Rajagopalan Bhaskaran

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.

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.

NMR and dynamical simulated annealing studies on the solution conformation of urotensin II

We determined the structure in solution of the vaso-constrictor hormone urotensin II (dodecapeptide) using nuclear magnetic resonance spectroscopy. Complete assignment of all proton resonances has been achieved and the structural information has been obtained from the interproton distance measurements derived from the nuclear Overhauser enhancement data. A combination of distance geometry and dynamical simulated annealing techniques was used to calculate the structure in solution. Nine resultant structures with fewer distance constraint violations were selected that satisfy the experimental restraints very well. The conformation of the molecule in the cyclic hexapeptide segment (core region) is well-defined whereas the N-terminal segment is disordered. This result correlates very well with the earlier predictions about the biologically active and inactive roles played by the core and the N-terminal segment respectively.

Three-dimensional structure in solution of griseoviridin, a group A antibiotic

The solution conformation of griseoviridin, a broad spectrum antibiotic, has been determined by 1H-NMR in deuterated dimethylsulfoxide. The structural determination is based on experimental data of NOE constraints Five structures were obtained from restrained molecular dynamics calculations, by imposing (the condition for) a minimum violation of distance constraints. These structures satisfy well the experimental restraints, with small values of NOE violation and total energies. On comparison with its crystal structure, a good agreement is noted with a backbone root-mean-square deviation value of 0.084 nm. However, a small variation between the structures is observed at the aminodecanoic acid part of the molecule.