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Classification

EC 3.4.24.34 This classification means that this enzyme:

• is a hydrolase: it hydrolyzes covalent bonds
• is an endopeptidase: it cleaves peptide bond
• cleaves interstitial collagens in the triple helical domain (at a site about three-fourths away from the N-terminus)

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.^[2] The difference between this classification and EC 3.4.24.7 is that this enzyme cleaves type III collagen more slowly than type I.

To see

[1] [2] ^[3] [3] [4] pockets [5]

Structure and domains

MMP-8 is composed of several domains: a propeptide, a catalytic domain, a hinge region, and a C-terminal hemopexinlike domain.^[4].

Propeptide

It corresponds to 79 aminoacids, from Phe21 to Met100. The sequence of residue is: FPVSSKEKNTKTVQDYLEKFYQLPSNQYQSTRKNGTNVIVEKLKEMQRFFGLNVTGKPNEETLDMMKKPRCGVPDSGGFM

Catalytic domain

Thanks to X-ray crystallography, the catalytic domain structure has been solved with 1,7 Å resolution (2OY4).This domain is composed of 157 residues, from Met86 to Gly242, organized in and .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.[6]

Ca2+ interactions

CA996 pocket interaction

The residues involved in the Ca996 interactions (coordinate bonds) are .

Zn2+ interactions

The zinc-binding motif HEXGHXXGXXH presents in the catalytic domain is characteristic for the protease activity of MMP-8.

Zn999 : the catalytic zinc

It 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 with the carbonyl and the hydroxyl oxygen of the hydroxamic acid moiety of the inhibitor. This discovery has been made thanks to the Pro-Leu-Gly-hydroxylamine inhibitor.^[5] On this you can only see the 3 His of MMP-8 with the Zn999. The fourth ligand of the catalytic zinc is a water molecule.

ZN999 pocket interaction

Zn998 : the structural zinc

The residues involved in the Zn998 interactions are . 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.^[6]

One of them is as well bound to the Glu residue thanks to a hydrogen bond. At first, the Gly 206 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. Then, the Ala 182 residue of the enzyme makes a hydrogen bond with the NH group of the substrate: this allows the substrate to enter the cavity of the catalytic site. The rest of the protein is stabilized by 4 hydrogen bonds with the amino acid located in the cavity.

The conserved cysteine present in the cysteine-switch motif (89-96) binds the catalytic zinc ion, thus inhibiting the enzyme. The dissociation of the cysteine from the zinc ion upon the activation-peptide release activates the enzyme.

Hemopexin domain

The hemopexin domain has two conserved cysteines that are disulfide bonded. Mutation of those cysteines to alanines ^[7] or reduction and alkylation destroys collagenolytic activity (K. Suzuki and H.Nagase, unpublished results).^[4]

[7]

This domain is localized outside of the catalytic domain shown in this page. It is essential for the substrate recognition of MMP-8 and the single catalytic domain of MMP-8 is not able to cleave collagen. When this hemopexin-like domain is removed, the protein loses its ability to cleave collagen. However, neutrophil collagenase is still able to cleave other substrate. 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.

Mechanism

To express collagenolytic activity, MMP-8 needs to have both the catalytic and hemopexin domains but it is not clear how the hemopexin domain help to cleave triple-helical collagens because it does not bind to collagen.^[8] Moreover, MMP-8 is secreted as inactive proproteins and then activated after a cleavage by extracellular proteinases. Indeed, it can't be activated without removal of the activation peptide. But recent evidences suggest that this protease activation is blocked by the formation of an intramolecular complex between the propeptide domain on the Cysteine residue and essential zinc atom in the catalytic domain. It is called the Cysteine-switch. 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 allow cleavage of the individual alpha chains of collagen, the binding of activated MMP-8 must partially unwind triple helix by inducing a conformational change in alpha chain. However, the molecular basis of this mechanism is not known.^[10]

The carbonyl group of the peptide bond coordinates with the active-site zinc. This displaces the water molecule from the zinc atom. The peptide hydrolysis is assisted by the carboxyl group of the glutamate, which 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. A pocket to the right of the active-site zinc, called the specificity pocket or , accommodates the side chain of the substrate residue, which becomes the new N-terminus after cleavage. The sizes of the S1′ pocket vary among the MMPs, and this is one of the major determining factors of substrate specificity.^[11]

Cleavage at Gly775–Ile776 or Leu776 in each alpha-chain of the molecule by the residues of the . ^[12] The cleavage 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]

Function

A major function of MMPs is thought to be the removal of ECM in tissue resorption.

Disease

Overexpression of MMP-8, or inadequate control by TIMPs, can be associated with a lot of pathological conditions: psoriasis, sclerosis, osteoarthritis, rheumatoid arthritis, osteoporosis, Alzheimer's disease, tumor growh and metastasis.^[14] Neutrophil collagenase or collagenase 2 (MMP-8) is unique among the family of matrix metalloproteinases (MMPs) because of its exclusive pattern of expression in inflammatory conditions.^[15]

Relevance

Structural highlights

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