Collagenases (molecular Biology Intelligence Unit) by Warren Hoeffler


1356cf1ae4baeb9.jpg Author Warren Hoeffler
Isbn 978-1570595622
File size 3.7 MB
Year 1999
Pages 259
Language English
File format PDF
Category biology


 

MEDICAL INTELLIGENCE UNIT 10 Collagenases Warren Hoeffler, Ph.D. Department of Dermatology Stanford University School of Medicine Stanford, California R.G. LANDES COMPANY AUSTIN, TEXAS U.S.A. MEDICAL INTELLIGENCE UNIT 10 Collagenases R.G. LANDES COMPANY Austin, Texas, U.S.A. Copyright ©1999 R.G. Landes Company All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Printed in the U.S.A. Please address all inquiries to the Publishers: R.G. Landes Company, 810 South Church Street, Georgetown, Texas, U.S.A. 78626 Phone: 512/ 863 7762; FAX: 512/ 863 0081 ISBN: 1-57059-562-3 While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein. Library of Congress Cataloging-in-Publication Data Collagenases / [edited by] Warren Hoeffler p. cm. -- (Medical intelligence unit) ISBN 1-57059-562-3 (alk. paper) 1. Collagenases. 2. Collagenases--Pathophysiology. I. Hoeffler, Warren, 1957- . II. Series. [DNLM: 1. Collagenases. QU 136 C697 1999] QP609.C64C645 1999 612'.01516--dc21 DNLM/DLC for Library of Congress 98-50414 CIP MEDICAL INTELLIGENCE UNIT 10 PUBLISHER’S NOTE Collagenases R.G. Landes Company produces books in six Intelligence Unit series: Medical, Molecular Biology, Neuroscience, Tissue Engineering, Biotechnology and Environmental. The authors of our books are acknowledged leaders in their fields. Topics are unique; almost without exception, no similar books exist on these topics. Our goal is to publish books in important and rapidly changing areas of bioscience for sophisticated researchers and clinicians. To achieve this goal, we have accelerated our publishing program to conform to the fast pace at which information grows in bioscience. Most of our books are published 90 to 120 days of receipt of Department ofwithin Dermatology the manuscript. We would like to thank readers for their Stanford University School of our Medicine continuing interest and welcome any comments or suggestions they Stanford, California may have for future books. Warren Hoeffler, Ph.D. Michelle Wamsley Production Manager R.G. Landes Company R.G. LANDES COMPANY AUSTIN, TEXAS U.S.A. CONTENTS 1. Structure of Collagenases and Strategies for Expression and Folding of the Recombinant Proteins ............................................... 1 Robert D. Gray Introduction ............................................................................................. 1 Matrix Metalloproteinase Structure ....................................................... 1 Collagenase Three-Dimensional Structure ............................................ 3 The Pexin Domain ................................................................................. 11 Bacterial Expression of Mammalian Proteins ...................................... 12 General Aspects of Protein Renaturation ............................................. 13 Expression and Folding of Recombinant MMPs ................................. 14 Neutrophil Collagenase ......................................................................... 15 Other MMPs .......................................................................................... 16 Mechanism of Collagenase Folding ...................................................... 18 Summary ................................................................................................ 18 2. Chondrocyte Expression of Collagenase 2 ............................................. 23 Ada A. Cole and Klaus E. Kuettner Introduction ........................................................................................... 23 Articular Cartilage ................................................................................. 23 MMPs in Cartilage ................................................................................. 25 Conclusions ........................................................................................... 31 3. Collagenase-3 ........................................................................................... 37 Identification, Characterization, and Physiological and Pathological Relevance ...................................................................... 37 Carlos López-Otín Identification and Structural Characterization of Human Collagenase-3. ................................................................. 37 Structure and Regulation of the Human Collagenase-3 Gene ............ 43 Physiological Significance of Human Collagenase-3 ........................... 45 Conclusions and Perspectives ............................................................... 49 4. Transcriptional Regulation of the Collagenase-1 (Matrix Metalloproteinase-1; MMP-1) Gene ........................................ 55 Joni L. Rutter and Constance E. Brinckerhoff Introduction ........................................................................................... 55 MMP-1 Biology and Biochemistry ....................................................... 