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Experimental Validation of the Predicted Binding Site of Escherichia coli K1 Outer Membrane Protein A to Human Brain Microvascular Endothelial Cells: Identification of Critical Mutations That Prevent E. coli Meningitis

Pascal, Tod A. and Abrol, Ravinder and Mittal, Rahul and Wang, Ying and Prasadarao, Nemani V. and Goddard, William A., III (2010) Experimental Validation of the Predicted Binding Site of Escherichia coli K1 Outer Membrane Protein A to Human Brain Microvascular Endothelial Cells: Identification of Critical Mutations That Prevent E. coli Meningitis. Journal of Biological Chemistry, 285 (48). pp. 37753-37761. ISSN 0021-9258. PMCID PMC2988380. http://resolver.caltech.edu/CaltechAUTHORS:20110105-080744616

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Abstract

Escherichia coli K1, the most common cause of meningitis in neonates, has been shown to interact with GlcNAc1–4GlcNAc epitopes of Ecgp96 on human brain microvascular endothelial cells (HBMECs) via OmpA (outer membrane protein A). However, the precise domains of extracellular loops of OmpA interacting with the chitobiose epitopes have not been elucidated. We report the loop-barrel model of these OmpA interactions with the carbohydrate moieties of Ecgp96 predicted from molecular modeling. To test this model experimentally, we generated E. coli K1 strains expressing OmpA with mutations of residues predicted to be critical for interaction with the HBMEC and tested E. coli invasion efficiency. For these same mutations, we predicted the interaction free energies (including explicit calculation of the entropy) from molecular dynamics (MD), finding excellent correlation (R^2 = 90%) with experimental invasion efficiency. Particularly important is that mutating specific residues in loops 1, 2, and 4 to alanines resulted in significant inhibition of E. coli K1 invasion in HBMECs, which is consistent with the complete lack of binding found in the MD simulations for these two cases. These studies suggest that inhibition of the interactions of these residues of Loop 1, 2, and 4 with Ecgp96 could provide a therapeutic strategy to prevent neonatal meningitis due to E. coli K1.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1074/jbc.M110.122804 DOIArticle
http://www.jbc.org/content/285/48/37753.abstractPublisherArticle
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2988380/PubMed CentralArticle
ORCID:
AuthorORCID
Pascal, Tod A.0000-0003-2096-1143
Abrol, Ravinder0000-0001-7333-6793
Goddard, William A., III0000-0003-0097-5716
Additional Information:© 2010 American Society for Biochemistry and Molecular Biology, Inc. Received March 12, 2010. Revision received September 5, 2010. First Published on September 17, 2010. This work was supported, in whole or in part, by National Institutes of Health Grants AI-040567 (to N. V. P.) and R01NS071112. This work was also supported by grants to Caltech from the National Science Foundation (CMMI-072870 and CTS-0608889). We thank Schrödinger, Inc. for access to the Phase Program for pharmacophore modeling. The computational facilities were provided by Army Research Office-Defense University Research Instrumentation Program and Office of Naval Research-Defense University Research Instrumentation Program.
Funders:
Funding AgencyGrant Number
NIHAI-040567
NIHRO1 NS071112
NSFCMMI-072870
NSFCTS-0608889
Department of Energy (DOE)UNSPECIFIED
Army Research Office (ARO)UNSPECIFIED
Office of Naval Research (ONR)UNSPECIFIED
Subject Keywords: * Computer Modeling * Heat Shock Protein * Molecular Dynamics * Mutagenesis Mechanisms * Protein-Protein Interactions * Bacterial Meningitis
Other Numbering System:
Other Numbering System NameOther Numbering System ID
WAG0895
PubMed Central ID:PMC2988380
Record Number:CaltechAUTHORS:20110105-080744616
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20110105-080744616
Official Citation:Pascal, T. A., R. Abrol, et al. (2010). "Experimental Validation of the Predicted Binding Site of Escherichia coli K1 Outer Membrane Protein A to Human Brain Microvascular Endothelial Cells." Journal of Biological Chemistry 285(48): 37753-37761.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:21590
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:25 Jan 2011 22:09
Last Modified:04 Jan 2019 16:42

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