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. 2020 May 3;6(5):e03865. doi: 10.1016/j.heliyon.2020.e03865

Thermal stability of human matrix metalloproteinases

Noemi Meraz-Cruz a,b, Felipe Vadillo-Ortega a,b, Aura M Jiménez-Garduño a,c, Alicia Ortega a,∗
PMCID: PMC7205746  PMID: 32395651

Abstract

Matrix metalloproteinases (MMP) are key players in the remodelling of the extracellular matrix under physiological and pathological conditions. Thermodynamic parameters of human recombinant metalloproteinases of the active (rMMP2, 3, 7, 8 and 9) and latent (rPro-MMP2, 3 and 9) forms were obtained by differential scanning calorimetry (DSC). Temperature by itself does not result in autocatalysis of recombinant MMP. The transitions observed by DSC correspond to structural domains of the monomeric protein. In this study, we show the domain organization of these proteins, where the thermal transition (Tm) of the main component is observed at 71.3 °C (ProMMP-2); 74.8 °C (ProMMP-8); 80.0 °C (ProMMP-3); 92.6 °C (ProMMP-9) and 98.3 °C (ProMMP-7). For MMP-3, this main Tm is related to the catalytic domain (CD). The isolated recombinant CD of MMP-3 unfolds as a single transition at Tm 83.4 °C, matching the more stable domain observed in the full-length active form of rMMP-3. The denaturation profile of rProMMP-3 shows the main transition at Tm 80 °C, a less stable domain before the propeptide domain (PD) cleavage. Our results indicate that the structural stability of MMP and particularly their CD are not substantially altered after cleavage of the PD. We propose that the thermodynamic parameters obtained by DSC are relevant for the functional study of MMP, particularly to reveal their contribution in complex biological samples in health and disease.

Keywords: Chemistry, Biological sciences, Proteins, Biochemistry, Metalloproteinases, Stromelysin, Collagenase, Matrilysin, Protein denaturation, Differential scanning calorimetry

Chemistry; Biological sciences; Proteins; Biochemistry; Metalloproteinases, Stromelysin, Collagenase, Matrilysin, Protein denaturation, Differential scanning calorimetry.

1. Introduction

Matrix metalloproteinases (MMP) are a group of zinc-dependent enzymes responsible for degradation of the extracellular matrix components in normal events such as embryogenesis [1], fetal membrane rupture during delivery [2], implantation [3], as well as in many pathological processes [4]. The MMP family members differ in their molecular weight, structural domain organization, substrate specificity, and regulation of functions [5]. The MMP are characterized by conserved functional and structural domains, such as the signal peptide, the propeptide domain (PD), the catalytic domain (CD), which contains the Zn2+-binding site [6], and the hemopexin domain (HD) [5]. All family members which are secreted as zymogens have a sequence of about 10 kDa (PD), which must be removed upon activation. There are however, membrane type MMPs (MT-MMPs), which share a common domain structure consisting of a signal peptide, a PD, CD, a hemopexin-like (HD) domain, a hinge (linker-1) and a stalk region (linker-2) [7]. In MMP, the proenzyme structure is maintained by the binding of Zn2+ to a conserved cysteine (Cys) residue located in a Cys switch motif <PRCGXPD> present in the PD. The Cys-Zn2+ interaction can be released by physical (chaotropic agents), chemical (HOCl, mercurials), or enzymatic (trypsin, plasmin) [8] treatments that result in the separation of the Zn2+ from the Cys residue leading to activation of the enzyme. The activity of MMP is tightly regulated due to their potential impact on the extracellular matrix degradation and tissue integrity. Structural and functional characteristics of these enzymes are compared and summarized in Table 1. The MMP structure has been studied by different techniques [9, 10], in the present study we analysed the thermodynamic parameters of rMMP from thermal denaturation to further understand the stability and domain organization of some representative members of this enzyme family, considering as representatives three of the four MMP subgroups, i.e., the interstitial collagenases (MMP-8), stromelysins (MMP-3), matrilysin (MMP-7), and gelatinases (MMP-2 and MMP-9). Differential scanning calorimetry (DSC) was used to investigate protein stability, which is directly related to protein structure and conformation [11]. Many factors are responsible for the folding and stability of native proteins, including hydrophobic interactions, hydrogen bonds, conformational entropy, and the physical environment. The Tm obtained from the DSC protein denaturation profile is an indicator of protein thermal stability, where a higher Tm means the protein is more stable. Enthalpy is determined by the integral of the ΔCp, which is obtained directly by DSC data. The rationale for this approach is to look for new tools to explore the biochemistry and biology of these enzymes, considering their thermal stabilities and their importance in health and disease.

