Structure and Function of Human Matrix Metalloproteinases

The extracellular matrix (ECM) is a macromolecules network, in which the most abundant molecule is collagen. This protein in triple helical conformation is highly resistant to proteinases degradation, the only enzymes capable of degrading the collagen are matrix metalloproteinases (MMPs). This resistance and maintenance of collagen, and consequently of ECM, is involved in several biological processes and it must be strictly regulated by endogenous inhibitors (TIMPs). The deregulation of MMPs activity leads to development of numerous diseases. This review shows MMPs complexity.


Extracellular Matrix-Collagen
The extracellular matrix (ECM) is a macromolecules network, composed of collagen, enzymes and proteins (Figure 1), that promote a structural and biochemical support.
The ECM has many components [1]: Fibers (collagen, elastin, laminin, and fibronectin), proteoglycans (syndecan-1 and aggrecan), glycoproteins (tenascin, vitronectin and entactin) and polysaccharides (hyaluronic acid) [2], that regulate cell migration, growth, and differentiation [3].  The collagen is the most abundant protein in ECM which gives structural support for cells [1,3]. Depending of mineralization degree, the tissues can be divided in rigid (bones) or compliant (sinews) or have a gradient between these two states (cartilage) [4]. Collagen can come in two main forms: fibrillar (type I, II, III, V, and XI) and non-fibrillar, the latter includes facit-fibril associated collagens with interrupted triple helix (type IX, XII, XIV, XIX, and XXI); short chain (type VIII and X); basement membrane (type IV); multiplexin (type XV and XVIII); MACIT-membrane associated collagens with interrupted triple-helix (type XIII and XVII) and others types (Type VI, VII, and VIII). The collagen protein consists of three α chains, in triple helix, where two chains are chemically similar (α1 and α2), with approximate dimensions of 300 × 1.5 nm [3,5]. The triple helix is divided into five D-segments with D1-D4 having a length of 67 nm and D5 equal to 0.46 nm [3,5]. The high glycine content is important for collagen helix stabilization as it allows collagen fibers to combine, facilitating hydrogen bridges and cross-link formation [5].
The collagen synthesis involves several steps [5] ( Figure 2). The mRNA is transcripted by ribosome, forming pre-propeptide. The following three stages occur in the endoplasmic reticulum: 1) The N-terminal signal sequence is removed; 2) hydroxylation of lysine and proline by prolyl hydroxylase and lysyl hydroxylases takes place, together with 3) glycosylation of lysine, forming the pro-collagen. This structure is a triple helix chain, but with the unwound terminals. The removal of these terminals occurs in extracellular medium, by collagen peptidases, forming tropocollagen. The microfibril collagen is formed by lysyl oxidase that packs together five tropocollagen chain [3], which have a characteristic image. At intervals of 67 nm, one zone has the roll of all tropocollagen and another zone has one less-"gap". The fibril collagen is composed of various microfibril collagen group and the alternating overlap and gap regions create the characteristic "bright and dark" Dbanding pattern [3].
(a) (b) Figure 2. Synthesis of microfibril collagen. (a) In intracellular medium, the mRNA is transcripted by ribosome, forming the pre-peptide, which is then processed in endoplasmic reticulum, forming procollagen. (b) In extracellular medium, the pro-collagen is processed by collagen peptidase, forming tropocollagen. For microfibril collagen formation, the tropocollagen is processed by lysil oxidase.
Its triple helix conformation makes collagen resistant to many proteases. The enzymes able to cleave this structure are capthesin K and enzymes with collagenolytic activity (MMPs-1, -2, -8, - 13, -14, and -18) [3,4]. The collagen type I, II and III have a specific cleavage sequence: (Gln/Leu)-Gly#(Ile/Leu)-(Ala/Pro), which is located at 3/4 of N-terminal and this is crucial for collagen In intracellular medium, the mRNA is transcripted by ribosome, forming the pre-peptide, which is then processed in endoplasmic reticulum, forming pro-collagen. (b) In extracellular medium, the pro-collagen is processed by collagen peptidase, forming tropocollagen. For microfibril collagen formation, the tropocollagen is processed by lysil oxidase.
Its triple helix conformation makes collagen resistant to many proteases. The enzymes able to cleave this structure are capthesin K and enzymes with collagenolytic activity (MMPs-1, -2, Figure 3. Collagen degradation. The enzyme with collagenolytic activity (collagenases) cleaves the triple helix at two fragments: 3/4 N-terminal and 1/4 C-terminal. Each chain (α1 and α2) has a specific cleavage sequence (# represents the cleavage site).
The ECM degradation is also an important process in development, morphogenesis, tissue repair and remodulation [8], and can affect the cellular behavior and phenotype [6,9]. The excessive degradation of ECM leads to metabolic and immune diseases, cancer and cardiovascular disorders (hypertension, atherosclerosis and aneurysm) [2,3,6].
