The three-dimensional structures of the zinc endopeptidases human neutrophil collagenase, adamalysin I1 from rattle snake venom, alkaline proteinase from Pseudomonas aeruginosa, and astacin from crayfish are topologically similar, with respect to a five-stranded 0-sheet and three a-helices arranged in typical sequential order. The four proteins exhibit the characteristic consensus motif HEXXHXXGXXH, whose three histidine residues are involved in binding of the catalytically essential zinc ion. Moreover, they all share a conserved methionine residue beneath the active site metal as part of a superimposable "Met-turn.'' This structural relationship is supported by a sequence alignment performed on the basis of topological equivalence showing faint but distinct sequential similarity. The alkaline proteinase is about equally distant (26% sequence identity) to both human neutrophil collagenase and astacin and a little further away from adamalysin I1 (17% identity). The pairs astacin/adamalysin 11, astacidhuman neutrophil collagenase, and adamalysin Whuman neutrophil collagenase exhibit sequence identities of 16%, 14%, and 13%, respectively. Therefore, the corresponding four distinct families of zinc peptidases, the astacins, the matrix metalloproteinases (matrixins, collagenases), the adamalysins/reprolysins (snake venom proteinases/reproductive tract proteins), and the serralysins (large bacterial proteases from Serratia, Erwinia, and Pseudomonas) appear to have originated by divergent evolution from a common ancestor and form a superfamily of proteolytic enzymes for which the designation "metzincins" has been proposed. There is also a faint but significant structural relationship of the metzincins to the thermolysin-like enzymes, which share the truncated zincbinding motif HEXXH and, moreover, similar topologies in their N-terminal domains.Keywords: crystal structure; metalloproteinase; molecular evolution; protein family; sequence alignment; topology is involved in metal binding (McKerrow, 1987;

Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases involved in tissue remodeling. They have also been implicated in various disease processes including tumour invasion and joint destruction and are therefore attractive targets for inhibitor design. For rational drug design, information of inhibitor binding at the atomic level is essential. Recently, we have published the refined high-resolution crystal structure of the catalytic domain of human neutrophil collagenase (HNC) complexed with the inhibitor Pro-Leu-Gly-NHOH, which is a mimic for the unprimed (P3-P1) residues of a bound peptide substrate. We have now determined two additional HNC complexes formed with the thiol inhibitor HSCH2CH(CH2Ph)CO-L-Ala-Gly-NH2 and another hydroxamate inhibitor, HONHCOCH(iBu)CO-L-Ala-Gly-NH2, which were both refined to R-values of 0.183/0.198 at 0.240/0.225-nm resolution. The inhibitor thiol and hydroxamate groups ligand the catalytic zinc, giving rise to a slightly distorted tetrahedral and trigonal-bipyramidal coordination sphere, respectively. The thiol inhibitor diastereomer with S-configuration at the P1' residue (corresponding to an L-amino acid analog) binds to HNC. Its peptidyl moiety mimics binding of primed (P1'-P3') residues of the substrate. In combination with our first structure a continuous hexapeptide corresponding to a peptide substrate productively bound to HNC was constructed and energy-minimized. Proteolytic cleavage of this Michaelis complex is probably general base-catalyzed as proposed for thermolysin, i.e. a glutamate assists nucleophilic attack of a water molecule. Although there are many structural and mechanistic similarities to thermolysin, substrate binding to MMPs differs due to the interactions beyond S1'-P1'. While thermolysin binds substrates with a kink at P1', substrates are bound in an extended conformation in the collagenases. This property explains the tolerance of thermolysin for D-amino acid residues at the P1' position, in contrast to the collagenases. The third inhibitor, HONHCOCH(iBu)CO-L-Ala-Gly-NH2, unexpectedly binds in a different manner than anticipated from its design and binding mode in thermolysin. Its hydroxamate group obviously interacts with the catalytic zinc in a favourable bidentate manner, but in contrast its isobutyl (iBu) side chain remains outside of the S1' pocket, presumably due to severe constraints imposed by the adjacent planar hydroxamate group. Instead, the C-terminal Ala-Gly-NH2 tail adopts a bent conformation and inserts into this S1' pocket, presumably in a non-optimized manner. Both the isobutyl side chain and the C-terminal peptide tail could be replaced by other, better fitting groups.(ABSTRACT TRUNCATED AT 250 WORDS)