Table 1.

Characteristics of MMP involved in extracellular matrix degradation.

Alternative
Name		 Molecular weight
(kDa)
 Molecular structure
Proenzyme Active enzyme
MMP-1
(P03956)		 Interstitial collagenase 54007 43000 Image 1
MMP-2
(P08253)		 Collagenase IV, Gelatinase A 73882 65000 Image 2
MMP-3
(P08254)		 Stromelysin 1, roteoglycanase, Transin 53977 45000 Image 3
MMP-7
(P09237)		 Matrilysin, Matrin 29677 19000 Image 4
MMP-8
(P22894)		 Neutrophil collagenase 53412 44000 Image 5
MMP-9
(P14780)		 Collagenase V, Gelatinase B 92000 83000 Image 6
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Domain arrangements:

Image 7 Signal peptide; Image 10 Fibronectin type domain; Image 13 Linker region.

Image 8 Propeptide; Image 11 Collagen-like domain.

Image 9 Catalytic domain; Image 12 Hemopexin-like domain.

2. RESULTS

For all rMMP used, a single protein band was present of the recombinant, as determined by SDS-PAGE, except for rMMP-9, which showed two bands. For SDS-PAGE, proteins were temperature denatured and no proteolysis during heating was observed.

2.1. Denaturation profile recombinant gelatinase A (MMP-2)

Figure 1A, shows the immunoblot of recombinant gelatinase A. With a band at 74 kDa corresponding to rPro-MMP-2 and a band at 65 kDa corresponding to rMMP-2, as previously reported [12]. Figure 1B, represents the predicted protein structures for Pro-MMP-2 and MMP-2. Full length ProMMP-2 contains all four domains; PD (Red ribbons), CD (Green ribbons), HD (Blue ribbons) and FD (fibronectine-like domain) (Yellow ribbons). Removal of the PD from rPro-MMP-2 yields a protein with different conformation, however, the conformations of HD and CD are conserved, as it is shown by the predicted structure. Figure 1C, shows the denaturation profile of rPro-MMP-2 and rMMP-2. After baseline correction, the denaturation profile for rPro-MMP-2 (upper case) shows two transitions, which unfold at Tm (I) 54.1 °C and Tm (II) 71.3 °C, indicating two structural domains that may have strong co-operative interactions, as it has been proposed to occurred for other monomeric proteins where the denaturation profiles yield more than one transitions [13, 14]. Active rMMP-2 (lower case), denatures as a single broad transition with average Tm (I) 67.5 °C. However, a second transition included in the broad transition can be fitted at Tm (II) 78 °C. The enthalpy contributions of rPro-MMP2 and rMMP-2 are similar, as can be seen in Table 2.

Figure 1.