The MMPs were first described in 1949 [20], as depolymerizing enzymes that facilitate tumor growth, by making connective tissue stroma, including small blood vessels that are more fluid [16,20,21]. In 1962, a MMP collagenase was isolated and characterized as an enzyme responsible for tadpole tail resorption [1,2,6,13,14,16,21,22], by Gross and Lapiere [22]. Over the next 20 years, several enzymes were purified, but it was in 1985 that the area has developed more significantly [16,21]. Taken together it has been demonstrated that MMPs are present in virus, archeabacteria, bacteria, plants, nematodes, and animals [6,13].
The MMPs were first described in 1949 [20], as depolymerizing enzymes that facilitate tumor growth, by making connective tissue stroma, including small blood vessels that are more fluid [16,20,21]. In 1962, a MMP collagenase was isolated and characterized as an enzyme responsible for tadpole tail resorption [1,2,6,13,14,16,21,22], by Gross and Lapiere [22]. Over the next 20 years, several enzymes were purified, but it was in 1985 that the area has developed more significantly [16,21]. Taken together it has been demonstrated that MMPs are present in virus, archeabacteria, bacteria, plants, nematodes, and animals [6,13].
The deregulation of MMP activity leads to the progression of various pathologies, that can be grouped into [1,6,14,18,19,24,25]: 1) Tissue destruction, 2) fibrosis, and 3) matrix weakening. The overexpression of MMPs is involves in several diseases (Table 1). MMPs have been recently recognized as biomarkers in several fields (diagnosis, monitoring, and treatment efficacy) [19], since their overexpression in diseases conditions is specific and elevated [19]. Huang et al. [26] related that MMP-9 represents a potential biomarker which is overexpressed in several types of tumors (colarectal carcinoma, breast, pancreatic, ovaria, cervical, osteosarcoma non-small cell lung cancer (NSCLC), and giant cell tumor of bone (GCTB)), which makes MMP-9 a preferential candidate for the early detection of these diseases [26]. The elevated levels of MMP-9 can be detected in plasma or blood, show triple protein levels compared to healthy patients [19,27,28]. The MMP-9 in contrast with other proposed biomarkers (MMP-1 and -3), also presents increased levels in specific risk group to atherosclerosis [19], as demonstrated by Rohde et al. [29]. Nilsson et al. [30] studied the MMPs-1, -3, -7, -10, and -12 in plasma and demonstrated that MMP-7 and -12 were elevated in type 2 diabetes, which is related to atherosclerosis and coronary events [30].
The MMPs are involved in the development and progression of atherosclerosis, which is correlated with cardiovascular diseases [19]. Their activity promotes the loss of collagen, elastin and other ECM proteins, inducing the necrotic core of atherosclerotic plaque that leads to myocardial infarction or a stroke [19]. Some studies show intracellular localization of MMP-2 in cardiac myocytes and colocalization of MMP-2 with troponin I in cardiac myofilaments [32]. The MMP-2 activity has also been detected in nuclear extracts from human heart and rat liver [32]. Poly ADP-ribose polymerase is a nuclear matrix enzyme involved in DNA repair and is susceptible to cleavage by MMP-2, in vitro its cleavage being blocked by MMPs inhibitors [32]. The presence of MMP-2 in nucleus could play a role in poly ADP-ribose polymerase degradation and affect DNA repair [32].
Collagenases (Appendix A, Table A1) cleave some ECM proteins and other soluble proteins, but the most important role of this type of MMPs is the cleavage of fibrillar collagen type I, II, III, IV and XI into two characteristic fragments, 1/4 C-terminal and 3/4 N -terminal [1,6,8,9,12,14,17,21]. This process takes place in two steps: first MMP unwind triple helical collagen and then hydrolyze the peptide bonds [1]. The hemopexin domain is essential for cleaving native fibrillar collagen while the catalytic domain can cleave non-collagen substrates [1].
Gelatinases are involved in physiological and pathological states, such as, embryonic growth and development, angiogenesis, vascular diseases, inflammatory, infective diseases, degenerative diseases of the brain and tumor progression [1]. Tumor metastasis is a process that involves the release of tumor cells, their migration through blood vessels, penetration into the blood and lymphatic system and their adhesion into the endothelial vessel and extravasation into tissue [11]. The activity of gelatinases is crucial for metastatic cell output and metastasis site entry [11]. Increased expression and activity of gelatinases have been described in malignant diseases such as breast, urogenital, brain, lung, skin and colorectal cancer [11].
Stromelysines (Appendix A, Table A3) have the same domain arrangement as collagenases, but do not cleave interstitial collagen [1]. MMP-3 and -10 are closely related by their structure and substrate specificity [1,8,9,17], while MMP-11 is distantly related [1]. The intracellular activation of MMP-1 is regulated by 10 amino acids insert, localized between the pro-and catalytic domains (RXRXKR), which is recognition by Golgi-associated proteinase furin.
The main characteristic of the matrilysins (Appendix A, Table A4) is the lack of hemopexin domain, present in the other MMPs [9,12,17,18]. This MMP group has a specific feature in the amino acid sequence with a threonine residue adjacent to the Zn 2+ -binding site [1].

