Substrate specificity determinants of human macrophage elastase (MMP-12) based on the 1.1 A crystal structure

The macrophage elastase enzyme (MMP-12) expressed mainly in alveolar macrophages has been identified in the mouse lung as the main destructive agent associated with cigarette smoking, which gives rise to emphysema, both directly via elastin degradation and indirectly by disturbing the proteinase/antiproteinase balance via inactivation of the alpha1-proteinase inhibitor (alpha1-PI), the antagonist of the leukocyte elastase. The catalytic domain of human recombinant MMP-12 has been crystallized in complex with the broad-specificity inhibitor batimastat (BB-94). The crystal structure analysis of this complex, determined using X-ray data to 1.1 A and refined to an R-value of 0.165, reveals an overall fold similar to that of other MMPs. However, the S-shaped double loop connecting strands III and IV is fixed closer to the beta-sheet and projects its His172 side-chain further into the rather hydrophobic active-site cleft, defining the S3 and the S1-pockets and separating them from each other to a larger extent than is observed in other MMPs. The S2-site is planar, while the characteristic S1'-subsite is a continuous tube rather than a pocket, in which the MMP-12-specific Thr215 replaces a Val residue otherwise highly conserved in almost all other MMPs. This alteration might allow MMP-12 to accept P1' Arg residues, making it unique among MMPs. The active-site cleft of MMP-12 is well equipped to bind and efficiently cleave the AlaMetPhe-LeuGluAla sequence in the reactive-site loop of alpha1-PI, as occurs experimentally. Similarities in contouring and particularly a common surface hydrophobicity both inside and distant from the active-site cleft explain why MMP-12 shares many substrates with matrilysin (MMP-7). The MMP-12 structure is an excellent template for the structure-based design of specific inhibitors for emphysema therapy and for the construction of mutants to clarify the role of this MMP.     

Crystal structures of the catalytic domain of human stromelysin-1 (MMP-3) and collagenase-3 (MMP-13) with a hydroxamic acid inhibitor SM-25453

Crystal structures of the catalytic domain of human stromelysin-1 (MMP-3) and collagenase-3 (MMP-13) with a hydroxamic acid inhibitor SM-25453 have been solved at 2.01 and 2.37A resolutions, respectively. The results revealed that the binding modes for this inhibitor to MMP-3 and -13 were quite similar. However, subtle comparative differences were observed at the bottom of S1' pockets, which were occupied with the guanidinomethyl moiety of the inhibitor. A remarkable feature of the inhibitor was the deep penetration of its long aliphatic chain into the S1' pocket and exposure of the guanidinomethyl moiety to the solvent.     

High-resolution solution structure of the catalytic fragment of human collagenase-3 (MMP-13) complexed with a hydroxamic acid inhibitor

The high-resolution solution structure of the catalytic fragment of human collagenase-3 (MMP-13) complexed with a sulfonamide derivative of a hydroxamic acid compound (WAY-151693) has been determined by multidimensional heteronuclear NMR. A total of 30 structures were calculated for residues 7-164 by means of hybrid distance geometry-simulated annealing using a total of 3280 experimental NMR restraints. The atomic rms distribution about the mean coordinate positions for the 30 structures is 0.43(+/-0.05) A for the backbone atoms, 0.80(+/-0.09) A for all atoms, and 0.47(+/-0.04) A for all atoms excluding disordered side-chains. The overall structure of MMP-13 is composed of a beta-sheet consisting of five beta-strands in a mixed parallel and anti-parallel arrangement and three alpha-helices where its overall fold is consistent with previously solved MMP structures. A comparison of the NMR structure of MMP-13 with the published 1.6 A resolution X-ray structure indicates that the major differences between the structures is associated with loop dynamics and crystal-packing interactions. The side-chains of some active-site residues for the NMR and X-ray structures of MMP-13 adopt distinct conformations. This is attributed to the presence of unique inhibitors in the two structures that encounter distinct interactions with MMP-13. The major structural difference observed between the MMP-13 and MMP-1 NMR structures is the relative size and shape of the S1' pocket where this pocket is significantly longer for MMP-13, nearly reaching the surface of the protein. Additionally, MMP-1 and MMP-13 exhibit different dynamic properties for the active-site loop and the structural Zn-binding region. The inhibitor WAY-151693 is well defined in the MMP-13 active-site based on a total of 52 distance restraints. The binding motif of WAY-151693 in the MMP-13 complex is consistent with our previously reported MMP-1:CGS-27023A NMR structure and is similar to the MMP-13: RS-130830 X-ray structure.     

