Covalent modification of matrix metalloproteinases by a photoaffinity probe: influence of nucleophilicity and flexibility of the residue in position 241.
A photoaffinity probe, developed for the specific labeling of matrix metalloproteinase (MMP) active sites, was recently shown to covalently modify a single residue in human MMP-12, namely, Lys(241), by reacting selectively with the side chain epsilon-amino group of that residue. The residue in position 241 of MMPs is not conserved; thus, variability in this position may be responsible for the dispersion in cross-linking yield observed between MMPs when labeled by this photoaffinity probe. By studying the pH dependence of the labeling properties of this probe toward different MMPs (MMP-12, MMP-3, MMP-9, and various mutants of human MMP-12) and identifying the site of covalent modification of MMP-3 by this probe, our new data demonstrated that the nucleophilicity of the residue in position 241 plays a key role in determining the cross-linking yield of MMP modification by the probe. However, these studies also reveal that subtle additional structural parameters, including local conformation and flexibility, of the residue in position 241 should also be taken into consideration, a property adding a further degree of complexity in our understanding of the photolabeling probe reactivity and in designing optimal photoaffinity probes for performing functional proteomic studies of zinc proteinases like MMPs.
Dabert-Gay
Anne-Sophie
AS
CEA, iBiTecS, Service d'Ingenierie Moleculaire des Proteines (SIMOPRO), CE-Saclay Gif/Yvette, F-91191, France.
Czarny
B
B
Lajeunesse
E
E
Thai
R
R
Nagase
H
H
Dive
V
V
D000818:Animals; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism; D006639:Histidine; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020782:Matrix Metalloproteinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009154:Mutation; D019920:Photoaffinity Labels / Q000737:chemistry* / Q000378:metabolism
D003432:Cross-Linking Reagents; D019920:Photoaffinity Labels; D006639:Histidine; D020782:Matrix Metalloproteinases