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Matrix metalloproteinase 1 (MMP-1) cleaves types I, II, and III collagen triple helices into 3 ⁄4 and 1 ⁄4 fragments. To understand the structural elements responsible for this activity, various lengths of MMP-1 segments have been introduced into MMP-3 (stromelysin 1) starting from the C-terminal end. MMP-3/MMP-1 chimeras and variants were overexpressed in Escherichia coli, folded from inclusion bodies, and isolated as zymogens. After activation, recombinant chimeras were tested for their ability to digest triple helical type I collagen at 25°C. The results indicate that the nine residues 183 RWTNNFREY191 located between the fifth ␤-strand and the second ␣-helix in the catalytic domain of MMP-1 are critical for the expression of collagenolytic activity. Mutation of Tyr191 of MMP-1 to Thr, the corresponding residue in MMP-3, reduced collagenolytic activity about 5-fold. Replacement of the nine residues with those of the MMP-3 sequence further decreased the activity 2-fold. Those variants exhibited significant changes in substrate specificity and activity against gelatin and synthetic substrates, further supporting the notion that this region plays a critical role in the expression of collagenolytic activity. However, introduction of this sequence into MMP-3 or a chimera consisting of the catalytic domain of MMP-3 with the hinge region and the C-terminal hemopexin domain of MMP-1 did not express any collagenolytic activity. It is therefore concluded that RWTNNFREY, together with the C-terminal hemopexin domain, is essential for collagenolytic activity but that additional structural elements in the catalytic domain are also required. These elements probably act in a concerted manner to cleave the collagen triple helix.Interstitial collagen types I, II, and III are the major structural proteins in connective tissues such as tendon, skin, bone, cartilage, and blood vessels. They consist of three ␣ chains with repeating Gly-X-Y triplets where X and Y are frequently Pro and Hyp, respectively. Each chain of the repeating tripeptide adopts a left-handed poly-Pro II helix conformation, and three left-handed chains then intertwine to form a right-handed superhelix (1-3). This triple helical conformation makes interstitial collagens resistant to most proteinases in vertebrates except for collagenases, cathepsin K (4), and neutrophil elastase (5). The action of cathepsin K is probably important in collagen breakdown in specialized environments such as during bone resorption in an acidic pH environment. Neutrophil elastase may degrade telopeptides of interstitial collagen (6) and the triple-helical region of type I collagen under inflammatory conditions, but the latter activity is much weaker than that of collagenase (5). Vertebrate collagenases, on the other hand, are synthesized by many cell types such as stromal fibroblasts, chondrocytes, keratinocytes, osteoblasts, endothelial cells, and macrophages in response to inflammatory cytokines, growth factors, cellular transformation, and other chemical and physical stimuli ...

The proteolytic activity of the matrix metalloproteinases (MMPs) involved in extracellular matrix degradation must be precisely regulated by their endogenous protein inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Disruption of this balance can result in serious diseases such as arthritis and tumor growth and metastasis. Knowledge of the tertiary structures of the proteins involved in such processes is crucial for understanding their functional properties and to interfere with associated dysfunctions. Within the last few years, several three-dimensional structures have been determined showing the domain organization, the polypeptide fold, and the main specificity determinants of the MMPs. Complexes of the catalytic MMP domains with various synthetic inhibitors enabled the structure-based design and improvement of high-affinity ligands, which might be elaborated into drugs. Very recently, structural information also became available for some TIMP structures and MMP-TIMP complexes, and these new data elucidated important structural features that govern the enzyme-inhibitor interaction.