Figure 1

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Analysis of rProMMP-2 and rMMP-2 structures. A) Immune-reactivity of anti MMP-2 antibody with the recombinant Pro-MMP-2 and MMP-2. B) Predicted model for ProMMP-2 (protein modelling based on SWISSPROT No. P08253). Blue ribbon is related to the C-terminal-Hemopexin domain (HD). Green ribbons are related with the N-terminal-catalytic domain (CD). Red ribbon is related to the pro-peptide domain, which is removed upon activation. Yellow ribbon is associated with the fibronectin-like domains (FD). C) DSC profiles of excess Cp as a function of increasing temperature of rPro-MMP-2 (upper panel) and rMMP-2 (lower panel): Experimental data (solid line curve) and best theoretical fit (dotted line curves), with transitions temperatures, labelled I and II.

Table 2.

Thermodynamic parameters of the recombinant MMPs irreversible unfolding.

MMP Peak Tm (°C) ΔH (kJ/mole)
ProMMP-3 I 41.2 ± 0.13 103.76
II 45.7 ± 0.18 157.70
III 80.0 ± 0.05 180.44
Active MMP-3 I 26.4 ± 0.55 172.00
II 83.9 ± 0.06 117.90
CD-MMP-3 I 83.4 ± 0.15 223.00
ProMMP-2 I 54.1 ± 0.08 129.19
II 71.3 ± 0.05 188.67
Active MMP-2 I 69.7 ± 0.22 182.01
II 77.6 ± 0.29 76.9
Active MMP-7 I 98.3 ± 0.27 57.73
Active MMP-8 I 32.0 ± 0.02 24.68
II 74.8 ± 0.37 229.70
ProMMP-9 I 92.6 ± 0.04 233.04
Active MMP-9 I 30.9 ± 0.15 24.26
II 40.7 ± 0.39 94.97
III 76.1 ± 0.04 592.45
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Tm and ΔH correspond to the theoretical fit (n = 2), refer to the absolute error of mean (±AEM) of two different runs.

2.2. Denaturation profile recombinant Stromelysin (MMP-3)

Figure 2A shows the immunoblot of recombinant Stromelysin, 54 and 45 kDa (rPro-MMP-3 and rMMP-3 respectively, as has been previously reported [17]. Figure 2B represents the predicted protein structure for rPro-MMP-3 with the PD (Red ribbons), the HD (Blue ribbons) and CD (Green ribbons). Removal of the PD to yield rMMP-3 conserves the conformation for the predicted CD structure. Figure 2C, shows the denaturation profile of rProMMP-3, rMMP-3 and the isolated catalytic domain rCD of MMP-3. The denaturation profile of rPro-MMP-3 has three defined transitions at Tm (I) 41.2 °C, (II) 45.7 °C and (III) 80 °C (upper case) and the active rMMP-3 shows two transitions at Tm (I) 26.4 y (II) 83.9 °C (Middle case). The unfolding profile of the rCD has a single transition observed at Tm (I) 83.4 °C (lower case). The Tm of rCD is also present in rMMP-3 and in rProMMP-3, indicating that the CD is more stable and structurally independent from the rest of the protein. The enthalpy contribution of each transition is listed in Table 2.

Figure 2.

Figure 2

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Analysis of rMMP-3 structure. A) Immune-reactivity of anti MMP-3 antibody with the recombinant Pro-MMP-3 and MMP-3. B) Predicted model for rProMMP-3 (protein modelling based on SWISSPROT No. P08254). Blue ribbon is related to the C-terminal Hemopexin domain. Green ribbon is related with the N-terminal Catalytic domain. Red ribbon is related to the pro-peptide domain (PD), which is removed upon enzyme activation. C) DSC profiles of excess Cp as a function of temperature of rPro-MMP-3 (upper panel); rMMP-3 (middle panel) and rCD-MMP3 (lower panel). Experimental data (solid line curve) and best theoretical fit (dotted line curves) with transitions temperatures, labelled I, II, and III.

