Novel Insights into the Catalytic Mechanism of Collagenolysis by Zn(II)-Dependent Matrix Metalloproteinase-1.

Collagen hydrolysis, catalyzed by Zn(II)-dependent matrix metalloproteinases (MMPs), is a critical physiological process. Despite previous computational investigations into the catalytic mechanisms of MMP-mediated collagenolysis, a significant knowledge gap in understanding remains regarding the influence of conformational sampling and entropic contributions at physiological temperature on enzymatic collagenolysis. In our comprehensive multilevel computational study, employing quantum mechanics/molecular mechanics (QM/MM) metadynamics (MetD) simulations, we aimed to bridge this gap and provide valuable insights into the catalytic mechanism of MMP-1. Specifically, we compared the full enzyme-substrate complex in solution, clusters in solution, and gas-phase to elucidate insights into MMP-1-catalyzed collagenolysis. Our findings reveal significant differences in the catalytic mechanism when considering thermal effects and the dynamic evolution of the system, contrasting with conventional static potential energy surface QM/MM reaction path studies. Notably, we observed a significant stabilization of the critical tetrahedral intermediate, attributed to contributions from conformational flexibility and entropy. Moreover, we found that protonation of the scissile bond nitrogen occurs via proton transfer from a Zn(II)-coordinated hydroxide rather than from a solvent water molecule. Following C-N bond cleavage, the C-terminus remains coordinated to the catalytic Zn(II), while the N-terminus forms a hydrogen bond with a solvent water molecule. Subsequently, the release of the C-terminus is facilitated by the coordination of a water molecule. Our study underscores the pivotal role of protein conformational dynamics at physiological temperature in stabilizing the transition state of the rate-limiting step and key intermediates, compared to the corresponding reaction in solution. These fundamental insights into the mechanism of collagen degradation provide valuable guidance for the development of MMP-1-specific inhibitors.

Mechanism of the Early Catalytic Events in the Collagenolysis by Matrix Metalloproteinase-1.

Metalloproteinase-1 (MMP-1) catalyzed collagen degradation is essential for a wide variety of normal physiological processes, while at the same time contributing to several diseases in humans. Therefore, a comprehensive understanding of this process is of great importance. Although crystallographic and spectroscopic studies provided fundamental information about the structure and function of MMP-1, the precise mechanism of collagen degradation especially considering the complex and flexible structure of the substrate, remains poorly understood. In addition, how the protein environment dynamically reorganizes at the atomic scale into a catalytically active state capable of collagen hydrolysis remains unknown. In this study, we applied experimentally-guided multiscale molecular modeling methods including classical molecular dynamics (MD), well-tempered (WT) classical metadynamics (MetD), combined quantum mechanics/molecular mechanics (QM/MM) MD and QM/MM MetD simulations to explore and characterize the early catalytic events of MMP-1 collagenolysis. Importantly the study provided a complete atomic and dynamic description of the transition from the open to the closed form of the MMP-1•THP complex. Notably, the formation of catalytically active Michaelis complex competent for collagen cleavage was characterized. The study identified the changes in the coordination state of the catalytic zinc(II) associated with the conformational transformation and the formation of catalytically productive ES complex. Our results confirm the essential role of the MMP-1 catalytic domain's α-helices (hA, hB and hC) and the linker region in the transition to the catalytically competent ES complex. Overall, the results provide unique mechanistic insight into the conformational transformations and associated changes in the coordination state of the catalytic zinc(II) that would be important for the design of effective MMP-1 inhibitors.

Suppression of cigarette smoke induced MMP1 expression by selective serotonin re-uptake inhibitors.

