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 former's considerably lower proteolytic activity. This relationship is consistent with the hypothesis that enzymes often trade stability for higher activity.
Collagenolysis is essential in extracellular matrix homeostasis, but its structural basis has long been shrouded in mystery. We have developed a novel docking strategy guided by paramagnetic NMR that positions a triple-helical collagen V mimic (synthesized with nitroxide spin labels) in the active site of the catalytic domain of matrix metalloproteinase-12 (MMP-12 or macrophage metalloelastase) primed for catalysis. The collagenolytically productive complex forms by utilizing seven distinct subsites that traverse the entire length of the active site. These subsites bury ϳ1,080 Å 2 of surface area, over half of which is contributed by the trailing strand of the synthetic collagen V mimic, which also appears to ligate the catalytic zinc through the glycine carbonyl oxygen of its scissile GϳVV triplet. Notably, the middle strand also occupies the full length of the active site where it contributes extensive interfacial contacts with five subsites. This work identifies, for the first time, the productive and specific interactions of a collagen triple helix with an MMP catalytic site. The results uniquely demonstrate that the active site of the MMPs is wide enough to accommodate two strands from collagen triple helices. Paramagnetic relaxation enhancements also reveal an extensive array of encounter complexes that form over a large part of the catalytic domain. These transient complexes could possibly facilitate the formation of collagenolytically active complexes via directional Brownian tumbling.Collagens comprise around 30% of the protein mass of the body (1) and resist general proteolysis. Matrix metalloproteinases (MMPs) 2 degrade collagen during cancer cell migration (2), angiogenesis (3, 4), destabilization of atherosclerotic plaques (5), and arthritis (6) and after myocardial infarction (7). For instance, plaques contain a collagen-rich cap and are sensitive to MMP-12 secreted by macrophages that makes them more vulnerable to rupture, precipitating myocardial infarction, or stroke (8).Collagen fibrils are bundles of staggered triple helices. Each triple helix comprises three extended strands twisted into a right-handed superhelix with hydrogen bonding between chains (9). Each collagen chain is a long series of GXY triplets in which the glycine is in the interior, X is often proline, and Y is often a stabilizing 4-hydroxyproline (Hyp). Because each chain is offset by one residue, the chains have been named leading, middle, and trailing (10). Lack of Pro and Hyp in four GXY triplets on the C-terminal side of the MMP scissile bond in interstitial collagens (types I, II, and III) was proposed to loosen the triple helix (1); this was later observed as 10-fold symmetry (11).Protease substrates are labeled by increasing distance from the scissile peptide bond with a prime indicating the C-terminal side: P 4 -P 3 -P-P 1 ϳP 1 Ј-P 2 Ј-P 3 Ј-P 4 Ј where ϳ denotes the scissile peptide bond. We refer to the residues on the N-terminal side of the scissile bond as "unprimed" and those on the C-terminal side as "primed." The...
Ketosamines are an important class of glycoconjugates widely employed in clinical diagnostics and implicated in development of diabetic complications, intestinal infections, or advanced cancer, as well as in food organoleptic and nutritional value. We report on the first preparation and structural characterization of 1-amino-1-deoxy-L-sorbose (L-sorbosamine, L-SorNH 2). The monosaccharide was synthesized from L-sorbose following a classic phenylosazone protocol. In aqueous solution, L-SorNH 2 assumes an anomeric equilibrium consisting of 89.3% α-pyranose, 3.7% β-pyranose, 3.8% α-furanose, 2.4% β-furanose, and 0.9% acyclic keto tautomer. The α-pyranose anomer in crystalline L-SorNH 2 × HCl adopts the 2 C 5 chair conformation, with bond lengths and valence angles comparing well with related sorbopyranose structures. All hydroxyl oxygen atoms, the ammonium group and chloride ion are involved in an extensive hydrogen bonding network which is formed by infinite chains with fused antidromic R 7 6 (14), R 5 4 (10), and R 4 3 (8) cycles. The Hirshfeld surface analysis suggests a significant contribution of the non-polar intermolecular contacts to the crystal structure, as well.
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