Mark O. Palmier

MMP-12 Catalytic Domain Recognizes Triple Helical Peptide Models of Collagen V with Exosites and High Activity

Matrix metalloproteinase (MMP)-12 (or metalloelastase) efficiently hydrolyzed the gelatinase-selective α1(V)436-447 fluorescent triple helical peptide (THP) when the substrate was submicromolar. The sequence of this THP was derived from collagen V, a component of collagen I fibrils. The hemopexin domains of MMP-12 and -9 each increased kcat/Km toward this substrate by decreasing Km, just as the hemopexin domain of MMP-1 enhances its triple helical peptidase activity. Non-fluorescent α1(V) THP subtly perturbed amide NMR chemical shifts of MMP-12 not only in the active site cleft but also at remote sites of the β-sheet and adjoining loops. The α1(V) THP protected MMP-12 from the NMR line broadening effects of Gd ·EDTA in the active site cleft and more dramatically in the V-B loop next to the primed subsites. Mutagenesis of the exosite in the V-B loop at Thr-205 and His-206 that vary among MMP sequences established that this site supports the high specific activity toward α1(V) fluorescent THP without affecting general MMP activity. Surprisingly the α1(V) THP also protected novel surfaces in the S-shaped metal-binding loop and β-strands III and V that together form a pocket on the remote side of the zinc binding site. The patterns of protection suggest bending of the triple helical peptide partly around the catalytic domain to reach novel exosites. Partial unwinding or underwinding of the triple helix could accompany this to facilitate its hydrolysis.

NMR and bioinformatics discovery of exosites that tune metalloelastase specificity for solubilized elastin and collagen triple helices

The catalytic domain of metalloelastase (matrix metalloproteinase-12 or MMP-12) is unique among MMPs in exerting high proteolytic activity upon fibrils that resist hydrolysis, especially elastin from lungs afflicted with chronic obstructive pulmonary disease or arteries with aneurysms. How does the MMP-12 catalytic domain achieve this specificity? NMR interface mapping suggests that α-elastin species cover the primed subsites, a strip across the β-sheet from β-strand IV to the II–III loop, and a broad bowl from helix A to helix C. The many contacts may account for the comparatively high affinity, as well as embedding of MMP-12 in damaged elastin fibrils in vivo. We developed a strategy called BINDSIght, for bioinformatics and NMR discovery of specificity of interactions, to evaluate MMP-12 specificity without a structure of a complex. BINDSIght integration of the interface mapping with other ambiguous information from sequences guided choice mutations in binding regions nearer the active site. Single substitutions at each of ten locations impair specific activity toward solubilized elastin. Five of them impair release of peptides from intact elastin fibrils. Eight lesions also impair specific activity toward triple helices from collagen IV or V. Eight sites map to the “primed” side in the III–IV, V–B, and S1′ specificity loops. Two map to the “unprimed” side in the IV–V and B–C loops. The ten key residues circumscribe the catalytic cleft, form an exosite, and are distinctive features available for targeting by new diagnostics or therapeutics.

An examination of the proteolytic activity for bovine pregnancy-associated glycoproteins 2 and 12

Abstract The pregnancy-associated glycoproteins (PAGs) represent a complex group of putative aspartic peptidases expressed exclusively in the placentas of species in the Artiodactyla order. The ruminant PAGs segregate into two classes: the ‘ancient’ and ‘modern’ PAGs. Some of the modern PAGs possess alterations in the catalytic center that are predicted to preclude their ability to act as peptidases. The ancient ruminant PAGs in contrast are thought to be peptidases, although no proteolytic activity has been described for these members. The aim of the present study was to investigate (1) if the ancient bovine PAGs (PAG-2 and PAG-12) have proteolytic activity, and (2) if there are any differences in activity between these two closely related members. Recombinant bovine PAG-2 and PAG-12 were expressed in a baculovirus expression system and the purified proteins were analyzed for proteolytic activity against a synthetic fluorescent cathepsin D/E substrate. Both proteins exhibited proteolytic activity with acidic pH optima. The k cat/K m for bovine PAG-2 was 2.7×105 m -1 s-1 and for boPAG-12 it was 6.8×104 m -1 s-1. The enzymes were inhibited by pepstatin A with a K i of 0.56 and 7.5 nm for boPAG-2 and boPAG-12, respectively. This is the first report describing proteolytic activity in PAGs from ruminant ungulates.