Globally, COPD remains a major cause of disability and death. In the United States alone, it is estimated that approximately 14 million people suffer from the disease. Given the high disease burden and requirement for chronic, long-term medical care associated with COPD, it is essential that new disease modifying agents are developed to complement the symptomatic therapeutics currently available. In the present report, we have identified a potentially novel therapeutic agent through the use of a high throughput screen based on the knowledge that cigarette smoke induces the proteolytic enzyme MMP1 leading to destruction of the lung in COPD. A construct utilizing the cigarette responsive promoter element of MMP-1 was conjugated to a luciferase reporter and utilized in an in vitro assay to screen the NIH Molecular Libraries Small Molecule Repository to identify putative targets that suppressed luciferase expression in response to cigarette smoke extract (CSE). Selective serotonin reuptake inhibitors potently inhibited luciferase expression and were further validated. SSRI treatment suppressed MMP-1 production in small airway epithelial cells exposed to (CSE) in vitro as well as in smoke exposed rabbits. In addition, SSRI treatment inhibited inflammatory cytokine production while rescuing cigarette smoke induced downregulation in vivo of the anti-inflammatory lipid transporter ABCA1, previously shown by our laboratory to be lung protective. Importantly, SSRI treatment prevented lung destruction in smoke exposed rabbits as measured by morphometry. These studies support further investigation into SSRIs as a novel therapeutic for COPD may be warranted.

Comparison of Different Reweighting Approaches for the Calculation of Conformational Variability of Macromolecules from Molecular Simulations.

Conformational variability and heterogeneity are crucial determinants of the function of biological macromolecules. The possibility of accessing this information experimentally suffers from severe under-determination of the problem, since there are a few experimental observables to be accounted for by a (potentially) infinite number of available conformational states. Several computational methods have been proposed over the years in order to circumvent this theoretically insurmountable obstacle. A large share of these strategies is based on reweighting an initial conformational ensemble which arises from, for example, molecular simulations of different qualities and levels of theory. In this work, we compare the outcome of three reweighting approaches based on radically different views of the conformational heterogeneity problem, namely Maximum Entropy, Maximum Parsimony and Maximum Occurrence, and we do so using the same experimental data. In this comparison we find both expected as well as unexpected similarities.

A Model for the Solution Structure of Human Fe(II)-Bound Acireductone Dioxygenase and Interactions with the Regulatory Domain of Matrix Metalloproteinase I (MMP-I).

The metalloenzyme acireductone dioxygenase (ARD) shows metal-dependent physical and enzymatic activities depending upon the metal bound in the active site. The Fe(II)-bound enzyme catalyzes the penultimate step of the methionine salvage pathway, converting 1,2-dihydroxy-5-(methylthio)pent-1-en-3-one (acireductone) into formate and the ketoacid precursor of methionine, 2-keto-4-thiomethyl-2-oxobutanoate, using O2 as the oxidant. If Ni(II) is bound, an off-pathway shunt occurs, producing 3-methylthiopropionate, formate, and carbon monoxide from the same acireductone substrate. The solution structure of the Fe(II)-bound human enzyme, HsARD, is described and compared with the structures of Ni-bound forms of the closely related mouse enzyme, MmARD. Potential rationales for the different reactivities of the two isoforms are discussed. The human enzyme has been found to regulate the activity of matrix metalloproteinase I (MMP-I), which is involved in tumor metastasis, by binding the cytoplasmic transmembrane tail peptide of MMP-I. Nuclear magnetic resonance titration of HsARD with the MMP-I tail peptide permits identification of the peptide binding site on HsARD, a cleft anterior to the metal binding site adjacent to a dynamic proline-rich loop.

The expression, purification, and substrate analysis of matrix metalloproteinases in Drosophila melanogaster.

Matrix metalloproteinases (MMPs) are evolutionarily conserved extracellular matrix proteinases. Genetic analysis of the Drosophila MMPs, Mmp1 and Mmp2, in vivo reveal that they play vital roles in tissue remodeling. Although the catalytic domain (CD) undertakes most MMP functions, few studies have sought to demonstrate the biochemical properties of the CDs of fly MMPs. Here, we identified the overexpression, purification, and refolding of the CDs of Drosophila Mmp1 and Mmp2 for biochemical studies. Zymography assays and substrate degradation analysis showed that both Mmp1-CD and Mmp2-CD were able to digest casein, gelatin, fibronectin, collagen (types I, IV, and V), while Mmp2-CD showed much higher degradation activity compared with Mmp1-CD. Moreover, human collagen III could be degraded by Mmp1-CD but not Mmp2-CD, and rat collagen I and laminin could be degraded by Mmp2-CD but not Mmp1-CD, suggesting that Drosophila Mmp1 and Mmp2 might have overlapping yet distinct substrate specificity. Using synthetic fluorescent substrates, we further demonstrated that the enzymatic activity of Mmp1-CD and Mmp2-CD could be inhibited by human tissue inhibitors of metalloproteinases (TIMPs). These results reveal the context of the cooperative yet distinct roles of Mmp1 and Mmp2 in tissue remodeling.

