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Matrix metalloproteinase-8
MMP-8, also called, Neutrophil collagenase or Collagenase 2, is a zinc-dependent and calcium-dependent enzyme mainly produced by neutrophils. It belongs to the collagens (MMP) family which is involved in the breakdown of extracellular matrix in embryonic development, reproduction, and tissue remodeling, as well as in disease processes. The gene coding this family is localized on the chromosome 11 of Homo sapiens with 467 residues.[1]
Here is the reloading for the initial structure of the catalytic domain of MMP-8.
ContentsClassificationEC 3.4.24.34 This classification means that this enzyme:
The metalloendopeptidase activity is defined by a mechanism in which water acts as a nucleophile, one or two metal ions hold the water molecule in place, and charged amino acid side chains are ligands for the metal ions.[1] The difference between this classification and EC 3.4.24.7 is that this enzyme cleaves type III collagen more slowly than type I. (On BRENDA, you can find all pieces of information about the MMP-8 enzyme like, for example, a list of different substrates or inhibitors)
Structure and domainsMMP-8 is composed of several domains: a propeptide, a catalytic domain, a hinge region, and a C-terminal hemopexin-like domain.[2]. PropeptideIt corresponds to 79 aminoacids, from Phe21 to Met100. The sequence of residue is: FPVSSKEKNTKTVQDYLEKFYQLPSNQYQSTRKNGTNVIVEKLKEMQRFFGLNVTGKPNEETLDMMKKPRCGVPDSGGFM Catalytic domainThanks to X-ray crystallography, the catalytic domain structure has been solved with 1,7 Å resolution (PDB ID : 2OY4).This domain is composed of 157 residues, from Met86 to Gly242, organized in three alpha helixes and five beta sheets.The protein folding and especially the zinc environment of the collagenase catalytic domain is very close to the astacins and the snake venom metalloproteinases. The catalytic domain alone has proteolytic activity against other protein substrates and synthetic substrates.[3] SubsitesBesides the catalytic site, the MMPs have other sites called subsites which can also interact with the substrates and inhibitors. Conventionally, the subsites on the left of the catalytic Zn2+ are designated as S1, S2, S3, ..., Sn and the ones on the right are known as S1', S2', etc. One of these subsites, the S1' pocket, is the main subsite for the substrate recognition. This pocket is variable in amino acid composition and depth and can be used to classify the MMPs. MMP-8 belongs to the class of the intermediate MMPs according to the depth of its S1' pocket.[4] This pocket is delimited by the Leu193, Val194, His197, Leu214, Tyr216, Tyr219, Ala220 and Arg222 residues.[5]. Moreover, this pocket is rich in hydrophobic amino acids, what is suitable for binding to the substrates of MMP-8.
Ca2+ interactionsThis enzyme binds 3 Ca ions, 2 of them in the catalytic domain, which are packed against the top of the beta sheet and have mostly a structural function, stabilizing the catalytic domain.[3] The residues involved in the Ca996 interactions (coordinate bonds) are two Gly residues (169 and 171) next to two Asp residues (137 and 173).
Zn2+ interactionsThe zinc-binding motif HEXGHXXGXXH presents in the catalytic domain is characteristic for the protease activity of MMP-8.  Zn999 : the catalytic zincIt is involved in the catalytic activity and is situated at the bottom of the active-site. This ion is penta-coordinated with: His197, His201 and His207 of MMP-8 and probably (according to the mechanism model described bellow) with a Gly residue of the substrate and a water molecule. On this link you can only see the 3 His of MMP-8 with the Zn999. Zn998 : the structural zincThe residues involved in the Zn998 interactions are an Asp residue (149) next to three His residues (147, 162 and 175). The glutamic acid adjacent to the first histidine is essential for catalysis. It should be noted that scientists were unable to exchange or remove this Zinc in their crystals, which is suggesting that there is a tight interaction with MMP-8.[3]
Hinge domainIt corresponds to a short linker region from G242 to P258, with the following sequence: GLSSNPIQPTGPSTPKP, between the catalytic and the hemopexin domains. The exact role of this domain isn't very well clear but it's known that autoproteolysis could occurred in MMP-8 leading to an unstable protein and different mutants[6] were made in the hinge region and shown that stability of MMP-8 could be increased, decreased or unchanged. Moreover, sequence alignements of collagenolytic MMPs in this hinge domain reveal that they all have the four prolines in the same positions, suggesting that these prolines could be important for the specific collagenolytic activity. Hemopexin domainThe hemopexin domain has two conserved cysteines that are disulfide bonded. Mutation of those cysteines to alanines [7] or reduction and alkylation destroy collagenolytic activity (K. Suzuki and H.Nagase, unpublished results).[2] It is essential for the substrate recognition of MMP-8 and the single catalytic domain of MMP-8 is not able to cleave collagen. However, neutrophil collagenase is still able to cleave other substrates. It seems that the collagen binds to two sites on MMP-8 : one in the catalytic site and another in the hemopexin domain. One hypothesis is that when the collagen binds to both sites, its helical structure is destabilized and unwound. Thus, the cleavage site of collagen is accessible and the cleavage reaction can occur.[8] Unfortunately, no structure of the full MMP-8 protein has been crystallized yet, but here you can see in orange the hemopexin domain of human pro-MMP1 which is very well conserved between these two proteins. In this article Matrix metalloproteinases: structures, evolution, and diversification,Irina Massova, Lakshmi P. Kotra, Rafael Fridman and Shahriar Mobashery, there are good pieces of information on conservations among the MMPs family.