Structure
Lovejoy et al. reported the first structure of MMP-inhibitor complex [33]. This structure reveals that the active site of MMP is a deep cavity and moreover that the catalytic domains of MMPs share a sequential similarity, where the percentage of similarity ranges between 33% (between MMP-21 and MMP23) and 86% (between MMP-3 and MMP-10) [33]. 3D structures of the catalytic domains of MMP-1 and -8 as well as structures of pro-MMP-3 and MMP-1 followed [33].

S 1 Pocket Selectivity
MMPs possess six pockets (S 1 , S 2 , S 3 , S 1 , S 2 , and S 3 ) [19] and the fragments of the substances and inhibitors are consequently named after the pocket that they interact with (P 1 , P 2 , P 3 , P 1 , P 2 , and P 3 ) [2,6]. The S 1 , S 2 , and S 3 pockets are unprimed pockets, which are localized on the right side of zinc ion [19]. The S 1 , S 2 , and S 3 pockets are the primed pockets, which are localized on the left side of zinc ion [2,19]. It has been demonstrated that the S 1 is the most variable pocket in MMPs [1,6,8,15,16], followed by S 2 , S 3 , S 1 , and S 3 pockets with an equivalent degree of variance, while the S 2 has the lowest variability. The S 2 and S 3 pockets are shallower than S 1 and are more exposed to solvent [1]. The S 3 pocket may also contribute to substrate specificity [1]. S 1 pocket is the most important since it is a determining factor for substrate specificity [1,2,8,16], Its cavity [1,19], formed by the Ω-loop [19] is a highly hydrophobic. By analyzing the depth of the different S 1 subsites, MMPs can be divided into three different subgroups [1,2,17,19,36] (Figure 10): the shallow, the intermedium and the deep pockets. This differentiation is related to the size of amino acids: MMP-1 and -7 have an Arg 214 and Tyr 214 residue, respectively [13,36], instead of a leucine present in MMP-2, -3, -8, -9, -10, -12, -13, and -14 [16,36], giving origin to shallow S 1 pocket [15].

Catalytic Mechanism
Over the past 30 years, the detailed structural characterization of different MMPs allowed disentangling of the individual catalytic steps that occur at the active site during proteolysis [23].
The catalytic activity requires catalytic zinc ion and a water molecule flanked by three conserved histidine and a conserved glutamate, with a conserved methionine acting as a hydrophobic base to support the structure surrounding the catalytic zinc ion [1,2,14] (Figure 12). In the initial transition states of the MMP-substrate interaction, Zn 2+ is penta-coordinated with a substrate's carbonyl oxygen atom, one oxygen atom from the glutamate-bound water and the three conserved histidine residues [1]. Hydrolysis of the peptide bond begins with the nucleophilic attack of the water coordinated with zinc to the carbonyl carbon of the substrate [13,14,17,18], subsequently proton transfer to the amine nitrogen occurs through the glutamic acid residue [2,8,12,13,15,16,18,21], promoting a gem-diol reaction intermediate [9,11,23] with a tetrahedral geometry [1,2,13]. This results in the breakdown of the substrate and the release of a water molecule [1]. The peptide is stabilized at the active site, by interaction between N-terminal residues and S1′ pocket [1,12], and by new hydrogen bonds formed between N-terminal, glutamate and water [2,9,11,13,15,16,18,23]. The two key steps in the catalytic process involve a structural rearrangement of the active site and the fate of the two obtained peptides [23]. Particularly relevant to the catalytic mechanism is the flexibility of the loop that forms the exterior of the S1′ pocket [23]. The internal flexibility of the catalytic domain plays an important role for enzymatic activity, but it is also the cause of drawbacks related to inhibitor selectivity. Pro-MMP-2 forms a complex with TIMP-2 through interactions of the hemopexin-like domain with the non-inhibitory C-terminal domain of TIMP-2 [1,2,8,17,18,25]. Formation of this complex is essential for pro-MMP-2 activation by MT1-MMP [8,17,18]. The complex then reaches the cell surface where it binds to the active site of MT1-MMP [1], via the free inhibitory N-terminal of TIMP-2 [2], orienting the pro-MMP-2 pro-domain adjacent to MT1-MMP [8,17,18]. With the aid of a second MT1-MMP [2], interactions between the MT1-MMP occur through their hemopexin-like domains, forming a quaternary tetrameric complex [1,17]: an MT1-MMP acts as a receptor for the pro-MMP-2-TIMP-2 complex and the other as a pro-MMP-2 activator [2,8,18]. Excess of TIMP-2 prevents this activation process by inhibiting the second MT1-MMP [2,8].

