Collagenases of the matrix metalloproteinase (MMP) family play major roles in morphogenesis, tissue repair, and human diseases, but how they recognize and cleave the collagen triple helix is not fully understood. Here, we report temperature-dependent binding of a catalytically inactive MMP-1 mutant (E200A) to collagen through the cooperative action of its catalytic and hemopexin domains. Contact between the two molecules was mapped by screening the Collagen Toolkit peptide library and by hydrogen/deuterium exchange. The crystal structure of MMP-1(E200A) bound to a triplehelical collagen peptide revealed extensive interactions of the 115-Å-long triple helix with both MMP-1 domains. An exosite in the hemopexin domain, which binds the leucine 10 residues C-terminal to the scissile bond, is critical for collagenolysis and represents a unique target for inhibitor development. The scissile bond is not correctly positioned for hydrolysis in the crystallized complex. A productive binding mode is readily modeled, without altering the MMP-1 structure or the exosite interactions, by axial rotation of the collagen homotrimer. Interdomain flexing of the enzyme and a localized excursion of the collagen chain closest to the active site, facilitated by thermal loosening of the substrate, may lead to the first transition state of collagenolysis. extracellular matrix | X-ray crystallography | protease T he interstitial collagens I, II, and III are the major structural proteins in connective tissues such as skin, bone, cartilage, tendon, and blood vessels (1). They consist of three α chains with repeating Gly-X-Y triplets (X and Y are often proline and hydroxyproline, respectively) that intertwine each other to form a triple helix of ∼300 nm in length (2). Interstitial collagens are resistant to most proteolytic enzymes, but vertebrate collagenases cleave them at a single site approximately three-quarters of the way from the N terminus of the triple helix, thus initiating collagenolysis (3). Owing to this unique activity, collagenases play important roles in embryo development, morphogenesis, tissue remodeling, wound healing, and human diseases, such as arthritis, cancer, and atherosclerosis (4, 5).Matrix metalloproteinase 1 (MMP-1) is a typical vertebrate collagenase (3). It consists of an N-terminal catalytic (Cat) domain containing an active-site zinc ion and a C-terminal hemopexin (Hpx) domain comprised of a four-bladed β-propeller, which are connected by a linker region (6, 7). Although the Cat domain can cleave a number of noncollagenous proteins including heat-denatured collagen (gelatin), its activity on native triplehelical collagen is negligible. The combination of the Cat and Hpx domains is required for MMP-1 to be able to degrade native collagen, and the same is true for all other collagenolytic MMPs, namely MMP-2, MMP-8, MMP-13, and MMP-14 (3). How collagenases interact with collagen and how the Hpx domain endows these enzymes with collagenolytic activity is not clearly understood. Another enigma of collagenolysis bec...
Microtubules form from longitudinally and laterally assembling tubulin α-β dimers. The assembly induces strain in tubulin, resulting in cycles of microtubule catastrophe and regrowth. This 'dynamic instability' is governed by GTP hydrolysis that renders the microtubule lattice unstable, but it is unclear how. We used a human microtubule nucleating and stabilizing neuronal protein, doublecortin, and high-resolution cryo-EM to capture tubulin's elusive hydrolysis intermediate GDP•Pi state, alongside the prehydrolysis analog GMPCPP state and the posthydrolysis GDP state with and without an anticancer drug, Taxol. GTP hydrolysis to GDP•Pi followed by Pi release constitutes two distinct structural transitions, causing unevenly distributed compressions of tubulin dimers, thereby tightening longitudinal and loosening lateral interdimer contacts. We conclude that microtubule catastrophe is triggered because the lateral contacts can no longer counteract the strain energy stored in the lattice, while reinforcement of the longitudinal contacts may support generation of force.
Mammalian prions propagate as distinct strains and are composed of multichain assemblies of misfolded host-encoded prion protein (PrP). Here, we present a near-atomic resolution cryo-EM structure of PrP fibrils present in highly infectious prion rod preparations isolated from the brains of RML prion-infected mice. We found that prion rods comprise single-protofilament helical amyloid fibrils that coexist with twisted pairs of the same protofilaments. Each rung of the protofilament is formed by a single PrP monomer with the ordered core comprising PrP residues 94–225, which folds to create two asymmetric lobes with the N-linked glycans and the glycosylphosphatidylinositol anchor projecting from the C-terminal lobe. The overall architecture is comparable to that of recently reported PrP fibrils isolated from the brain of hamsters infected with the 263K prion strain. However, there are marked conformational variations that could result from differences in PrP sequence and/or represent distinguishing features of the distinct prion strains.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.