Induction of macrophage elastase (MMP-12) gene expression by statins

The statins (including mevastatin and lovastatin) are a widely prescribed class of serum-cholesterol lowering drugs that function by inhibiting 3-hydroxymethylglutaryl coenzyme A (HMG CoA) reductase activity and cellular sterol synthesis. Statins are also widely being appreciated for their inhibitory effects upon inflammation, primarily mediated through direct regulation of inflammatory gene expression. Here we report that statins are also capable of increasing the expression of macrophage elastase (MMP-12). The induction of MMP-12 in mouse macrophages by statins is specific for HMG CoA reductase inhibition, rescued by mevalonate and not observed after inhibition of subsequent steps in the cholesterol biosynthetic pathway. Modulation of cholesterol metabolism may lead to changes in MMP-12 expression and subsequent impacts during physiological and pathophysiological states. We conclude that statins, in addition to their previously described anti-inflammatory properties, may promote the production of some proteinases from activated macrophages.     

Proteinase-activated receptor-1 regulation of macrophage elastase (MMP-12) secretion by serine proteinases

The serine proteinases plasmin and thrombin convert proenzyme matrix metalloproteinases (MMPs) into catalytically active forms. In addition, we demonstrate that plasmin(ogen) and thrombin induce a significant increase in secretion of activated murine macrophage elastase (MMP-12) protein. Active serine protease is responsible for induction, as demonstrated by the absence of MMP-12 induction in plasminogen(Plg)-treated urokinase-type plasminogen activator-deficient macrophages. Since increased MMP-12 protein secretion was not accompanied by an increase in MMP-12 mRNA, we examined post-translational mechanisms. Protein synthesis was not required for early release of MMP-12 but was required for later secretion of activated enzyme. Immunofluorescent microscopy demonstrated basal expression in macrophages that increased following serine proteinase exposure. Inhibition of MMP-12 secretion by hirudin and pertussis toxin demonstrated a role for the thrombin G protein-coupled receptor (protease-activated receptor 1 (PAR-1)). PAR-1-activating peptides were able to induce MMP-12 release. Investigation of signal transduction pathways involved in this response demonstrate the requirement for protein kinase C, but not tyrosine kinase, activity. These data demonstrate that plasmin and thrombin regulate MMP-12 activity through distinct mechanisms: post-translational secretion of preformed MMP-12 protein, induction of protein secretion that is protein kinase C-mediated, and extracellular enzyme activation. Most importantly, we show that serine proteinase MMP-12 regulation in macrophages occurs via the protein kinase C-activating G protein-coupled receptor PAR-1.     

Over-expression and refolding of isotopically labeled recombinant catalytic domain of human macrophage elastase (MMP-12) for NMR studies

Human macrophage elastase (MMP-12) plays an important role in inflammatory processes and is involved in a number of physiological or pathological situations, such as conversion of plasminogen into angiostatin, allergic airway inflammation, vascular remodeling or alteration, as well as emphysema, and has been justified as a novel drug target. Here, we report the over-expression in Escherichia coil, purification and refolding of MMP-12 catalytic domain for NMR studies. The primary sequence of expressed protein was identified by means of MALDI-TOF MS, and was confirmed by the MALDI-TOF MS data of trypsin-digested peptides. A significantly optimized protocol has been worked out to prepare 15N and/or 13C-labeled MMP-12 catalytic domain, and the yield of the purified protein is estimated to 10-12 mg from 0.5L of M9 minimal media. Finally, the 15N-1H HSQC spectrum of uniformly 15N-labeled MMP-12 catalytic domain indicates the presence of well-ordered and properly folded protein in a monomeric form.     

