The proteolysis of collagen triple-helical structure (collagenolysis) is a poorly understood yet critical physiological process. Presently, matrix metalloproteinase 1 (MMP-1) and collagen triple-helical peptide models have been utilized to characterize the events and calculate the energetics of collagenolysis via NMR spectroscopic analysis of 12 enzyme-substrate complexes. The triple-helix is bound initially by the MMP-1 hemopexin-like (HPX) domain via a four amino acid stretch (analogous to type I collagen residues 782–785). The triple-helix is then presented to the MMP-1 catalytic (CAT) domain in a distinct orientation. The HPX and CAT domains are rotated with respect to one another compared with the X-ray “closed” conformation of MMP-1. Back-rotation of the CAT and HPX domains to the X-ray closed conformation releases one chain out of the triple-helix, and this chain is properly positioned in the CAT domain active site for subsequent hydrolysis. The aforementioned steps provide a detailed, experimentally-derived, and energetically favorable collagenolytic mechanism, as well as significant insight into the roles of distinct domains in extracellular protease function.

The structures of the catalytic domain of matrix metalloproteinase 12 in the presence of acetohydroxamic acid and N-isobutyl-N-[4-methoxyphenylsulfonyl]glycyl hydroxamic acid have been solved by x-ray diffraction in the crystalline state at 1.0 and 1.3-Å resolution, respectively, and compared with the previously published x-ray structure at 1.2-Å resolution of the adduct with batimastat. The structure of the N-isobutyl-N-[4-methoxyphenylsulfonyl]glycyl hydroxamic acid adduct has been solved by NMR in solution. The three x-ray structures and the solution structure are similar but not identical to one another, the differences being sizably higher in the loops. We propose that many of the loops show a dynamical behavior in solution on a variety of time scales. Different conformations of some flexible regions of the protein can be observed as ''frozen'' in different crystalline environments. The mobility in solution studied by NMR reveals conformational equilibria in accessible time scales, i.e., from 10 ؊5 s to ms and more. Averaging of some residual dipolar couplings is consistent with further motions down to 10 ؊9 s. Finally, local thermal motions of each frozen conformation in the crystalline state at 100 K correlate well with local motions on the picosecond time scale. Flexibility͞con-formational heterogeneity in crucial parts of the catalytic domain is a rule rather than an exception in matrix metalloproteinases, and its extent may be underestimated by inspection of one x-ray structure. Backbone flexibility may play a role in the difficulties encountered in the design of selective inhibitors, whereas it may be a requisite for substrate binding and broad substrate specificity. macrophage metalloelastase ͉ protein mobility ͉ solution structure ͉ x-ray structure

Triplex with a twist: Through metadynamics calculations, the thrombin binding aptamer (TBA) has been shown to adopt a stable G‐triplex structural motif, in addition to the usual G‐quadruplex (see scheme). An 11‐mer oligonucleotide was also shown to form a stable G‐triplex, whose structural and thermodynamic properties have been characterized.