Homocysteine Disrupts Balance between MMP-9 and Its Tissue Inhibitor in Diabetic Retinopathy: The Role of DNA Methylation.

High homocysteine is routinely observed in diabetic patients, and this non-protein amino acid is considered as an independent risk factor for diabetic retinopathy. Homocysteine biosynthesis from methionine forms S-adenosyl methionine (SAM), which is a major methyl donor critical in DNA methylation. Hyperhomocysteinemia is implicated in increased oxidative stress and activation of MMP-9, and in diabetic retinopathy, the activation of MMP-9 facilitates capillary cell apoptosis. Our aim was to investigate the mechanism by which homocysteine activates MMP-9 in diabetic retinopathy. Human retinal endothelial cells, incubated with/without 100 µM homocysteine, were analyzed for MMP-9 and its tissue inhibitor Timp1 expressions and interactions, and ROS levels. Timp1 and MMP-9 promoters were analyzed for methylated and hydroxymethylated cytosine levels (5mC and 5hmC respectively) by the DNA capture method, and DNA- methylating (Dnmt1) and hydroxymethylating enzymes (Tet2) binding by chromatin immunoprecipitation. The results were confirmed in retinal microvessels from diabetic rats receiving homocysteine. Homocysteine supplementation exacerbated hyperglycaemia-induced MMP-9 and ROS levels and decreased Timp1 and its interactions with MMP-9. Homocysteine also aggravated Dnmts and Tets activation, increased 5mC at Timp1 promoter and 5hmC at MMP-9 promoter, and suppressed Timp1 transcription and activated MMP-9 transcription. Similar results were obtained from retinal microvessels from diabetic rats receiving homocysteine. Thus, hyperhomocysteinemia in diabetes activates MMP-9 functionally by reducing Timp1-MMP-9 interactions and transcriptionally by altering DNA methylation-hydroxymethylation of its promoter. The regulation of homocysteine could prevent/slow down the development of retinopathy and prevent their vision loss in diabetic patients.

Synthesis, Molecular Docking, and In Vitro Boron Neutron Capture Therapy Assay of Carboranyl Sinomenine.

In comparison with pristine sinomenine and carborane precursors, the calculations of molecular docking with matrix metalloproteinases (MMPs) and methylcarboranyl-n-butyl sinomenine showed improved interactions. Accordingly, methylcarboranyl-n-butyl sinomenine shows a high potential in the treatment of rheumatoid arthritis (RA) in the presence of slow neutrons. The reaction of potassium salt of sinomenie, which is generated from the deprotonation of sinomenine (1) using potassium carbonate in a solvent of N,N-dimethyl formamide, with 4-methylcarboranyl-n-butyl iodide, (2) forms methylcarboranyl-n-butyl sinomenine (3) in 54.3% yield as a new product. This new compound was characterized by 1H, 13C, and 11B NMR spectroscopy, FT-IR spectroscopy, and elemental analyses to confirm its molecular composition. In addition to molecular docking interactions with MMPs, the in vitro killing effects of 3, along with its toxicity measurements, exhibited its potential to be the new drug delivery agent for boron neutron capture synovectomy (BNCS) and boron neutron capture therapy (BNCT) for the treatment of rheumatoid arthritis (RA) and cancers in the presence of slow neutrons, respectively.

Effects of flexibility of the α2 chain of type I collagen on collagenase cleavage.

