Hydrogen-Deuterium Exchange and Hydroxyl Radical Footprinting for Mapping Hydrophobic Interactions of Human Bromodomain
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J. Am. Soc. Mass Spectrom. (2019) DOI: 10.1007/s13361-019-02316-1
RESEARCH ARTICLE
Hydrogen-Deuterium Exchange and Hydroxyl Radical Footprinting for Mapping Hydrophobic Interactions of Human Bromodomain with a Small Molecule Inhibitor Ke Sherry Li,1 Elizabeth T. Schaper Bergman,1 Brett R. Beno,2 Richard Y.-C. Huang,3 Ekaterina Deyanova,3 Guodong Chen,3 Michael L. Gross1 1
Department of Chemistry, Washington University, St. Louis, MO 63130, USA Molecular Structure & Design, Research and Development, Bristol-Myers Squibb, Princeton, NJ 08540, USA 3 Pharmaceutical Candidate Optimization, Research and Development, Bristol-Myers Squibb, Princeton, NJ 08540, USA 2
Abstract. Mass spectrometry (MS)–based protein footprinting, a valuable structural tool in map76-83 N140 ping protein-ligand interaction, has been extenKHQFAWPF P82 sively applied to protein-protein complexes, W81 showing success in mapping large interfaces. L92 Here, we utilized an integrated footprinting strategy incorporating both hydrogen-deuterium exchange (HDX) and hydroxyl radical footprinting modification of reporter (i.e., fast photochemical oxidation of proteins (FPOP)) for molecular-level characterization of the interaction of human bromodomain-containing protein 4 (BRD4) with a hydrophobic benzodiazepine inhibitor. HDX does not provide strong evidence for the location of the binding interface, possibly because the shielding of solvent by the small molecule is not large. Instead, HDX suggests that BRD4 appears to be stabilized by showing a modest decrease in dynamics caused by binding. In contrast, FPOP points to a critical binding region in the hydrophobic cavity, also identified by crystallography, and, therefore, exhibits higher sensitivity than HDX in mapping the interaction of BRD4 with compound 1. In the absence or under low concentrations of the radical scavenger, FPOP modifications on Met residues show significant differences that reflect the minor change in protein conformation. This problem can be avoided by using a sufficient amount of proper scavenger, as suggested by the FPOP kinetics directed by a dosimeter of the hydroxyl radical. Keywords: Protein, Small-molecule binding, Mass spectrometry, Fast photochemical oxidation of proteins (FPOP), Hydrogen-deuterium exchange, Benzyl (1-methyl-6-phenyl-4Hbenzo[f][1,2,4]triazolo[4,3a][1,4]diazepin-4-yl)carbamate, Human bromodomain-containing protein 4 (BRD4) modification of hBRD4
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Received: 31 March 2019/Revised: 24 July 2019/Accepted: 6 August 2019
Introduction
D
espite the rapid development of protein-based therapeutic biologics, small molecules are still remarkably dominant in development pipelines of the biopharmaceutical industry,
Electronic supplementary material The online version of this article (https:// doi.org/10.1007/s13361-019-02316-1) contains supplementary material, which is available to authorized users. Correspondence to: Michael Gross; e-mail: [email protected]
making up over 90% of the therapeutics in use [1]. Smallmolecule drugs have relatively stable chemical pr
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