Apparent tradeoff of higher activity in MMP-12 for enhanced stability and flexibility in MMP-3.

The greater activity of MMP-12 than MMP-3 toward substrates from protein fibrils has been quantified. Why is MMP-12 the more active protease? We looked for behaviors associated with the higher activity of MMP-12 than MMP-3, using nuclear magnetic resonance to monitor backbone dynamics and residue-specific stabilities of their catalytic domain. The proteolytic activities are likely to play important roles in inflammatory diseases of arteries, lungs, joints, and intestines. Nuclear magnetic resonance line broadening indicates that regions surrounding the active sites of both proteases sample conformational substates within milliseconds. The more extensive line broadening in MMP-3 suggests greater sampling of conformational substates, affecting the full length of helix B and beta-strand IV forming the active site, and more remote sites. This could suggest more excursions to functionally incompetent substates. MMP-3 also has enhanced subnanosecond fluctuations in helix A, in the beta-hairpin of strands IV and V, and before and including helix C. Hydrogen exchange protection in the EX2 regime suggests that MMP-3 possesses 2.8 kcal/mol higher folding stability than MMP-12(E219A). The beta-sheet of MMP-3 appears to be stabilized still more. The higher stability of MMP-3 relative to MMP-12 coincides with the formers considerably lower proteolytic activity. This relationship is consistent with the hypothesis that enzymes often trade stability for higher activity.

Inactivation of N-TIMP-1 by N-terminal acetylation when expressed in bacteria.

The high-affinity binding of tissue inhibitors of metalloproteinases (TIMPs) to matrix metalloproteinases (MMPs) is essential for regulation of the turnover of the extracellular matrix during development, wound healing, and progression of inflammatory diseases, such as cancer, atherosclerosis, and arthritis. Bacterially expressed N-terminal inhibitory domains of TIMPs (N-TIMPs) have been used extensively for biochemical and biophysical study of interactions with MMPs. Titration of N-TIMP-1 expressed in E. coli indicates, however, that only about 42% of the protein is active as an MMP inhibitor. The separation of inactive from fully active N-TIMP-1 has been achieved both by MMP affinity and by high-resolution cation exchange chromatography at an appropriate pH, based on a slight difference of charge. Purification by cation exchange chromatography with a Mono S column enriches the active portion of N-TIMP-1 to >95%, with K(i) of 1.5 nM for MMP-12. Mass spectra reveal that the inactive form differs from active N-TIMP-1 in being N-terminally acetylated, underscoring the importance of the free alpha-NH(2) of Cys1 for MMP inhibition. N(alpha)-acetylation of the CTCVPP sequence broadens the N-terminal sequence motifs reported to be susceptible to alpha-amino acetylation by E. coli N-acetyl transferases.

Solubilized elastin substrate for continuous fluorimetric assay of kinetics of elastases.

Elastolysis is central to progression of emphysema and aortic aneurysms. Characterization of steady-state enzyme kinetics of elastolysis is fettered by the insolubility of mature elastin and the polydispersity of solubilized elastin. We prepared a fluor-tagged, 100-kDa fraction (fEln-100) from commercial α-elastin. It is soluble, less heterogeneous in mass, cross-linked like mature elastin, and likely to retain the capacity of α-elastin to self-assemble. fEln-100 has introduced the ability to compare quantitatively the apparent k(cat) and K(m) of elastases. For example, metalloelastase (MMP-12) displays higher apparent affinity for fEln-100, while MMP-2 displays faster catalytic turnover.