56 MMP-1 Structure, Organization, and Location .................................. 57 Transcription of MMP-1: Basal/Constitutive and TPA-induced Expression ........................................................... 59 Transcriptional Regulation by Cytokines and Growth Factors .......... 61 Role of PEA3 Sites and AP-1 Sites: The Concept of Cooperativity .... 62 Inhibition of MMP-1 Transcription ..................................................... 64 Summary and Conclusion .................................................................... 65 5. Interpreting Transcriptional Control Elements .................................... 73 Warren Hoeffler The Promise of Transcription ‘Factorology’ ........................................ 73 Functional Assays for Transcriptional Activation Largely Unavailable ........................................................................... 74 DNA Binding Assays Are Helpful, but Inconclusive ........................... 75 Molecular Mechanisms of Transcriptional Activation ........................ 76 TFIIIC as a Prototypical Transcriptional Activator ............................. 78 Enhancer Binding Protein AP-1 (aka Jun/Fos) ................................... 81 Other Influences on Transcriptional Control ...................................... 86 Summary ................................................................................................ 87 6. Activation and Induction of Collagenases ............................................. 91 Kazuki Nabeshima, Hiroaki Kataoka, Bryan P. Toole and Masashi Koono Introduction ........................................................................................... 91 Procollagenase Activation ..................................................................... 92 Collagenase Induction by Cell-Cell Interactions ................................. 99 EMMPRIN ........................................................................................... 102 Conclusions and Perspectives ............................................................. 106 7. Role of Reactive Oxygen Species in the Induction of Collagenases, and Other MMPs—Pathogenic Implications for Photoaging and Tumor Progression ........................................................................ 115 Meinhard Wlaschek and Karin Scharffetter-Kochanek Introduction ......................................................................................... 115 The Role of UV-induced Reactive Oxygen Species (ROS) and Collagenases in Photoaging and Photocarcinogenesis ........... 117 The Regulation of Matrix-Degrading Metalloproteases by Ultraviolet Irradiation (UV) Induced Reactive Oxygen Species (ROS) .......... 118 Conclusions ......................................................................................... 122 8. Integrins as Regulators of Collagenase Expression ............................. 127 Terhi Lehtinen and Jyrki Heino Introduction ......................................................................................... 127 Extracellular Matrix Receptor Integrins ............................................. 128 Osteogenic Cells Inside Collagen Lattices as a Model for Cell–Matrix Interaction ............................................................ 130 Integrins as Regulators of MMP Expression ...................................... 135 Integrin signaling leading to altered gene expression ........................ 137 Integrin Induced MMP Expression in Health and Disease ............... 140 9. Pharmacological Inhibition of Collagenases ....................................... 147 Dennis H. Oh and Warren Hoeffler Introduction ......................................................................................... 147 Familiar Drugs in Novel Applications ................................................ 149 Novel Active Site Protease Inhibitors ................................................. 153 Oligonucleotides .................................................................................. 159 10. Collagenase in Embryonic Development and Postnatal Remodeling of Connective Tissues .............................. 171 Stephen M. Krane and Weiguang Zhao MMPs with Collagenase Activity ........................................................ 171 Creating Mutations in Collagen that Confer Resistance to Collagenase .................................................................................. 172 Transgenic Mice with Collagenase Resistant Collagen ...................... 172 Characterizing Effects on Joints and Bone ......................................... 179 Characterization of Collagenase Cleavage ......................................... 181 Concluding Remarks ........................................................................... 182 11. The Role of Interstitial Collagenases in Tumor Progression .............. 189 Eric W. Howard Introduction: Tumor Progression and the Role of Interstitial Collagenase ................................................................ 