Cleavage of collagen by collagenases such as matrix metalloproteinase 1 (MMP-1) is a key step in development, tissue remodeling, and tumor proliferation. The abundant heterotrimeric type I collagen composed of two α1(I) chains and one α2(I) chain is efficiently cleaved by MMP-1 at a unique site in the triple helix, a process which may be initiated by local unfolding within the peptide chains. Atypical homotrimers of the α1(I) chain, found in embryonic and cancer tissues, are very resistant to MMP cleavage. To investigate MMP-1 cleavage, recombinant homotrimers were constructed with sequences from the MMP cleavage regions of human collagen chains inserted into a host bacterial collagen protein system. All triple-helical constructs were cleaved by MMP-1, with α2(I) homotrimers cleaved efficiently at a rate similar to that seen for α1(II) and α1(III) homotrimers, while α1(I) homotrimers were cleaved at a much slower rate. The introduction of destabilizing Gly to Ser mutations within the human collagenase susceptible region of the α2(I) chain did not interfere with MMP-1 cleavage. Molecular dynamics simulations indicated a greater degree of transient hydrogen bond breaking in α2(I) homotrimers compared with α1(I) homotrimers at the MMP-1 cleavage site, and showed an extensive disruption of hydrogen bonding in the presence of a Gly to Ser mutation, consistent with chymotrypsin digestion results. This study indicates that α2(I) homotrimers are susceptible to MMP-1, proves that the presence of an α1(I) chain is not a requirement for α2(I) cleavage, and supports the importance of local unfolding of α2(I) in collagenase cleavage.

Efficient protease based purification of recombinant matrix metalloprotease-1 in E. coli.

MMP1 is an essential enzyme for tissue remodeling both in normal and pathological states. We report a method of purifying activated human MMP1 in E. coli without using urea or 4-Aminophenylmercuric acetate (APMA). Instead, a non-ionic detergent, Triton X-100, was used in the lysis buffer to solubilize MMP1 followed by the protease activities of both trypsin and MMP1 to digest E. coli proteins and activate pro-MMP1. Identity of activated MMP1 was confirmed by Western blot using anti-human MMP1 antibodies, whereas the mass was determined to be 43 kD using matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF-MS). Collagen and gelatin degradation by purified MMP1 were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) of degraded FITC-labeled type-1 collagen and gelatin zymogram. Broad-spectrum protease activity of purified MMP1 was also confirmed by lysis of native E. coli proteins. Inexpensive high throughput purification of recombinant human MMP1 in E. coli will enable easier MMP1 production for diverse applications.

Thermodynamic Basis of Selectivity in the Interactions of Tissue Inhibitors of Metalloproteinases N-domains with Matrix Metalloproteinases-1, -3, and -14.

The four tissue inhibitors of metalloproteinases (TIMPs) are potent inhibitors of the many matrixins (MMPs), except that TIMP1 weakly inhibits some MMPs, including MMP14. The broad-spectrum inhibition of MMPs by TIMPs and their N-domains (NTIMPs) is consistent with the previous isothermal titration calorimetric finding that their interactions are entropy-driven but differ in contributions from solvent and conformational entropy (ΔSsolv, ΔSconf), estimated using heat capacity changes (ΔCp). Selective engineered NTIMPs have potential applications for treating MMP-related diseases, including cancer and cardiomyopathy. Here we report isothermal titration calorimetric studies of the effects of selectivity-modifying mutations in NTIMP1 and NTIMP2 on the thermodynamics of their interactions with MMP1, MMP3, and MMP14. The weak inhibition of MMP14 by NTIMP1 reflects a large conformational entropy penalty for binding. The T98L mutation, peripheral to the NTIMP1 reactive site, enhances binding by increasing ΔSsolv but also reduces ΔSconf However, the same mutation increases NTIMP1 binding to MMP3 in an interaction that has an unusual positive ΔCp This indicates a decrease in solvent entropy compensated by increased conformational entropy, possibly reflecting interactions involving alternative conformers. The NTIMP2 mutant, S2D/S4A is a selective MMP1 inhibitor through electrostatic effects of a unique MMP-1 arginine. Asp-2 increases reactive site polarity, reducing ΔCp, but increases conformational entropy to maintain strong binding to MMP1. There is a strong negative correlation between ΔSsolv and ΔSconf for all characterized interactions, but the data for each MMP have characteristic ranges, reflecting intrinsic differences in the structures and dynamics of their free and inhibitor-bound forms.