2.3. Denaturation profile of recombinant Matrilysin (MMP-7)

Figure 3A, shows the SDS-PAGE of recombinant Matrilysin, the active form of rMMP-7. A single protein band was observed at 19 kDa, as previously reported for MMP-7 [18]. Figure 2B, represents the CD (Green ribbons) of the predicted protein structure for the smallest member of the MMP family, when compared with MMP-2 and MMP-3. Figure 3C, shows the denaturation profile of rMMP-7, which denatures as a single sharp transition at Tm 98.3 °C, the highest thermal stability observed among all rMMP analysed in this study. The rMMP-7 unfolds with a lower enthalpy compared with the rest of rMMP, see Table 2. Due to the high thermal stability of rMMP-7 it could be possible to relate the denaturation of the full-length rMMP-7 to the structure corresponding to the CD in other MMP.

Figure 3.

Figure 3

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Analysis of rMMP-7 structure. A) SDS-PAGE of rMMP-7 determined by silver staining as described under Material and Methods. B) Predicted model for ProMMP-7 (protein modelling based on SWISSPROT No. P09237). MMP-7 is the catalytic domain represented by green ribbon in the rest of the MMP. C) DSC denaturation profiles of rMMP-7, excess Cp as a function of increasing temperature. Experimental data (solid line curve) and best theoretical fit (dotted line curves).

2.4. Denaturation profile of recombinant MMP-8

Figure 4A, shows the immunoblot of the active rMMP-8 with a single band of 54 kDa, as has been reported [19]. Figure 4B, represents the predicted protein structure of the active form, where the HD (Blue ribbons) and CD (Green ribbons) represent the two separated structural domains predicted for this protein. Figure 4C shows the denaturation profile of rMMP-8, where two transitions are observed at Tm (I) 32 °C and (II) 74.8 °C. The enthalpy contribution of component II, is like that observed for the transition of higher Tm obtained for the other MMP studied, see Table 2.

Figure 4.

Figure 4

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Analysis of rMMP-8 structure. A) SDS-PAGE of rMMP-8. B) Predicted model for ProMMP-8 (protein modelling based on SWISSPROT No. P22894). Blue ribbon is related to the C-terminal Hemopexin domain. Green ribbon is related with the N-terminal, catalytic domain. C) DSC profiles of excess Cp as a function of temperature of rPro-MMP-8. Experimental data (solid line curve) and best theoretical fit (dotted line curves).

2.5. Denaturation profile of recombinant MMP-9

Figure 5A, shows the immunoblot of anti-MMP-9 as a single protein of 92 kDa, which corresponds to recombinant Pro-MMP-9 [20]. However, the active form of the protein rMMP-9 showed two bands [21], in this study, 56% corresponded to the 83 kDa protein as has been previously reported, which results from MMP-2 cleavage [21], and 44% of the rMMP-9 sample, has a 65 kDa protein, which has been also previously reported to result from MMP-3 cleavage [22]. Figure 5B, represents the predicted protein structures for Pro-MMP-9 and MMP-9. Full length ProMMP-9 contains all four domains; PD (Red ribbons), CD (Green ribbons), HD (Blue ribbons) and FD (Yellow ribbons). Removal of the PD from rPro-MMP-2 yields a protein with different conformation, however, the conformations of HD and CD are conserved, as it is shown by the predicted structure. Figure 5C shows, the denaturation profile of the rPro-MMP-9 (upper case) with a single transition Tm 92 °C, whereas the denaturation profile of the rMMP-9 shows three transitions at Tm (I) 31 °C, (II) 41 °C and (III) 76 °C. Although it seems like the contribution of the proteins detected as rMMP-9 in the immunoblot are almost of the same proportion, the denaturation profile reveals three uneven enthalpy contributions of the observed transitions. The enthalpy contribution of rMMP-9 component III, is two-fold higher in comparison to the related component (CD) of rMMP-2, rMMP-3, and rMMP-8 (see Table 2), suggesting that the two proteins observed by immunoblot for rMMP-9, may unfold under the same transition.

Figure 5.

