1), 128-142. https://doi.org/10.1016/j.jmb.2011.10.049

Molecular basis for the activation of a catalytic asparagine residue in a self-cleaving bacterial autotransporter. / Barnard, Travis J.; Gumbart, James; Peterson, Janine H.; Noinaj, Nicholas; Easley, Nicole C.; Dautin, Nathalie; Kuszak, Adam J.; Tajkhorshid, Emad; Bernstein, Harris D.; Buchanan, Susan K.

In: Journal of Molecular Biology, Vol. 415, No. 1, 06.01.2012, p. 128-142.

Research output: Contribution to journalArticle

Barnard, TJ, Gumbart, J, Peterson, JH, Noinaj, N, Easley, NC, Dautin, N, Kuszak, AJ, Tajkhorshid, E, Bernstein, HD & Buchanan, SK 2012, 'Molecular basis for the activation of a catalytic asparagine residue in a self-cleaving bacterial autotransporter', Journal of Molecular Biology, vol. 415, no. 1, pp. 128-142. https://doi.org/10.1016/j.jmb.2011.10.049
Barnard, Travis J. ; Gumbart, James ; Peterson, Janine H. ; Noinaj, Nicholas ; Easley, Nicole C. ; Dautin, Nathalie ; Kuszak, Adam J. ; Tajkhorshid, Emad ; Bernstein, Harris D. ; Buchanan, Susan K. / Molecular basis for the activation of a catalytic asparagine residue in a self-cleaving bacterial autotransporter. In: Journal of Molecular Biology. 2012 ; Vol. 415, No. 1. pp. 128-142.
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