THE RECOMBINANT CATALYTIC DOMAIN OF HUMAN NEUTROPHIL COLLAGENASE LACKS TYPE-I COLLAGEN SUBSTRATE-SPECIFICITY

SCHNIERER S, KLEINE T, GOTE T, HILLEMANN A, KNAUPER V, Tschesche H. THE RECOMBINANT CATALYTIC DOMAIN OF HUMAN NEUTROPHIL COLLAGENASE LACKS TYPE-I COLLAGEN SUBSTRATE-SPECIFICITY. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS. 1993;191(2):319-326

THE X-RAY CRYSTAL-STRUCTURE OF THE CATALYTIC DOMAIN OF HUMAN NEUTROPHIL COLLAGENASE INHIBITED BY A SUBSTRATE-ANALOG REVEALS THE ESSENTIALS FOR CATALYSIS AND SPECIFICITY

BODE W, REINEMER P, HUBER R, KLEINE T, SCHNIERER S, Tschesche H. THE X-RAY CRYSTAL-STRUCTURE OF THE CATALYTIC DOMAIN OF HUMAN NEUTROPHIL COLLAGENASE INHIBITED BY A SUBSTRATE-ANALOG REVEALS THE ESSENTIALS FOR CATALYSIS AND SPECIFICITY. EMBO JOURNAL. 1994;13(6):1263-1269.Matrix metalloproteinases are a family of zinc endopeptidases involved in tissue remodelling. They have been implicated in various disease processes including tumour invasion and joint destruction. These enzymes consist of several domains, which are responsible for latency, catalysis and substrate recognition. Human neutrophil collagenase (PMNL-CL, MMP-8) represents one of the two 'interstitial' collagenases that cleave triple helical collagens types I, II and III. Its 163 residue catalytic domain (Met80 to Gly242) has been expressed in Escherichia coli and crystallized as a non-covalent complex with the inhibitor Pro-Leu-Gly-hydroxylamine. The 2.0 angstrom crystal structure reveals a spherical molecule with a shallow active-site cleft separating a smaller C-terminal subdomain from a bigger N-terminal domain, composed of a rive-stranded beta-sheet, two alpha-helices, and bridging loops. The inhibitor mimics the unprimed (P1-P3) residues of a substrate; primed (P1'-P3') peptide substrate residues should bind in an extended conformation, with the bulky P1' side-chain fitting into the deep hydrophobic S1' subsite. Modelling experiments with collagen show that the scissile strand of triple-helical collagen must be freed to fit the subsites. The catalytic zinc ion is situated at the bottom of the active-site cleft and is penta-coordinated by three histidines and by both hydroxamic acid oxygens of the inhibitor. In addition to the catalytic zinc, the catalytic domain harbours a second, non-exchangeable zinc ion and two calcium ions, which are packed against the top of the beta-sheet and presumably function to stabilize the catalytic domain. The polypeptide folding and in particular the zinc environment of the collagenase catalytic domain bear a close resemblance to the astacins and the snake venom metalloproteinases

PREPARATION OF ACTIVE RECOMBINANT TIMP-1 FROM ESCHERICHIA-COLI INCLUSION-BODIES AND COMPLEX-FORMATION WITH THE RECOMBINANT CATALYTIC DOMAIN OF PMNL-COLLAGENASE