Figure 5

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Analysis of rMMP-9 structure. A) Immune-reactivity of anti MMP-9 antibody to recombinant Pro-MMP-9 and MMP-9. B) Predicted model for Pro-MMP-9 (protein modelling based on SWISSPROT No. P14780). Blue ribbon is related to the C-terminal Hemopexin domain. Green ribbon is related with the N-terminal catalytic domain. Red ribbon is related to the pro-peptide domain, which is removed upon activation. Yellow ribbon is related to the fibronectin-like domains. C) DSC profiles of excess Cp as a function of increasing temperature, for rPro-MMP-9 (upper panel) and rMMP-9 (lower panel). Experimental data (solid line curve) and best theoretical fit (dotted line curves).

2.6. Correlation between DSC denaturation profile parameters and structural characteristics

After calculating thermodynamic parameters from DSC for each metalloproteinase analysed in this study, we aimed to correlate those parameters with other structural ones that could explain the differences in protein stability. Figure 6 shows that if ordered by molecular weight, MMPs could be nicely fitted into graphs describing their thermodynamic properties. MMP-7 is the smallest molecule analysed which showed the highest Tm value, and lowest ΔH. These values are attributable to a very high degree of protein stability (high Tm), and flexibility (low ΔH) [23]. If we calculate CpTm (Cp at Tm) of each active form of MMP using Kirchhoffs law (Cp = ΔH/ΔT) we can attribute a specific CpTm value to each metalloproteinase. In the case of MMP-7 the value is below one (CpTm = 0.59). MMP-7 is also known to belong to the “shallow” S1’ pocket MMP type. S1’ pocket belongs to the selectivity region of the catalytic domain and its geometry and electrostatic features are key players for inhibitors design [24], here we found that our correlation and CpTm value also distinguishes between the S1’ pocket MMP type. A shallower pocket could be associated to a more stable conformation since it has fewer gaps between atoms. In the case of MMP-3 classified as a “deep” S1’ pocket, CpTm increases to 1.4, deeper pockets are surely associated to some degree of “less stable” conformations, and we found it correlated to a lower Tm and higher ΔH, compared to MMP-7. Finally, all the members of the “intermediate” S1' pocket show CpTm values over 2 (MMP-8 CpTm = 3; MMP-2 CpTm = 2,65; MMP-9 CpTm = 7,79) and showed the lower Tm values.

Figure 6.

Figure 6

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Correlation between molecular weight and thermodynamic DSC parameters. Black squares correspond to Tm (°C) axis and white squares correspond to ΔH (kJ/mol) axis. For Tm series, a polynomial regression was performed (y = 0, 0004 × 3 - 0, 0529 × 2 + 1, 4952x + 86, 279, R2 = 0, 99345); for ΔH exponential regression was performed (y = 27, 959 e 0, 0347x. R2 = 0, 92439). MMPs were ordered per molecular weight and divided into three groups: S1 shallow (MMP-7), S1 deep (MMP-3) and S1 intermediate (MMP-8, MMP-2 and MMP-9), these structural features correlated to Tm and ΔH parameters.

3. DISCUSSION

The catalytic activity of MMP is a temperature-dependent processes, which have been previously described for MMP-9, MMP-1 and MMP-3 [25, 26, 27]. There are no evidences, including the present results, which indicate a temperature-dependence activation for auto proteolytic activity of any of the MMP used in this study. Heating rProMMP or rMMP at a rate 1 °C/min up to 95 °C for SDS-PAGE analysis, did not result in proteolysis in any of the MMP tested. Therefore, the different transitions observed during the thermal denaturation of a monomeric MMP, are associated to different structural domains. There are many examples of protein denaturation of human enzymes and enzymes from other mammals, where Tm is over 50 °C [14, 28]. Membrane proteins with enzymatic activity unfold at much lower temperatures especially in mammals [29], for example, SERCA1, has a Tm at 49–50 °C, a protein which we have been working more extensively [15, 30].