Identification of Conserved Water Sites in Protein Structures for Drug Design.

Identification of conserved waters in protein structures is a challenging task with applications in molecular docking and protein stability prediction. As an alternative to computationally demanding simulations of proteins in water, experimental cocrystallized waters in the Protein Data Bank (PDB) in combination with a local structure alignment algorithm can be used for reliable prediction of conserved water sites. We developed the ProBiS H2O approach based on the previously developed ProBiS algorithm, which enables identification of conserved water sites in proteins using experimental protein structures from the PDB or a set of custom protein structures available to the user. With a protein structure, a binding site, or an individual water molecule as a query, ProBiS H2O collects similar proteins from the PDB and performs local or binding site-specific superimpositions of the query structure with similar proteins using the ProBiS algorithm. It collects the experimental water molecules from the similar proteins and transposes them to the query protein. Transposed waters are clustered by their mutual proximity, which enables identification of discrete sites in the query protein with high water conservation. ProBiS H2O is a robust and fast new approach that uses existing experimental structural data to identify conserved water sites on the interfaces of protein complexes, for example protein-small molecule interfaces, and elsewhere on the protein structures. It has been successfully validated in several reported proteins in which conserved water molecules were found to play an important role in ligand binding with applications in drug design.

Effects of Mutations on Structure-Function Relationships of Matrix Metalloproteinase-1.

Matrix metalloproteinase-1 (MMP-1) is one of the most widely studied enzymes involved in collagen degradation. Mutations of specific residues in the MMP-1 hemopexin-like (HPX) domain have been shown to modulate activity of the MMP-1 catalytic (CAT) domain. In order to reveal the structural and conformational effects of such mutations, a molecular dynamics (MD) study was performed of in silico mutated residues in the X-ray crystallographic structure of MMP-1 complexed with a collagen-model triple-helical peptide (THP). The results indicate an important role of the mutated residues in MMP-1 interactions with the THP and communication between the CAT and the HPX domains. Each mutation has a distinct impact on the correlated motions in the MMP-1•THP. An increased collagenase activity corresponded to the appearance of a unique anti-correlated motion and decreased correlated motions, while decreased collagenase activity corresponded both to increased and decreased anti-correlated motions.

The effect of pomegranate juice on clinical signs, matrix metalloproteinases and antioxidant status in patients with knee osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is one of the commonest forms of musculoskeletal disorders that leads to joint degeneration and has a major impact on patients' quality of life. Experimental and in vitro studies have suggested the protective roles of pomegranate juice (PJ) as a rich antioxidant source for mitigating cartilage inflammation. In this interventional study, 38 patients with knee OA were randomly divided into two groups: PJ or control for 6 weeks to evaluate the effect of this intervention on clinical signs, inflammation and antioxidant status. RESULTS: Significant decreases in Western Ontario and McMaster Universities Osteoarthritis index (WOMAC) total score (P = 0.01), stiffness score (P = 0.00) and physical function score (P = 0.01) were observed in PJ group after the intervention. The means of serum levels of matrix metalloproteinase (MMP)-13 was significantly decreased (P = 0.02) and glutathione peroxidase was increased in the intervention group compared with the control group after the study period (P = 0.02). CONCLUSIONS: According to the findings of this clinical trial, PJ consumption can improve physical function and stiffness, decrease breakdown cartilage enzymes and increase antioxidant status in patients with knee OA. © 2016 Society of Chemical Industry.

Collagenolytic Matrix Metalloproteinase Activities toward Peptomeric Triple-Helical Substrates.