Catalytic Mechanism
Over the past 30 years, the detailed structural characterization of different MMPs allowed disentangling of the individual catalytic steps that occur at the active site during proteolysis [23].
The catalytic activity requires catalytic zinc ion and a water molecule flanked by three conserved histidine and a conserved glutamate, with a conserved methionine acting as a hydrophobic base to support the structure surrounding the catalytic zinc ion [1,2,14] (Figure 12). In the initial transition states of the MMP-substrate interaction, Zn 2+ is penta-coordinated with a substrate's carbonyl oxygen atom, one oxygen atom from the glutamate-bound water and the three conserved histidine residues [1]. Hydrolysis of the peptide bond begins with the nucleophilic attack of the water coordinated with zinc to the carbonyl carbon of the substrate [13,14,17,18], subsequently proton transfer to the amine nitrogen occurs through the glutamic acid residue [2,8,12,13,15,16,18,21], promoting a gem-diol reaction intermediate [9,11,23] with a tetrahedral geometry [1,2,13]. This results in the breakdown of the substrate and the release of a water molecule [1]. The peptide is stabilized at the active site, by interaction between N-terminal residues and S 1 pocket [1,12], and by new hydrogen bonds formed between N-terminal, glutamate and water [2,9,11,13,15,16,18,23]. The two key steps in the catalytic process involve a structural rearrangement of the active site and the fate of the two obtained peptides [23]. Particularly relevant to the catalytic mechanism is the flexibility of the loop that forms the exterior of the S 1 pocket [23]. The internal flexibility of the catalytic domain plays an important role for enzymatic activity, but it is also the cause of drawbacks related to inhibitor selectivity. Cells 2020, 9, x FOR PEER REVIEW 12 of 19 Figure 12. Catalytic mechanism.

Conclusions
MMPs are the most important enzymes for ECM maintenance. They are also involved in several biological and pathological diseases. However, MMPs remain a challenge for science, due to their high complexity, both in terms of regulation and activity. To that end, understanding of their role in the development of diseases and their mode of action require further studies. The development synthetic inhibitors in particular, critically depend on the full understanding of the structural details about S1′ pocket in MMPs and its interaction with the substrate.

Conclusions
MMPs are the most important enzymes for ECM maintenance. They are also involved in several biological and pathological diseases. However, MMPs remain a challenge for science, due to their high complexity, both in terms of regulation and activity. To that end, understanding of their role in the development of diseases and their mode of action require further studies. The development synthetic inhibitors in particular, critically depend on the full understanding of the structural details about S 1 pocket in MMPs and its interaction with the substrate.

Fibronectin, gelatin and proteoglycans
Organs: brain, kidney, pancreas and lung Brain tumor (astrocytomas and glioblastomas) and tumor progression and angiogenesis.
Identified in 1999 and cloned from a human brain cDNA library. MMP-24 can activate MMP-2 latent form. MMP-24 is neuro-specific and contribute to neuronal circuit formation and plasticity. It has a role in the development of dermal neuro-immune synapses.  MMP-27 is classified as stromelysin and holds 51.6% structural homology with MMP-10.
Tissue homeostasis and repair, osteoarthritis and rheumatoid arthritis. -