MechanismMMP-8 is secreted as inactive proprotein and then activated after a cleavage by extracellular proteinases. Indeed, it can't be activated without removal of the activation peptide. After this protease activation, recent evidences suggest that, there is formation of an intramolecular complex between the Cysteine residue (Cys91) of the propeptide domain and essential zinc atom in the catalytic domain. It is called the Cysteine-switch: it inhibits the action of MMP-8. This discovery is unprecedented in enzymology and offers the opportunity for multiple modes of physiological activation of MMP-8. Moreover, since conditions in different cells and tissues may match those necessary to effect one of these activation modes for a given MMP, this may offer metabolic flexibility in the control of MMP activation.[9] To express collagenolytic activity, MMP-8 needs to have both the catalytic and hemopexin domains. The linker peptide can position the hemopexin domain in such a way that it bends over the active site of the catalytic domain. But understanding how the Hemopexin domain assists in the cleavage of collagen is elusive.[10] Thus, the collagen would be captured between these two domains. However, the active site cannot accommodate the entire triple helix in a native state. The linker peptide would, by means of its collagen-like conformation, change the quaternary structure of the captured collagen. Interactions between proline residues of the collagenase and a specific region of the collagen would generate a “proline zipper,” resulting in destabilization of the cleavage site area of the collagen. After destabilization, one chain of the triple helix fits in the specificity pocket or S1' pocket to the right of the active-site zinc. At first, the Gly residue of the substrate binds the active site thanks to the Zn2+ atom. When it binds it takes the place of unstable water molecules and establishes stabilizing interactions with the active site thanks to its C terminal part.[11] The carboxyl group of the glutamate (GLU 198 in the active site green link above) serves as a general base to draw a proton from the displaced water molecule, thereby facilitating the nucleophilic attack of the water molecule on the carbonyl carbon of the peptide scissile bond. Then, the Alanine residue of the active site makes a hydrogen bond with the NH group of the substrate. Moreover, this NH group becomes the new N-terminus after cleavage.[12] The cleavage is at Gly775–Ile776 or Leu776 in each alpha-chain of the collagen molecule[6] and takes place at neutral pH. It generates fragments that spontaneously lose their helical conformation, denature to gelatin, and become soluble. The gelatin is then susceptible to attack by gelatinases and other proteases.[13] There is no crystallized complex of MMP-8 and the collagen, however you can see here the complex of MMP-1 and a triple-helical collagen peptide, MMP-1 being very close to MMP-8 it gives an idea of the MMP8-collagen complex.
Inhibitors of MMP-8Endogenous inhibitorsThe tissue inhibitors of metalloproteinases (TIMPs) are specific inhibitors of the whole family of MMPs proteins. Currently, four TIMPs were identified (TIMP-1, TIMP-2, TIMP-3, TIMP-4). They are 21 to 29kDa proteins, contain 2 subdomains (N-ter and C-ter) and have a "wedge-like" shape. This is the N-ter domain which interacts with the catalytic domains of MMPs and impedes their proteolytic activity.[12] The Cys1 residue is crucial for the inhibitor effect of TIMPs because it can interact with the catalytic zinc of the MMPs. The result is a chelation of the zinc by the N-terminal amino group and the carbonyl group of Cys1.[14] Moreover, this interaction triggers the expulsion of the water molecule which was bound to the zinc. No structures of MMP-8 with TIMPs are available on the Protein Data Bank. However, the mechanism of inhibition is common to all the MMPs. To see the interaction between the catalytic domain of MMP-3 and TIMP-1 (in green) click here.[15] Synthetic inhibitorsBecause endogenous TIMPs have a broad spectrum of action over MMPs, researches were conducted to produce engineered TIMPs and modify their affinity for MMPs. For instance, mutation of the Thr2 of TIMP-1 modifies the specificity of this inhibitor as this residue interacts with the S1' pocket of the MMPs.[16] Besides the modification of TIMPs, the research for MMPs inhibitors resulted in the discovery of molecules with the ability to interact with the catalytic domain of MMPs. The first developed synthetic inhibitors were molecules that mimic the natural substrates of MMPs combined with a zinc chelating groupement.[17] Numerous range of compounds such as hydroxamate, thiol, pyrimidine and phosphorus based-molecules were developed. Those inhibitors inhibit the activity of MMPs by chelating the catalytic zinc like N-hydroxyurea for MMP-8.[18] Recently, new range of inhibitors which do not chelate the catalytic zinc were developed. Those compounds target the selectivity regions for substrates of the MMPs rather than binding to the catalytic zinc. For instance, they can interact with the S1' pocket and induce a conformational change like new inhibitors of MMP-8.[19]
FunctionA major function of MMPs is thought to be the removal of ECM in tissue resorption. Because of their recognized roles in disease (see below) the MMPs have long been considered as pharmacological targets, but their multiplicity, associated with their variable expressions in different tissues and their apparently overlapping substrate specificities, have presented considerable challenges to those hoping to design suitable therapeutic inhibitors.
DiseaseInvolvementOverexpression of MMP-8, or inadequate control by TIMPs, can be associated with a lot of pathological conditions[20] because they facilitates penetration of anatomical barriers.
What makes this MMP unique is its exclusive pattern of expression in inflammatory conditions: the density of the interstitial collagen network increases in inflamed tissue.[25] Therapeutic potential
You can find very interesting pieces of information concerning MMPs related diseases and therapeutic potentials in a nature paper: "Is there new hope for therapeutic matrix metalloproteinase inhibition, Roosmarijn E. Vandenbroucke & Claude Libert".
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