Use of hydrogen/deuterium exchange mass spectrometry and mutagenesis as a tool to identify the binding region of inhibitors targeting the human mitotic kinesin Eg5

Brier, S., Lemaire, D., DeBonis, S., Kozielski, F. and Forest, E. (2006) Use of hydrogen/deuterium exchange mass spectrometry and mutagenesis as a tool to identify the binding region of inhibitors targeting the human mitotic kinesin Eg5. Rapid Communications in Mass Spectrometry, 20(3), pp. 456-462. (doi:10.1002/rcm.2329)

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Publisher's URL: http://dx.doi.org/10.1002/rcm.2329

Abstract

An experimental procedure associating both hydrogen/deuterium exchange mass spectrometry (H/D-MS) and mutagenesis was developed to identify the protein-binding region of small inhibitors targeting the motor domain of the human mitotic kinesin Eg5. All the tested inhibitors decrease the deuterium incorporation rate of the same peptides corresponding to the following secondary structure elements: loop L5/helix alpha 2 (region Tyr125-Glu145) and strand beta 5/helix alpha 3 (region Ile202-Leu227). Replacement of these two regions by the equivalent ones from N. crassa conventional kinesin heavy chain completely abolishes the modification of the deuterium incorporation rate by the inhibitors as well as their effects on the basal ATPase activity. The six tested inhibitors thus share a common binding site on Eg5. The strategy reported here allows the regions of a protein involved in ligand binding to be rapidly pinpointed and can be applied to other proteins and used as a general in vitro screening procedure to identify compounds targeting specific binding regions

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Kozielski, Professor Frank
Authors: Brier, S., Lemaire, D., DeBonis, S., Kozielski, F., and Forest, E.
College/School:College of Medical Veterinary and Life Sciences > Institute of Cancer Sciences
Journal Name:Rapid Communications in Mass Spectrometry
ISSN:0951-4198

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