189 Participation of Collagenase in Proteolytic Cascades ........................ 193 Collagenases in Carcinomas ................................................................ 193 Regulation of Collagenase Inhibition—The Role of TIMPs in Cancer .......................................................................................... 197 Role of Collagenase in Angiogenesis .................................................. 198 Conclusions ......................................................................................... 200 12. The Role of Collagenase in Wound Healing ........................................ 207 Mona Ståhle-Bäckdahl Introduction ......................................................................................... 207 Collagenase Expression in Skin Wounds ........................................... 209 Regulation of Collagenase During Re-epithelialization .................... 210 Role of Collagenase in Wound Healing ............................................. 215 13. Matrix Metalloproteinases in the Pathogenesis of Lung Injury ......... 221 Annie Pardo and Moisés Selman Cell Types and Extracellular Matrix in the Normal Lung ................. 221 Matrix Metalloproteinases in Acute Lung Injury .............................. 224 Metalloproteinases in Chronic Lung Injury ....................................... 227 14. Collagenase and Aging .......................................................................... 241 Michael D. West Introduction to Aging ......................................................................... 241 The Molecular Biology of Cellular Aging ........................................... 244 Conclusions ......................................................................................... 249 Index ................................................................................................................ 253 EDITORS Warren Hoeffler Department of Dermatology Stanford University Medical School Stanford, California, U.S.A. Chapter 5, 9 CONTRIBUTORS Constance E. Brinckerhoff Department of Medicine Department of Biochemistry Dartmouth Medical School Hanover, New Hampshire, U.S.A. Chapter 4 Eric W. Howard Department of Pathology University of Oklahoma Health Sciences Center Oklahoma City, Oklahoma, U.S.A. Chapter 11 Ada A. Cole Departments of Biochemistry Rush Medical College Rush-Presbyterian-St. Luke's Medical Center Chicago, Illinois, U.S.A. Chapter 2 Hiroaki Kataoka Department of Pathology Miyazaki Medical College Miyazaki, Japan Chapter 6 Robert D. Gray Department of Biochemistry and Molecular Biology Department of Ophthalmology and Visual Sciences University of Louisville School of Medicine Louisville, Kentucky, U.S.A. Chapter 1 Jyrki Heino MediCity Research Laboratory and Department of Medical Biochemistry University of Turku Turku, Finland Department of Biological and Environmental Science University of Jyväskylä Jyväskylä, Finland Chapter 8 Masashi Koono Department of Pathology Miyazaki Medical College Miyazaki, Japan Chapter 6 Stephen M. Krane Department of Medicine Harvard Medical School Massachusetts General Hospital Boston, Massachusetts, U.S.A. Chapter 10 Klaus E. Kuettner Departments of Biochemistry and Orthopedic Surgery Rush Medical College Rush-Presbyterian-St. Lukeís Medical Center Chicago, Illinois, U.S.A. Chapter 2 Terhi Lehtinen MediCity Research Laboratory and Department of Medical Biochemistry University of Turku Turku, Finland, Department of Biological and Environmental Science University of Jyväskylä Jyväskylä, Finland Chapter 8 Carlos López-Otín Departamento Bioquímica y Biología Molecular Facultad de Medicina Universidad de Oviedo Oviedo, Spain Chapter 3 Kazuki Nabeshima Department of Pathology Miyazaki Medical College Miyazaki, Japan Chapter 6 Dennis H. Oh Department of Dermatology Stanford University Medical Center Stanford, California, U.S.A. Chapter 9 Annie Pardo Facultad de Ciencias, Universidad Nacional Autónoma de México. México City, México Chapter 13 Joni L. Rutter Department of Pharmacology and Toxicology Dartmouth Medical School Hanover, New Hampshire, U.S.A. Chapter 4 Karin Scharffetter-Kochanek Department of Dermatology University of Cologne Cologne, Germany Chapter 7 Moisés Selman Instituto Nacional de Enfermedades Respiratorias México City, México Chapter 13 Mona Ståhle-Bäckdahl Department of Dermatology Karolinska Hospital Stockholm, Sweden Chapter 12 Bryan P. Toole Department of Anatomy and Cell Biology Tufts University School of Medicine Boston, Massachusetts, U.S.A. Chapter 6 Michael D. West Geron Corporation Menlo Park, California, U.S.A. Chapter 14 Meinhard Wlaschek Department of Dermatology University of Cologne Cologne, Germany Chapter 7 Weiguang Zhao Department of Medicine Harvard Medical School Massachusetts General Hospital Boston, Massachusetts, U.S.A. Chapter 10 PREFACE H ow we think about a subject has a lot to do with what context we place that subject in. In the case of collagenases, a very important set of enzymes with key roles in development, normal physiology, and pathogenesis, the discussion is usually only an addendum to