Crystal structure of human BACE2 in complex with a hydroxyethylamine transition-state inhibitor

BACE2 is a membrane-bound aspartic protease of the A1 family with a high level of sequence homology to BACE1. While BACE1 is involved in the generation of amyloid plaques in Alzheimer's disease by cleaving Abeta-peptides from the amyloid precursor protein, the physiological function of BACE2 is not well understood. BACE2 appears to be associated with the early onset of dementia in patients with Down's syndrome, and it has been shown to be highly expressed in breast cancers. Further, it may participate in the function of normal and abnormal processes of human muscle biology. Similar to other aspartic proteases, BACE2 is expressed as an inactive zymogen requiring the cleavage of its pro-sequence during the maturation process. We have produced mature BACE2 by expression of pro-BACE2 in Escherichia coli as inclusion bodies, followed by refolding and autocatalytic activation at pH 3.4. Using a C and N-terminally truncated BACE2 variant, we were able to crystallize and determine the crystal structure of mature BACE2 in complex with a hydroxyethylamine transition-state mimetic inhibitor at 3.1 angstroms resolution. The structure of BACE2 follows the general fold of A1 aspartic proteases. However, similar to BACE1, its C-terminal domain is significantly larger than that of the other family members. Furthermore, the structure of BACE2 reveals differences in the S3, S2, S1' and S2' active site substrate pockets as compared to BACE1, and allows, therefore, for a deeper understanding of the structural features that may facilitate the design of selective BACE1 or BACE2 inhibitors.     

Crystal structure of human thymidine phosphorylase in complex with a small molecule inhibitor

Human thymidine phosphorylase (HTP), also known as platelet-derived endothelial cell growth factor (PD-ECGF), is overexpressed in certain solid tumors where it is linked to poor prognosis. HTP expression is utilized for certain chemotherapeutic strategies and is also thought to play a role in tumor angiogenesis. We determined the structure of HTP bound to the small molecule inhibitor 5-chloro-6-[1-(2-iminopyrrolidinyl) methyl] uracil hydrochloride (TPI). The inhibitor appears to mimic the substrate transition state, which may help explain the potency of this inhibitor and the catalytic mechanism of pyrimidine nucleotide phosphorylases (PYNPs). Further, we have confirmed the validity of the HTP structure as a template for structure-based drug design by predicting binding affinities for TPI and other known HTP inhibitors using in silico docking techniques. This work provides the first structural insight into the binding mode of any inhibitor to this important drug target and forms the basis for designing novel inhibitors for use in anticancer therapy.     

Crystal structure of human MMP9 in complex with a reverse hydroxamate inhibitor

Matrix metalloproteinases (MMPs) and their inhibitors are important in connective tissue re-modelling in diseases of the cardiovascular system, such as atherosclerosis. Various members of the MMP family have been shown to be expressed in atherosclerotic lesions, but MMP9 is consistently seen in inflammatory atherosclerotic lesions. MMP9 over-expression is implicated in the vascular re-modelling events preceding plaque rupture (the most common cause of acute myocardial infarction). Reduced MMP9 activity, either by genetic manipulation or through pharmacological intervention, has an impact on ventricular re-modelling following infarction. MMP9 activity may therefore represent a key mechanism in the pathogenesis of heart failure. We have determined the crystal structure, at 2.3 A resolution, of the catalytic domain of human MMP9 bound to a peptidic reverse hydroxamate inhibitor as well as the complex of the same inhibitor bound to an active-site mutant (E402Q) at 2.1 A resolution. MMP9 adopts the typical MMP fold. The catalytic centre is composed of the active-site zinc ion, co-ordinated by three histidine residues (401, 405 and 411) and the essential glutamic acid residue (402). The main differences between the catalytic domains of various MMPs occur in the S1' subsite or selectivity pocket. The S1' specificity site in MMP9 is perhaps best described as a tunnel leading toward solvent, as in MMP2 and MMP13, as opposed to the smaller pocket found in fibroblast collagenase and matrilysin. The present structure enables us to aid the design of potent and specific inhibitors for this important cardiovascular disease target.     