In this study, we observed important modifications in the thermodynamic parameters of proenzyme and active forms of MMP; 1) The predicted structures for active and inactive forms of MMP, did not present major changes in the structures related to the HD and CD after the removal of the PD fragment. 2) Pro-MMP and MMP shown a well-defined structural domain, with a high Tm over 70 °C.

Removal of the pro-peptide is required for MMP activation, resulting in protein conformational changes that can be detected by DSC. Gelatinases; ProMMP-9 and ProMMP-2, once activated, revealed a higher and lower temperature transitions, confirming that relevant conformational changes occurred after PD removal, even though the predicted structures show minor changes. The only data reported of calorimetric studies on MMP, corresponds to MMP-2 [31]. A thermal denaturation of this enzyme shows a single broad transition at Tm 72 °C, which is like the transition observed in this study for ProMMP-2, with Tm at 71.3 °C. The small difference between our results can be explained by an incomplete baseline subtraction done in the MMP-2 DSC analysis done in the referred study [31].

The fact that the catalytic domain of MMP-3, unfolds at Tm 83.4 °C, the same transition temperature for one of the components present in the active form of MMP-3 (Tm 83.9 °C), strongly suggests that they corresponded to the same structural domain. The conformation of the CD within the protein, is not affected after PD and HD removal.

To further support the idea that the CD in the rest of the MMP studied corresponded to the transition observed at higher Tm, the MMP-7, which it is only composed of the catalytic domain, had a single transition at Tm 98.3 °C. The high Tm and low enthalpy of rMMP-7 give this protein a restricted temperature range in which the native state of the protein is stable once the Tm is reached, implying that is structural conformation is one of the most stable among the MMP. Differential Scanning Calorimetry add further information on the role of conformational changes during MMP activation and substrate interaction. A restriction of DSC to analyse complex biological samples is the need for highly purified forms of the enzymes for DSC studies, a situation that could be overcome with the use of standardized values of thermal denaturation, such as those presented in this paper, as a reference to identify the specific contribution of enzyme domains in a complex mix of cellular products as it has been previously seen in chorioallantoid membrane during labour [2].

The fact that MMP catalytic domain has a very high Tm, which is not substantially affected after removal of the propeptide domain has provided us insight not only in the understanding of the conformational changes that occur under activation of the pro enzyme, but also give some indication to understand the mechanism of collagenolysis.

4. Experimental procedures

4.1. Reagents and antibodies

Human recombinant active (rMMP) and proenzymes (rPro-MMP) were purchased from Calbiochem (EMD Biosciences, SanDiego, CA) including active MMP-2 and proMMP-2, catalytic domain of MMP-3, active MMP-3 and proMMP-3, active MMP-7, active MMP-8, and active MMP-9 and, proMMP-9. Monoclonal antibodies against MMP-9 (Ab-3), MMP-8 (Ab-1), MMP-3 (Ab-1), were purchased from Calbiochem. Antibody against MMP-2 (Ab-11) was obtained from Pharmingen (BD Biosciences, San Jose, CA). For all MMPs the SDS-PAGE shows a single band except for MMP-9 where two bands were presents as indicated in the technical report.

4.2. Electrophoresis and immunoblotting

Protein content of rMMP was determined by Bradford assay per manufacturer's instructions (Sigma-Aldrich, St Louis, MO). Equal amounts of protein (0.5 μg/sample) were resolved on 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and heated in a thermal cycler at a heating rate of 1ºC/min up to 95 °C. The SDS-PAGE was transferred to nitrocellulose membranes (Amersham Biosciences, NJ, USA). After several washes with PBST, the membranes were incubated with the appropriate secondary antibodies conjugated with horseradish peroxidase (goat anti-mouse 1:3000, BD Biosciences or goat anti-rabbit 1:40,000, Jackson Immunoresearch, WestGrove, PA, USA). Detection of complexes was performed using ECL per the manufacturer's recommendations (Amersham Biosciences, NJ, USA). Densitometry analysis was performed with UVP BioImaging System (UVP, Upland, CA). The relative abundance of MMP bands was determined by image analysis using arbitrary measures with UVP Bioimaging System (UVP, Upland, CA). Molecular weight (MW) of each band was calculated using protein standards (Invitrogen, Life Technologies, Inc. Carlsbad, CA, USA). Profile of MMP was determined by differential scanning calorimetry (DSC).

