Molecular Determinants of Matrix Metalloproteinase-12 Covalent Modification by a Photoaffinity Probe

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 ⑀ 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 ⑀ 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.

A Pan Photoaffinity Probe for Detecting Active Forms of Matrix Metalloproteinases

“…Probe 1 was shown to label hMMP-12 by covalent modification of the side chain e amino group of Lys241. [19,20] This result is in agreement with the well-known efficiency of the nitrene intermediate, resulting from the photoactivation of the azide, to react with nucleophiles. [19][20][21] This preference explains why probe 1 mostly targets only two MMPs-MMP-12 and MMP-3, containing a Lys and a His residue, respectively, at position 241-with high efficiency and other MMPs less so, because position 241 in highly variable in MMPs.…”

“…[19,20] This result is in agreement with the well-known efficiency of the nitrene intermediate, resulting from the photoactivation of the azide, to react with nucleophiles. [19][20][21] This preference explains why probe 1 mostly targets only two MMPs-MMP-12 and MMP-3, containing a Lys and a His residue, respectively, at position 241-with high efficiency and other MMPs less so, because position 241 in highly variable in MMPs. A high level of sensitivity for detecting MMPs would be highly desirable, however, because difficulty in detecting active forms of MMPs has been proposed to be a consequence of the low levels of expression of MMP active forms, well below the current limits of ABP detection.…”

“…The identification of the residue modified by probe 1 in MMP-12 and MMP-3 was successfully achieved by microsequencing and mass spectrometry after isolation of the covalent adducts and their digestion by proteases. [19,20] Despite several attempts by similar approaches, we were unable to identify the residues in MMP-9 and MMP-12 modified by probe 2. To gain indirect insight, we tested probe 2 with a series of MMP-12 mutants in which the lysine in position 241 was replaced by the different residues (His, Ala, Arg, Thr, Gln) present at position 241 in the other MMPs selected for this study.…”

“…This study was motivated by the differences in reactivity expected between azide versus trifluorodiazirine groups in modifying the active sites in MMP residues. [17] In this respect, it is of interest to note that whereas probe 1 was shown mostly to crosslink MMP-3 and MMP-12, [19,20] by targeting the histidine and lysine side chains, respectively, present at position 241 in these MMPs, in contrast these two MMPs are the least modified by probe 2.…”

Insights from Selective Non-phosphinic Inhibitors of MMP-12 Tailored to Fit with an S1′ Loop Canonical Conformation

“…NEP inhibition assays were performed using Mca-Arg-Pro-Pro-Gly-Phe-Ser-ProDpa-OH from Enzo Life Sciences, as substrate (5 mM, K m ϭ 2 mM) and human NEP (0.5 nM) from R & D Systems. MMPs inhibition assays were performed using Mca-ProLeu-Gly-Leu-Dpa-Ala-Arg-NH 2 , as substrate (13 mM, K m ϭ 8.5 mM) and human MMPs (nanomolar range concentration) from R & D Systems (15), except for human MMP-12 that was produced and purified as described previously (35). The substrate and enzyme concentrations for the experiments were chosen so as to remain well below 10% of substrate utilization and to observe the initial rates.…”