that on the larger group of enzymes in their category, matrix metalloproteinases. As information about these enzymes has undergone explosive growth in the last few years, any textbook attempting to cover this diverse group of enzymes ironically has diminished room for considering collagenases. The time has come to give this topic its own book, and to consider more broadly the diverse roles these enzymes play in (primarily human) biology. The authors contributing to this volume have all made major contributions to our understanding of collagenases, and each has recounted some of their own work here. They were encouraged to tell their story from their own personal perspectives, resulting is a more engaging description of the work. In the interests of putting together a more coherent volume devoted to these enzymes the authors were also challenged to consider related work, and how their area of interest fits into a larger picture. The topics for the chapters were chosen to give appropriate emphasis to the major themes associated with collagenases. The opening chapter describes the structural characteristics of collagenases, and is followed by individual chapters devoted to collagenases 1, 2, and 3. Transcriptional regulation of these genes are detailed and put into perspective of the current knowledge base. Since collagenases are uniquely regulated posttranslationally, various methods of activation are considered, followed by how these mechanisms come into play in normal physiological functions, and as part of certain pathologies. Interference with the activities of these enzymes for pharmacologic benefit is addressed, and the role of collagenases in wound healing and in cancer receives special attention. Even the ultimate pathology from which we collectively suffer, aging, is also discussed. In short, the information in this book cuts across a unique sampling of various medical fields, with the common theme that they all share an interest in this very important group of enzymes. Although technical detail is presented, and well documented in the references, more general perspectives are also clearly presented throughout. Since collagens are principle components comprising so much of our bodies, an understanding of the enzymes that orchestrate their constructive modeling, as well as decay, should be an area of importance to anyone interested in the body. Warren Hoeffler, Ph.D. CHAPTER 1 Structure of Collagenases and Strategies for Expression and Folding of the Recombinant Proteins Robert D. Gray Introduction T he development of recombinant DNA technology and the attendant ability to express virtually any protein in quantities sufficient for biophysical studies is an essential component of modern structural and mechanistic biology. Indeed, most, if not all of the threedimensional structures of the matrix metalloproteinases (MMPs) currently in the literature were derived from recombinant proteins obtained by expression in Escherichia coli. This approach is necessary because culture of mammalian tissues or cells generally produces only relatively small amounts of the enzymes. Expression of heterologous proteins in E. coli, however, often is accompanied by problems that must be overcome to ensure that a native structure is being studied. Potential problems include low yields of the target protein, precipitation of the expressed protein within the cell, lack of processing and proteolytic modification such as removal of C and N-terminal residues to produce proteins with heterogeneous termini. The purpose of this article is first to review structural aspects of the collagenases and second, to review systems that have been utilized for heterologous expression of the collagenases and other MMPs. The article is divided into the following parts: (a) a summary of MMP nomenclature and primary structural domains as a reference point for collagenases; (b) aspects of collagenase three-dimensional structure; (c) an overview of protein expression in E. coli; (d) an examination of general aspects of protein folding; (e) a discussion of specific recombinant collagenases and other MMPs. All of the examples discussed relate to MMPs expressed in E. coli; eukaryotic expression is not covered. Matrix Metalloproteinase Structure General Aspects The MMPs comprise a group of zinc endopeptidases that degrade proteins of the extracellular matrix. The enzymes share several functional characteristics including the ability to degrade at least one protein of the extracellular matrix, secretion into the extracellular matrix as proenzymes which must be activated to express proteolytic activity, and inhibition Collagenases, edited by Warren Hoeffler. ©1999 R.G. Landes Company. Collagenases 2 Table 1.1. The matrix metalloproteinase family Group Collagenases Interstitial collagenase PMN collagenase Gelatinases Gelatinase A Gelatinase B Stromelysins Stromelysin-1 Stromelysin-2 Stromelysin-3 Others Matrilysin MMP# Other Names MMP-1 MMP-8 EC# 3.4.24.7 3.4.24.34 MMP-2 MMP-9 72 kD gelatinase 92 kD gelatinase 3.4.24.24 3.4.24.35 MMP-3 Transin (rat) procollagenase activator Transin-2 (rat) 3.4.24.17 punctuated metallopreteinase (PUMP); uterine MP Metalloelastase (mouse) Collagenase-3 Membrane-type 3.4.24.23 MMP-10 MMP-11 MMP-7 MMP-12 MMP-13 MMP-14 3.4.24.22 Adapted from www.bioscience.org/molglanc/mmp.htm by specific MMP inhibitors, the tissue inhibitors of metalloproteinases (TIMPs).1 Historically, three groups of MMPs have been recognized based on substrate specificity: collagenases, which act uniquely on interstitial triple helical collagens (types I, II and III); gelatinases, which rapidly degrade denatured collagens and basement membrane (type IV) collagen; and stromelysins, which cleave the core polypeptide of proteoglycans among other proteins of the extracellular matrix. Two additional groups of MMPs have recently been proposed to accommodate those enzymes which do not fit in the classical scheme.2 MMP nomenclature is summarized in Table 1.1. Primary Structure The MMPs are members of a family of metallopeptidases, the metzincins, so named because of the presence of a catalytically essential zinc ion and a conserved methionine residue within the active site.3,4 Other members of this family include astacin, meprins, snake venom metalloproteinases (MPs) and the serralysins, which are bacterial metalloproteinases. Amino acid and DNA3,4 sequence analysis reveals the modular structure of the MMPs.2,5 The polypeptide chain of each family member starts with a hydrophobic N-terminal leader sequence that directs the protein to the secretory pathway. The leader sequence, which is removed prior to secretion, is followed by a propeptide that maintains enzymatic latency by coordination of a cysteine thiol to the catalytic zinc. The catalytic domain (CD) follows; it contains binding pockets for the substrate that direct the scissile peptide bond to the active site Zn2+ which is ligated to three histidine residues within an HEXGHXXGXXH motif. The conserved Glu is presumed to function as a general base in activating a water molecule that hydrolyzes the substrate peptide bond. Collagenases and Strategies for Expression and Folding of the Recombinant Proteins 3 The smallest member of the MMP family, promatrilysin, consists of the propeptide and the CD only. All other MMPs include, as a minimum, a C-terminal domain that exhibits sequence homology to the plasma protein hemopexin. This so-called pexin domain (PD) connects to the CD via a proline-rich hinge region of variable length (5-50 residues). The gelatinases also contain fibronectin-like gelatin-binding modules and gelatinase B contains in addition an α(2)-collagen-like sequence. From the standpoint of folding of the recombinant protein, it should be noted that the three-dimensional structure of the only full-length MMP published, porcine synovial collagenase, reveals that the catalytic and pexin regions appear to be independent structural domains.5 The crystal structure of the separated CD and PD of human collagenases suggests that these domains are independent folding units as well. Collagenase Three-Dimensional Structure X-ray crystallography and NMR have recently provided detailed models of the collagenases and several related MMPs. Most of these were determined using CDs inhibited by zinc-binding substrate analogues. Initial efforts were focused on fibroblast collagenase,6-9 neutrophil collagenase10 and stromelysin-1.11 Subsequently, structures of full-length porcine fibroblast collagenase,5 matrilysin12 and the PDs of collagenase-313 and 72 kDa gelatinase14 were published. The following section summarizes some of the available structural information with an emphasis on comparing human fibroblast and neutrophil collagenases. I have relied on the published structures of Lovejoy et al6,15 and Spurlino et al8 for MMP-1 and especially on the extensive work of the group at the Max-Planck-Institute10,13,16-18 for MMP-8 and MMP-13 for insights into comparisons between MMP-1 and MMP-8. Catalytic Domain Folding Topology Collagenases from fibroblasts and neutrophils exhibit similar, but nonidentical folding patterns as illustrated in the topological diagrams in Figure 1.1. The fibroblast CD (residues 100-269) consists of three α-helices (A-C), five β-strands (I-V) and their connecting loops. Strands I, II, III and V run parallel to each other while strand IV is oriented in an antiparallel direction. Helix A is positioned closest to strands I and II, while helix B, which provides the scaffolding for the putative catalytic Glu and two of the three His residues that ligate the catalytic zinc lies next to strand III. Helix C is positioned adjacent to strand IV. The neutrophil enzyme (residues 80-242) is folded in a similar fashion except that helices B and C are arranged in a slightly different topology from that of the fibroblast enzyme: in MMP-1, helix C is positioned more closely to strand III, while in MMP-8, it is closer to strand IV. Ribbon diagrams depicting the CD of fibroblast and neutrophil collagenases are shown in Figure 1.2. For MMP-1 and MMP-8, the CD are slightly oblate spheroids in overall shape.7,8,10 