Unexpected active-site flexibility in the structure of human neutrophil elastase in complex with a new dihydropyrimidone inhibitor

Human neutrophil elastase (HNE), a trypsin-type serine protease, is of pivotal importance in the onset and progression of chronic obstructive pulmonary disease (COPD). COPD encompasses a group of slowly progressive respiratory disorders and is a major medical problem and the fifth leading cause of death worldwide. HNE is a major target for the development of compounds that inhibit the progression of long-term lung function decline in COPD patients.Here, we present the three-dimensional structure of a potent dihydropyrimidone inhibitor (DHPI) non-covalently bound to HNE at a resolution of 2.0 Å. The inhibitor binds to the active site in a unique orientation addressing S1 and S2 subsites of the protease. To facilitate further analysis of this binding mode, we determined the structure of the uncomplexed enzyme at a resolution of 1.86 Å. Detailed comparisons of the HNE:DHPI complex with the uncomplexed HNE structure and published structures of other elastase:inhibitor complexes revealed that binding of DHPI leads to large conformational changes in residues located in the S2 subsite. The rearrangement of residues Asp95-Leu99B creates a deep, well-defined cavity, which is filled by the P2 moiety of the inhibitor molecule to almost perfect shape complementarity. The shape of the S2 subsite in complex with DHPI clearly differs from all other observed HNE structures. The observed structural flexibility of the S2 subsite is a key feature for the understanding of the binding mode of DHPIs in general and the development of new HNE selective inhibitors.     

Macrophage elastase (MMP-12) in expanding murine adipose tissue

Background

Matrix metalloproteinases (MMPs) are known to play a role in adipose tissue development, but little information is available on the role of individual proteinases. Expansion of adipose tissue is associated with an increased macrophage content. Macrophage elastase (MMP-12) has an important role in macrophage infiltration, which induces pro-inflammatory effects in adipose tissue.

Methods

The role of MMP-12 was investigated in adipose tissues of MMP-12 deficient and wild-type control mice kept on normal chow or on high fat diet for 15 weeks.

Results

MMP-12 deficiency had no significant effect on total body weight or on subcutaneous (SC) or gonadal (GON) adipose tissue mass. Adipocyte and blood vessel size and density in SC and GON adipose tissues of obese mice were also comparable in MMP-12 deficient and control mice. Macrophage infiltration in SC and GON adipose tissues was not affected by MMP-12 deficiency, but the amount of crown-like structures (CLS) was significantly lower. MMP-12 deficiency did not affect elastin content in the extracellular matrix of SC or GON adipose tissue.

Conclusions

Adipose tissue mass and composition in mice with nutritionally induced obesity was not markedly affected by MMP-12 deficiency, except for an apparently lower degree of CLS.

General Significance

MMP-12 does not seem to be essential for macrophage infiltration in adipose tissue, but contributes to the formation of CLS surrounding moribund adipocytes.     

The refined 2.3 A crystal structure of human leukocyte elastase in a complex with a valine chloromethyl ketone inhibitor

The stoichiometric complex formed between human leukocyte elastase and a synthetic MeO-Suc-Ala-Ala-Pro-Val chloromethyl ketone inhibitor was co-crystallized and its X-ray structure determined, using Patterson search methods. Its structure has been crystallographically refined to a final R value of 0.145 (8.0 and 2.3 A). The enzyme structure is very similar to that recently observed in a complex formed with the ovomucoid third domain from turkey [(1986) EMBO J. 5,2453-2458]. The rms deviation of all alpha-carbon atoms is 0.32 A. The peptidic inhibitor is bound in a similar overall conformation as the ovomucoid binding segment. Covalent bonds are formed between Val-P1 of the inhibitor and His-57 NE2 and Ser-195 OG of the enzyme. The carbonyl carbon is tetrahedrally deformed to a hemiketal. The valine side chain is arranged in the S1 pocket in the g-conformation.     