KLEINE T, BARTSCH S, BLASER J, et al. PREPARATION OF ACTIVE RECOMBINANT TIMP-1 FROM ESCHERICHIA-COLI INCLUSION-BODIES AND COMPLEX-FORMATION WITH THE RECOMBINANT CATALYTIC DOMAIN OF PMNL-COLLAGENASE. BIOCHEMISTRY. 1993;32(51):14125-14131.TIMP-1 is a member of the family of tissue inhibitors of metalloproteinases involved in regulating the activity of extracellular matrix degrading metalloproteinases. The TIMP-1 cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR) amplification of the corresponding mRNA from human fibroblasts. Cloning and expression of the TIMP-1 cDNA were performed in Escherichia coli. In the host vector system chosen, rTIMP-1 is stored intracellularly in its denatured, insoluble form in inclusion bodies. We report a new method for the purification and renaturation of rTIMP-1 from E. coli inclusion bodies to an active inhibitor of matrix metalloproteinases (80% yield), presumably containing the correct assignment of the six disulfide bonds. A resin with the covalently bound recombinant catalytic domain of the PMNL-collagenase as the affinity ligand provided an effective means for the separation of correctly folded, active rTIMP-1 from inactive forms with mismatched disulfides. TIMP-1 and TIMP-2, the two most extensively examined members of the family of tissue inhibitors of metalloproteinases, are known to form a complex with the activated forms of most matrix metalloproteinases and the latent forms of the 92-kDa and 72-kDa gelatinases, respectively. In this study, we report on the complex formation of the recombinant catalytic domain of the PMNL-collagenase with TIMP-1, nonglycosylated recombinant TIMP-1, and recombinant TIMP-2. The K(i) values for the different inhibitors were determined in a kinetic assay using a fluorogenic substrate peptide. In this assay, rTIMP-2 had a more effective inhibitory capability against the recombinant catalytic domain of the PMNL-collagenase than TIMP-1. As for the PMNL-collagenase, the N-terminal catalytic domain is sufficient for enzyme-inhibitor interaction and binding

FUNCTION AND STRUCTURE OF HUMAN-LEUKOCYTE COLLAGENASE

Tschesche H, KNAUPER V, KLEINE T, et al. FUNCTION AND STRUCTURE OF HUMAN-LEUKOCYTE COLLAGENASE. JOURNAL OF PROTEIN CHEMISTRY. 1994;13(5):460-461

STRUCTURAL IMPLICATIONS FOR THE ROLE OF THE N-TERMINUS IN THE SUPERACTIVATION OF COLLAGENASES - A CRYSTALLOGRAPHIC STUDY

REINEMER P, GRAMS F, HUBER R, et al. STRUCTURAL IMPLICATIONS FOR THE ROLE OF THE N-TERMINUS IN THE SUPERACTIVATION OF COLLAGENASES - A CRYSTALLOGRAPHIC STUDY. FEBS LETTERS. 1994;338(2):227-233.For the collagenases PMNL-CL and FIB-CL, the presence of the N-terminal Phe(79) correlates with an increase in proteolytic activity. We have determined the X-ray crystal structure of the recombinant Phe(79)-Gly(242) Catalytic domain of human neutrophil collagenase (PMNL-CL, MMP-8) using the recently solved model of the Met(80)-Gly(242) form for phasing and subsequently refined it to a final crystalographic R-factor of 18.0% at 2.5 Angstrom resolution. The PMNL-CL catalytic domain is a spherical molecule with a flat active site cleft separating a smaller C-terminal subdomain from a bigger N-terminal domain, that harbours two zinc ions, namely a 'structural' and a 'catalytic' zinc, and two calcium ions. The N-terminal segment prior to Pro(86), which is disordered in the Met(80)-Gly(242) form, packs against a concave hydrophobic surface made by the C-terminal helix. The N-terminal Phe(79) ammonium group makes a salt link with the side chain carboxylate group of the strictly conserved Asp(232). Stabilization of the catalytic site might be conferred via strong hydrogen bonds made by the adjacent, likewise strictly conserved Asp(233) With the characteristic 'Met-turn', which forms the base of the active site residues

Inhibition of Matrix Metalloproteinases in Rheumatoid Arthritis and the Crystallographic Binding Mode of a Peptide Inhibitor

Tschesche H, BLÄSER J, KLEINE T, et al. Inhibition of Matrix Metalloproteinases in Rheumatoid Arthritis and the Crystallographic Binding Mode of a Peptide Inhibitor. In: Inhibition of Matrix Metalloproteinases: Therapeutic Potential. Annals of the New York Academy of Sciences. Vol 732. New York Acad. of Sciences; 1994: 400-402