The extended substrate binding site consists of a groove that runs across one face of the molecule. From the viewpoint in Figure 1.2, the substrate binding site appears as a cleft in the lower right-hand side of both structures. It is demarcated on the top by strand IV, on its back side by helix B, and at the bottom by the loop connecting helices B and C. The bound inhibitors (Fig. 1.3 for chemical structures) that are positioned to the amino side (Fig. 1.2A and B) or carboxyl side (Fig. 1.2C) of the scissile peptide bond ligate the catalytic zinc of each enzyme through their hydroxamate or thiol functional groups. The zinc electrophile is situated at the bottom of the catalytic site where it is coordinated to His218, His222 and His228 in MMP-1 and His197, His201 and His207 in MMP-8. In both enzymes, the two His residues in helix B project their Nε2 atoms toward the zinc. Helix B terminates at Gly204 in MMP-8 and at Gly225 in MMP-1; a turn at this position redirects the polypeptide backbone such that His207 or His228 completes the triad of nitrogenous residues that provide nearly perfect tetrahedral coordination of the metal ion. Collagenases 4 A B Fig. 1.1. Topology cartoon representing the folding pattern of human collagenase catalytic domain (panel A) and human neutrophil collagenase catalytic domain (panel B). The diagrams were generated with the computer program TOPS65 as modified by Westhead and which is available on the internet at http://tops.ebi.ac.uk/tops/. The triangles represent β-strands and the circles represent helices. Strand direction (N to C) is indicated by the direction of the triangle: upward pointing triangles represent strands pointing toward the viewer and downward pointing triangles indicate strands pointing away from the viewer. Helix direction is indicated by the position of the connecting line: center lines indicate upward pointing helices and edge connectors show downward pointing helices. The diagrams were generated from structural data in the Protein Data Bank (PDB codes 1hfc8 and 1jap10 for MMP-1 and MMP-8, respectively). Helices are labeled A-C and strands are labeled I-V. The diagrams depict the major similarities in secondary structure and folding of the two catalytic domains as well as the different placement of helix C with respect to strands III and IV. Collagenases and Strategies for Expression and Folding of the Recombinant Proteins 5 Fig 1.2 A. (above) Ribbon diagrams showing the structure of inhibitor complexes of the catalytic domains of MMP-1 and MMP-8. Panel A shows the complex of MMP-1 with NHOH–Leu– Phe–NMe (Fig. 1.3) and was generated from data of Spurlino et al8 from Protein Data Base structure 1hfc. The amino and carboxyl termini of the molecule are indicated along with the strand and helix designations. The catalytic zinc (magenta sphere at right center) is shown ligated to three His residues of the protein and the hydroxamate group of the inhibitor. The putative catalytic Glu is shown projecting from helix B above the catalytic zinc, and the side chain of the Met at the bottom of the zinc binding site within the Met turn is also shown. The structural zinc (magenta sphere at upper right) and the calcium (light blue sphere, upper right) are shown in their binding sites where they fasten the large loop structure to the body of the protein. Panel B (see next page) shows a similar view of MMP-8 catalytic domain (data of Grams et al,19 PDB 1ja0, complexed to HSBzPp–Ala–Gly–NH2. The inhibitor is ligated via its thiol to the catalytic zinc and the P1' benzyl group lies within the S1' binding pocket. The structural zinc and calcium ion is depicted in the top center and top right–hand side of the diagram. The second calcium ion is depicted in the top left–hand side of the diagram. The positions of the catalytic Glu and zinc site Met are also shown. The (carboxyl) prime–side of the substrate binding region is shown by the extended inhibitor. Panel C (see next page) also shows MMP-8 bound to the inhibitor ProLeuGlyNHOH (blue structure) to illustrate the extended substrate binding site on the amino side of the cleavage site. The P3 Pro of the inhibitor resides in a narrow cleft defined by the side chains of His162, Phe164 and Ser151. The data are from Bode et al,10 PDB 1jap. This figure as well as Figures 1.4 and 1.5 were generated from the referenced structural data using the Swiss Protein Viewer66 with rendering in conjunction with Quickdraw3D and POV–Ray. 6 Fig 1.2 B. Fig 1.2 C. Collagenases Collagenases and Strategies for Expression and Folding of the Recombinant Proteins Fig. 1.3. Structure of the three peptide–based metal–coordinating inhibitors in Figure 1.2. 