Although collagenolytic matrix metalloproteinases (MMPs) possess common domain organizations, there are subtle differences in their processing of collagenous triple-helical substrates. In this study, we have incorporated peptoid residues into collagen model triple-helical peptides and examined MMP activities toward these peptomeric chimeras. Several different peptoid residues were incorporated into triple-helical substrates at subsites P3, P1, P1', and P10' individually or in combination, and the effects of the peptoid residues were evaluated on the activities of full-length MMP-1, MMP-8, MMP-13, and MMP-14/MT1-MMP. Most peptomers showed little discrimination between MMPs. However, a peptomer containing N-methyl Gly (sarcosine) in the P1' subsite and N-isobutyl Gly (NLeu) in the P10' subsite was hydrolyzed efficiently only by MMP-13 [nomenclature relative to the α1(I)772-786 sequence]. Cleavage site analysis showed hydrolysis at the Gly-Gln bond, indicating a shifted binding of the triple helix compared to the parent sequence. Favorable hydrolysis by MMP-13 was not due to sequence specificity or instability of the substrate triple helix but rather was based on the specific interactions of the P7' peptoid residue with the MMP-13 hemopexin-like domain. A fluorescence resonance energy transfer triple-helical peptomer was constructed and found to be readily processed by MMP-13, not cleaved by MMP-1 and MMP-8, and weakly hydrolyzed by MT1-MMP. The influence of the triple-helical structure containing peptoid residues on the interaction between MMP subsites and individual substrate residues may provide additional information about the mechanism of collagenolysis, the understanding of collagen specificity, and the design of selective MMP probes.

Metal Ion Dependence of the Matrix Metalloproteinase-1 Mechanism.

Matrix metalloproteinase-1 (MMP-1) plays crucial roles in disease-related physiologies and pathological processes in the human body. We report here solution studies of MMP-1, including characterization of a series of mutants designed to bind metal in either the catalytic site or the structural site (but not both). Circular dichroism and fluorescence spectroscopy of the mutants demonstrate the importance of the structural Zn(II) in maintaining both secondary and tertiary structure, while UV-visible, nuclear magnetic resonance, electron paramagnetic resonance, and extended X-ray absorption fine structure show its presence influences the catalytic metal ion's coordination number. The mutants allow us to demonstrate convincingly the preparation of a mixed-metal analogue, Co(C)Zn(S)-MMP-1, with Zn(II) in the structural site and Co(II) in the catalytic site. Stopped-flow fluorescence of the native form, Zn(C)Zn(S)-MMP-1, and the mixed-metal Co(C)Zn(S)-MMP-1 analogue shows that the internal fluorescence of a nearby Trp residue is modulated with catalysis and can be used to monitor reactivity under a number of conditions, opening the door to substrate profiling.

Expression of matrix metalloproteinases in bovine luteal cells induced by prostaglandin F2α, interferon γ and tumor necrosis factor α.

We recently demonstrated that luteal cells flow out from the ovary via lymphatic vessels during luteolysis. However, the regulatory mechanisms of the outflow of luteal cells are not known. Matrix metalloproteinases (MMPs) can degrade the extracellular matrix and basal membrane, and tissue inhibitors of matrix metalloproteinases (TIMPs) inhibit the activity of MMPs. To test the hypothesis that MMP expression in luteal cells is regulated by luteolytic factors, we investigated the effects of prostaglandin F2α (PGF), interferon γ (IFNG) and tumor necrosis factor α (TNF) on the mRNA expression of MMPs and TIMPs in cultured luteal cells. Luteal cells obtained from the CL at the mid-luteal stage (days 8-12 after ovulation) were cultured with PGF (0.01, 0.1, 1 µM), IFNG (0.05, 0.5, 5 nM) and TNF (0.05, 0.5, 0.5 nM) alone or in combination for 24 h. PGF and IFNG significantly increased the expression of MMP-1 mRNA. In addition, 1 µM PGF in combination with 5 nM IFNG stimulated MMP-1 and MMP-9 mRNA expression significantly more than either treatment alone. In contrast, IFNG significantly decreased the level of MMP-14 mRNA. The mRNA expression of TIMP-1, which preferentially inhibits MMP-1, was suppressed by 5 nM INFG. One µM PGF and 5 nM IFNG suppressed TIMP-2 mRNA expression. These results suggest a new role of MMPs: luteal MMPs stimulated by PGF and IFNG break down the extracellular matrix surrounding luteal cells, which accelerates detachment from the CL during luteolysis, providing an essential prerequisite for outflow of luteal cells from the CL to lymphatic vessels.