Crystal structure of phosphodiesterase 4D and inhibitor complex(1)

Cyclic nucleotide phosphodiesterases (PDEs) regulate physiological processes by degrading intracellular second messengers, adenosine-3′,5′-cyclic phosphate or guanosine-3′,5′-cyclic phosphate. The first crystal structure of PDE4D catalytic domain and a bound inhibitor, zardaverine, was determined. Zardaverine binds to a highly conserved pocket that includes the catalytic metal binding site. Zardaverine fills only a portion of the active site pocket. More selective PDE4 inhibitors including rolipram, cilomilast and roflumilast have additional functional groups that can utilize the remaining empty space for increased binding energy and selectivity. In the crystal structure, the catalytic domain of PDE4D possesses an extensive dimerization interface containing residues that are highly conserved in PDE1, 3, 4, 8 and 9. Mutations of R358D or D322R among these interface residues prohibit dimerization of the PDE4D catalytic domain in solution.     

X-ray crystal structure of the complex of human leukocyte elastase (PMN elastase) and the third domain of the turkey ovomucoid inhibitor.

Orthorhombic crystals diffracting beyond 1.7 A resolution, have been grown from the stoichiometric complex formed between human leukocyte elastase (HLE) and the third domain of turkey ovomucoid inhibitor (OMTKY3). The crystal and molecular structure has been determined with the multiple isomorphous replacement technique. The complex has been modeled using the known structure of OMTKY3 and partial sequence information for HLE, and has been refined. The current crystallographic R-value is 0.21 for reflections from 25 to 1.8 A resolution. HLE shows the characteristic polypeptide fold of trypsin-like serine proteinases and consists of 218 amino acid residues. However, several loop segments, mainly arranged around the substrate binding site, have unique conformations. The largest deviations from the other vertebrate proteinases of known spatial structure are around Cys168. The specificity pocket is constricted by Val190, Val216 and Asp226 to preferentially accommodate medium sized hydrophobic amino acids at P1. Seven residues of the OMTKY3-binding segment are in specific contact with HLE. This interaction and geometry around the reactive site are similar as observed in other complexes. It is the first serine proteinase glycoprotein analysed, having two sugar chains attached to Asn159 and to residue 109.     

Crystal structure of the stromelysin-3 (MMP-11) catalytic domain complexed with a phosphinic inhibitor mimicking the transition-state

Stromelysin-3 (ST3) is a matrix metalloproteinase (MMP-11) whose proteolytic activity plays an important role in tumorigenicity enhancement. In breast cancer, ST3 is a bad prognosis marker: its expression is associated with a poor clinical outcome. This enzyme therefore represents an attractive therapeutic target.The topology of matrix metalloproteinases (MMPs) is remarkably well conserved, making the design of highly specific inhibitors difficult. The major difference between MMPs lies in the S(1)' subsite, a well-defined hydrophobic pocket of variable depth. The present crystal structure, the first 3D-structure of the ST3 catalytic domain in interaction with a phosphinic inhibitor mimicking a (d, l) peptide, clearly demonstrates that its S(1)' pocket corresponds to a tunnel running through the enzyme. This open channel is filled by the inhibitor P(1)' group which adopts a constrained conformation to fit this pocket, together with two water molecules interacting with the ST3-specific residue Gln215. These observations provide clues for the design of more specific inhibitors and show how ST3 can accommodate a phosphinic inhibitor mimicking a (d, l) peptide.The presence of a water molecule interacting with one oxygen atom of the inhibitor phosphinyl group and the proline residue of the Met-turn suggests how the intermediate formed during proteolysis may be stabilized. Furthermore, the hydrogen bond distance observed between the methyl of the phosphinic group and the carbonyl group of Ala182 mimics the interaction between this carbonyl group and the amide group of the cleaved peptidic bond. Our crystal structure provides a good model to study the MMPs mechanism of proteolysis.     