7 8 Collagenases The zinc is positioned below the catalytic Glu, whose carboxyl group points toward it. The crystal structure of the fibroblast enzyme reveals a water molecule in this region that may be H-bonded to Glu219 such that the water’s oxygen atom could serve as the nucleophile positioned to attack the scissile carbonyl in the hydrolytic step.15 The characteristic Met residue at the base of the zinc binding site is found in a 1,4 turn consisting of homologous-AlaLeu-Met-Tyr- sequences in the two enzymes. Figure 1.2 also shows the positions of structural zinc and calcium ions. The noncatalytic zinc is bound in the upper right-hand region of the molecule within a tetrahedral site composed of His168, Asp170, His183, His196 (MMP-1) or His147, Asp149, His162, His175 in MMP-8. MMP-1 has one calcium binding site within the S-shaped loop connecting strands III and IV (residues175-181). Binding is mediated by the carboxyls of Asp175, Asp198 and Glu201 along with three peptide carbonyls. MMP-8, on the other hand, has two calcium ions. As with MMP-1, one calcium is bound to the carboxyls of Asp177, Glu180 and Asp 154 and three backbone carbonyls within the loop connecting strands III and IV, and a second binds at the beginning of strand V where Asp173 provides one ligating carboxyl group and the remaining ligands consist of backbone carbonyls and a water molecule. Evidently, the role of the structural zinc and calcium is to fasten the long loop connecting strands III and IV (which comprises the upper lip of the substrate binding groove) to the main body of the molecule. Thus these ions probably function in maintaining the conformational integrity of the substrate binding site. Substrate and Inhibitor Binding Site Potential enzyme-substrate binding interactions are suggested in protein structures containing tightly bound inhibitors. These inhibitors (Fig. 1.3) are substrate analogues that incorporate a metal binding moiety such as a carboxyl, thiol or hydroxamate group in place of the scissile peptide bond. The metal binding group may be flanked by peptides designed to interact with the S (amino side) or S' (carboxyl side) subsites of the enzyme. In the structures shown, the inhibitors position themselves in an extended conformation within the substrate binding groove (Fig. 1.2). The metal binding functionality ligates the catalytic zinc at the open coordination position vacated by dissociation of the cysteinyl SH group provided by the propeptide. Inhibitor binding is stabilized by H-bonds with the backbone of the enzyme as well as by extensive interactions with the P1' residue and less extensive contacts with the P3 residue. Interactions with P1, P2' and P3' appear to be less extensive or absent altogether. These structures reveal that in both the fibroblast and neutrophil enzymes, a primary determinant of substrate specificity lies within the S1' subsite, which consists of a rather expansive hydrophobic pocket that can easily accommodate the side chain of Ile or Leu found at the P1' position of collagen substrates. In MMP-8, the opening of this pocket is ringed by polar groups,16 which can H-bond to inhibitors. The S1' pocket itself is lined with hydrophobic side chains (Leu 193 and Leu 214) while Arg222, whose guanidinium group is H-bonded to backbone oxygen atoms, defines the bottom of the pocket (Fig. 1.4). InterestFig 1.4. (opposite) Close–up view of the S1' binding pocket of MMP-1 and MMP-8. These diagrams are from the same data as figures 1.2A and 1.2B. In panel A, the structure in gold is the inhibitor NHOH–Leu–Phe–NMe. Panel B shows MMP-8 with the P1' residue (HSBzPp portion only) ligated through its sulfur (yellow sphere) to the catalytic zinc (magenta sphere). The bottom of the S1' site is formed by Arg222 in MMP-8; in MMP-1, the homologous residue is Ser243 (Panel A). In MMP-1, the side chain of Arg214 projects into the S1' site. In both enzymes, strand IV forms the upper edge of the site and helix B forms the back side. Red atoms are oxygen, blue are nitrogen and gray are carbons. Hydrogen atoms are not shown. Collagenases and Strategies for Expression and Folding of the Recombinant Proteins Fig 1.4 A. Fig 1.4 B. 9 Collagenases 10 Fig 1.5 A. Fig 1.5 B.

Author Warren Hoeffler Isbn 978-1570595622 File size 3.7 MB Year 1999 Pages 259 Language English File format PDF Category Biology Book Description: FacebookTwitterGoogle+TumblrDiggMySpaceShare Collagenases are an important subfamily of matrix metalloproteinases utilizing native collagens as a substrate. This book reviews a current knowledge of the structures and functions of collagenases and the roles they play in development, tumor metastasis, and tissue remodeling during wound healing. This review comes at a time when new collagenases are being discovered, such as collagenases, rather than all matrix metalloproteinases so that a more thorough examination of the function of this crucial set of enzymes in a variety of systems can be achieved. Because of the great deal of work that has gone into understanding this group of enzymes, the findings in this area may also serve as useful paradigms for work in related areas.     Download (3.7 MB) Connective Tissue: Histophysiology, Biochemistry, Molecular Biology Matrix Metalloproteases: Methods and Protocols Matrix Metalloproteinase Biology Cell Death Signaling In Cancer Biology And Treatment DNA Methods in Food Safety Load more posts

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