4.3. Differential scanning calorimetry (DSC)

A high-resolution Microcal VP-DSC (MicroCal LLC, Northampton, MA, USA) was used to obtain all scans. Recombinant proteins were diluted in a buffer containing: 0.1 M KCl, 0.02 M Tris-Maleate at pH 7.0, followed by overnight dialysis against the same buffer. Proteins and reference solutions were carefully degassed under vacuum for 5 min before loading the cells (0.56 ml). The unfolding profile of rPro-MMP-2, rPro-MMP-3 and rPro-MMP-9, and the rMMP active forms; rMMP-2, 3, 7, 8 and 9, were heated at a rate of 1 °C/min from 10 to 100 °C per established protocols [11, 13, 15]. Denaturation was completely irreversible after scanning up to 100 °C. DSC scans were deconvoluted assuming irreversible denaturation. The transition temperature (Tm) is defined as the temperature of half denaturation, and corresponds to the temperature at half the area under the individual peaks after the DSC scans were deconvoluted. This procedure requires that denaturation can be approximated by a two-state reaction of the form

N→kD

that obeys pseudo first-order kinetics, and where the temperature dependence of the rate constant k is given by the Arrhenius relation, as described previously [13, 15]. These assumptions have been shown to hold for the SERCA and PMCA enzymes in skeletal muscle [13, 14], several mammal tissues proteins [15] and for Colicines [16]. The fraction of each component denatured as a function of increasing temperature at a constant rate (fD) is given as,

fD[T(t)]=1−exp{−(RTc2EAv)exp(EA(T−Tc)RTc2)}

where Tc is the temperature at which k = 1, t is time, and v is the scan rate. The derivative of fD as a function of temperature is proportional to the excess Cp. The curves of excess Cp as a function of temperature were deconvoluted into individual components using a recursive minimization routine [13].

4.4 Protein Structure Predictions.Swiss-Model repository, a database of available annotated three-dimensional comparative protein structure models generated by fully automated homology-modelling pipeline from Swiss-model [32] (http://swissmodel.expasy.org/repository) and SwissPdb Viewer (http://expasy.org/spdbv7) were utilized for protein modelling of MMPs. For all cases, the NH2-terminal is oriented in the upper-right side of the molecule that corresponds to the catalytic Domain (CD) -labelled green-, the COOH-terminal which is oriented in the lower-right side of the molecule that corresponds to the hemopexin domain (HD)-labelled blue-, the propeptide domain (PD) -labelled red- and the fibronectin-like domain (FD) -labelled yellow.

Declarations

Author contribution statement

N. Meraz-Cruz: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.

A. Ortega: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

F. Vadillo-Ortega: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

A. Jiménez-Garduño: Analyzed and interpreted the data; Wrote the paper.

Funding statement

F. Vadillo-Ortega was supported by Consejo Nacional de Ciencia y Tecnología, México (C01-7036). A. Ortega was supported by Universidad Nacional Autónoma de México (DGAPA-IN218215 and DGAPA-IN-IN219119).

Competing interest statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.

Acknowledgements

Funding for this project was obtained from CONACyT grant C01-7036, Consejo Nacional de Ciencia y Tecnología, Mexico (to F. Vadillo-Ortega), grants DGAPA-IN218215 and DGAPA-IN- IN219119 (Dirección General del Personal Académico, Universidad Nacional Autónoma de México (UNAM)) (to A. Ortega). We thank M.C. Ibrahim Ramirez for reviewing the manuscript.

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