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 http://resolver.caltech.edu/CaltechAUTHORS:20110105-080744616
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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.
|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.|
|Subject Keywords:||* Computer Modeling * Heat Shock Protein * Molecular Dynamics * Mutagenesis Mechanisms * Protein-Protein Interactions * Bacterial Meningitis|
|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.|
|Deposited By:||Tony Diaz|
|Deposited On:||25 Jan 2011 22:09|
|Last Modified:||26 Dec 2012 12:48|
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