title: Molecular determinants of matrix metalloproteinase-12 covalent modification by a photoaffinity probe: insights into activity-based probe development and conformational variability of matrix metalloproteinases.
abstract: Mass spectroscopy, microsequencing, and site-directed mutagenesis studies have been performed to identify in human matrix metalloelastase (hMMP-12) residues covalently modified by a photoaffinity probe, previously shown to be able to covalently label specifically the active site of matrix metalloproteinases (MMPs). Results obtained led us to conclude that photoactivation of this probe in complex with hMMP-12 affects a single residue in human MMP-12, Lys(241), through covalent modification of its side chain epsilon NH(2) group. Because x-ray and NMR studies of hMMP-12 indicate that Lys(241) side chain is highly flexible, our data reveal the existence of particular Lys(241) side-chain conformation in which the epsilon NH(2) group points toward the photolabile group of the probe, an event explaining the high levels of cross-linking yield between hMMP-12 and the probe. Lys(241) is not conserved in MMPs, thus differences in cross-linking yields observed with this probe between MMP members may be linked to the residue variability observed at position 241 in this family.
authors.affiliation: Service d'Ingénierie Moléculaire de Protéines, CE-Saclay, Gif/Yvette 91191, Cedex, France.
authors.forename: Anne-Sophie
authors.initials: AS
authors.lastname: Dabert-Gay
authors.forename: Bertrand
authors.initials: B
authors.lastname: Czarny
authors.forename: Laurent
authors.initials: L
authors.lastname: Devel
authors.forename: Fabrice
authors.initials: F
authors.lastname: Beau
authors.forename: Evelyne
authors.initials: E
authors.lastname: Lajeunesse
authors.forename: Sarah
authors.initials: S
authors.lastname: Bregant
authors.forename: Robert
authors.initials: R
authors.lastname: Thai
authors.forename: Athanasios
authors.initials: A
authors.lastname: Yiotakis
authors.forename: Vincent
authors.initials: V
authors.lastname: Dive
mesh_terms: D020134:Catalytic Domain / Q000502:physiology; D006801:Humans; D008239:Lysine / Q000737:chemistry* / Q000235:genetics; D013058:Mass Spectrometry; D053508:Matrix Metalloproteinase 12 / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D019920:Photoaffinity Labels / Q000737:chemistry*
chemical_list: D019920:Photoaffinity Labels; C507122:MMP12 protein, human; D053508:Matrix Metalloproteinase 12; D008239:Lysine
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