Crystal structures of allosamidin derivatives in complex with human macrophage chitinase

The pseudotrisaccharide allosamidin is a potent family 18 chitinase inhibitor with demonstrated biological activity against insects, fungi, and the Plasmodium falciparum life cycle. The synthesis and biological properties of several derivatives have been reported. The structural interactions of allosamidin with several family 18 chitinases have been determined by x-ray crystallography previously. Here, a high resolution structure of chitotriosidase, the human macrophage chitinase, in complex with allosamidin is presented. In addition, complexes of the allosamidin derivatives demethylallosamidin, methylallosamidin, and glucoallosamidin B are described, together with their inhibitory properties. Similar to other chitinases, inhibition of the human chitinase by allosamidin derivatives lacking a methyl group is 10-fold stronger, and smaller effects are observed for the methyl and C3 epimer derivatives. The structures explain the effects on inhibition in terms of altered hydrogen bonding and hydrophobic interactions, together with displaced water molecules. The data reported here represent a first step toward structure-based design of specific allosamidin derivatives.     

The crystal structure of human quinolinic acid phosphoribosyltransferase in complex with its inhibitor phthalic acid

Quinolinic acid (QA), a biologically potent but neurodestructive metabolite is catabolized by quinolinic acid phosphoribosyltransferase (QPRT) in the first step of the de novo NAD⁺ biosynthesis pathway. This puts QPRT at the junction of two different pathways, that is, de novo NAD⁺ biosynthesis and the kynurenine pathway of tryptophan degradation. Thus, QPRT is an important enzyme in terms of its biological impact and its potential as a therapeutic target. Here, we report the crystal structure of human QPRT bound to its inhibitor phthalic acid (PHT) and kinetic analysis of PHT inhibition of human QPRT. This structure, determined at 2.55 Å resolution, shows an elaborate hydrogen bonding network that helps in recognition of PHT and consequently its substrate QA. In addition to this hydrogen bonding network, we observe extensive van der Waals contacts with the PHT ring that might be important for correctly orientating the substrate QA during catalysis. Moreover, our crystal form allows us to observe an intact hexamer in both the apo‐ and PHT‐bound forms in the same crystal system, which provides a direct comparison of unique subunit interfaces formed in hexameric human QPRT. We call these interfaces “nondimeric interfaces” to distinguish them from the typical dimeric interfaces observed in all QPRTs. We observe significant changes in the nondimeric interfaces in the QPRT hexamer upon binding PHT. Thus, the new structural and functional features of this enzyme we describe here will aid in understanding the function of hexameric QPRTs, which includes all eukaryotic and select prokaryotic QPRTs. Proteins 2014; 82:405–414. © 2013 Wiley Periodicals, Inc.     

The crystal structure of the complex of non-peptidic inhibitor of human neutrophil elastase ONO-6818 and porcine pancreatic elastase

The crystal structure of a new inhibitor of human neutrophil elastase (HNE), N-[2-[5-(tert-butyl)-1,3,4-oxadiazol-2-yl]-(IRS)-1-(methylethyl)-2-oxoethyl]-2-(5-amino-6-oxo-2-phenyl-6H-pyrimidin-1-ly)acetamide (ONO-6818, 1) complexed to porcine pancreatic elastase (PPE) has been determined at 1.86 A resolution. Analytical results provided evidence of a 1:1 complex in which the electrophilic ketone of 1 covalently bound to O gamma of Ser195 at the active site of PPE. The role of the unique electron-withdrawing ketone of 1 has been elucidated.     

Crystal structure of human factor VIIa/tissue factor in complex with peptide mimetic inhibitor

The 3D structure of human factor VIIa/soluble tissue factor in complex with a peptide mimetic inhibitor, propylsulfonamide-D-Thr-Met-p-aminobenzamidine, is determined by X-ray crystallography. As compared with the interactions between thrombin and thrombin inhibitors, the interactions at S2 and S3 sites characteristic of factor VIIa and factor VIIa inhibitors are revealed. The S2 site has a small pocket, which is filled by the hydrophobic methionine side chain in P2. The small S3 site fits the small size residue, D-threonine in P3. The structural data and SAR data of the peptide mimetic inhibitor show that these interactions in the S2 and S3 sites play an important role for the improvement of selectivity versus thrombin. The results will provide valuable information for the structure-based drug design of specific inhibitors for FVIIa/TF.