Structural insights into triple-helical collagen cleavage by matrix metalloproteinase 1.

Collagenases of the matrix metalloproteinase (MMP) family play major roles in morphogenesis, tissue repair, and human diseases, but how they recognize and cleave the collagen triple helix is not fully understood. Here, we report temperature-dependent binding of a catalytically inactive MMP-1 mutant (E200A) to collagen through the cooperative action of its catalytic and hemopexin domains. Contact between the two molecules was mapped by screening the Collagen Toolkit peptide library and by hydrogen/deuterium exchange. The crystal structure of MMP-1(E200A) bound to a triple-helical collagen peptide revealed extensive interactions of the 115-Å-long triple helix with both MMP-1 domains. An exosite in the hemopexin domain, which binds the leucine 10 residues C-terminal to the scissile bond, is critical for collagenolysis and represents a unique target for inhibitor development. The scissile bond is not correctly positioned for hydrolysis in the crystallized complex. A productive binding mode is readily modeled, without altering the MMP-1 structure or the exosite interactions, by axial rotation of the collagen homotrimer. Interdomain flexing of the enzyme and a localized excursion of the collagen chain closest to the active site, facilitated by thermal loosening of the substrate, may lead to the first transition state of collagenolysis.

Examination of matrix metalloproteinase-1 in solution: a preference for the pre-collagenolysis state.

Catalysis of collagen degradation by matrix metalloproteinase 1 (MMP-1) has been proposed to critically rely on flexibility between the catalytic (CAT) and hemopexin-like (HPX) domains. A rigorous assessment of the most readily accessed conformations in solution is required to explain the onset of substrate recognition and collagenolysis. The present study utilized paramagnetic NMR spectroscopy and small angle x-ray scattering (SAXS) to calculate the maximum occurrence (MO) of MMP-1 conformations. The MMP-1 conformations with large MO values (up to 47%) are restricted into a relatively small conformational region. All conformations with high MO values differ largely from the closed MMP-1 structures obtained by x-ray crystallography. The MO of the latter is ~20%, which represents the upper limit for the presence of this conformation in the ensemble sampled by the protein in solution. In all the high MO conformations, the CAT and HPX domains are not in tight contact, and the residues of the HPX domain reported to be responsible for the binding to the collagen triple-helix are solvent exposed. Thus, overall analysis of the highest MO conformations indicated that MMP-1 in solution was poised to interact with collagen and then could readily proceed along the steps of collagenolysis.

Mutations in the catalytic domain of human matrix metalloproteinase-1 (MMP-1) that allow for regulated activity through the use of Ca2+.

Conditionally active proteins regulated by a physiological parameter represent a potential new class of protein therapeutics. By systematically creating point mutations in the catalytic and linker domains of human MMP-1, we generated a protein library amenable to physiological parameter-based screening. Mutants screened for temperature-sensitive activity had mutations clustered at or near amino acids critical for metal binding. One mutant, GVSK (Gly(159) to Val, Ser(208) to Lys), contains mutations in regions of the catalytic domain involved in calcium and zinc binding. The in vitro activity of GVSK at 37 °C in high Ca(2+) (10 mm) was comparable with MMP-1 (wild type), but in low Ca(2+) (1 mm), there was an over 10-fold loss in activity despite having similar kinetic parameters. Activity decreased over 50% within 15 min and correlated with the degradation of the activated protein, suggesting that GVSK was unstable in low Ca(2+). Varying the concentration of Zn(2+) had no effect on GVSK activity in vitro. As compared with MMP-1, GVSK degraded soluble collagen I at the high but not the low Ca(2+) concentration. In vivo, MMP-1 and GVSK degraded collagen I when perfused in Zucker rat ventral skin and formed higher molecular weight complexes with α2-macroglobulin, an inhibitor of MMPs. In vitro and in vivo complex formation and subsequent enzyme inactivation occurred faster with GVSK, especially at the low Ca(2+) concentration. These data suggest that the activity of the human MMP-1 mutant GVSK can be regulated by Ca(2+) both in vitro and in vivo and may represent a novel approach to engineering matrix-remodeling enzymes for therapeutic applications.