Solvent water interactions within the active site of the membrane type I matrix metalloproteinase

Matrix metalloproteinases (MMP) are an important family of proteases which catalyze the degradation of extracellular matrix components. While the mechanism of peptide cleavage is well established, the process of enzyme regeneration, which represents the rate limiting step of the catalytic cycle, remains unresolved. This step involves the loss of the newly formed N-terminus (amine) and C-terminus (carboxylate) protein fragments from the site of catalysis coupled with the inclusion of one or more solvent waters. Here we report a novel crystal structure of membrane type I MMP (MT1-MMP or MMP-14), which includes a small peptide bound at the catalytic Zn site via its C-terminus. This structure models the initial product state formed immediately after peptide cleavage but before the final proton transfer to the bound amine; the amine is not present in our system and as such proton transfer cannot occur. Modeling of the protein, including earlier structural data of Bertini and coworkers [I. Bertini, et al., Angew. Chem., Int. Ed., 2006, 45, 7952–7955], suggests that the C-terminus of the peptide is positioned to form an H-bond network to the amine site, which is mediated by a single oxygen of the functionally important Glu240 residue, facilitating efficient proton transfer. Additional quantum chemical calculations complemented with magneto-optical and magnetic resonance spectroscopies clarify the role of two additional, non-catalytic first coordination sphere waters identified in the crystal structure. One of these auxiliary waters acts to stabilize key intermediates of the reaction, while the second is proposed to facilitate C-fragment release, triggered by protonation of the amine. Together these results complete the enzymatic cycle of MMPs and provide new design criteria for inhibitors with improved efficacy.Financial support was provided by the Max Planck Gesellschaft and the Cluster of Excellence RESOLV (EXC 1069) funded by the Deutsche Forschungsgemeinschaft. I. S. is supported by the Binational Science Foundation (BSF) and Israel Science Foundation (ISF). I. S. and M. H. are also supported by the ERC Advanced Grant 695437 THz-Calorimetry. M. G. is an Awardee of the Weizmann Institute of Science National Postdoctoral Award Program for Advancing Women in Science and a recipient of an A. v. Humboldt Fellowship. N. C. acknowledges the support of the Australian Research Council: Future Fellowship (FT140100834). Open Access funding provided by the Max Planck Society

Structural and spectroscopic characterization of the catalytic domain of human membrane type 1 matrix metalloproteinase

In dieser Arbeit wird ein Mitglied dieser Proteinfamilie, die Typ 1 Membran Metalloproteinase (MT1-MMP oder MMP-14) untersucht. Dazu wurden vier Herangehensweisen entwickelt, um die enzymatische Aktivität von MT1-MMP besser verstehen zu können. Die Bandbreite der Methoden erstreckt sich von detaillierten Strukturanalysen des aktiven Zentrums mit Hilfe von Röntgen Kristallstrukturanalyse, bis hin zu Untersuchungen der enzymatischen Aktivität bei der die Substrataufnahme des Proteins im Detail untersucht wurde. In den letzten beiden Kapiteln werden abschließend drei neue spektroskopische Herangehensweisen zur Untersuchung der katalytischen Aktivität von MT1-MMP beschrieben. Bei der ersten dieser Methoden handelt es sich um Hochdruckspektroskopie gekoppelt mit einer externen Störung (z. B. durch Temperatur), die es erlaubt das Verständnis, welches dem katalytischen Mechanismus der Metalloproteasen im Bezug auf Löslichkeitsverhalten, Energie- und Volumenänderungen zugrunde liegt besser zu verstehen.In this thesis one member of this family, the membrane type 1 matrix metalloproteinase (MT1-MMP or MMP-14) is examined. Four general approaches were employed to better understand the enzymatic activity of MT1-MMP. These are described in the chapters 2 to 5, each representing a self-contained study. These range from detailed structural analysis of the active site of the protein using Xray crystallography, to new assays for enzymatic activity where substrate delivery to the protein is tightly controlled. In the final two chapters three new spectroscopic approaches for studying the catalysis of MT1-MMP are described. The first of these is high pressure spectroscopy which allows solvation, energetics and volume changes to be study in detail

Structural Studies of Matrix Metalloproteinase by X-Ray Diffraction.

Matrix Metalloproteinases (MMPs) are a family of proteolytic enzymes whose endopeptidase activity is dependent on the presence of specific metal ions. MT1-MMP (or MMP-14), which has been implicated in tumor progression and cellular invasion, contains a membrane-spanning region located C-terminal to a hemopexin-like domain and an N-terminal catalytic domain. We recombinantly expressed the catalytic domain of human MT1-MMP in E. coli and purified it from inclusion bodies using a refolding protocol that yielded significant quantities of active protein. Crystals of MT1-MMP were obtained using the vapour diffusion method. Here, we describe the protocols used for crystallization and the data analysis together with the resulting diffraction pattern

A Caged Substrate Peptide for Matrix Metalloproteinases

Based on the widely applied fluorogenic peptide FS-6 (Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2; Mca = methoxycoumarin-4-acetyl; Dpa = N-3-(2,4-dinitrophenyl)l-α,β-diaminopropionyl) a caged substrate peptide Ac-Lys-Pro-Leu-Gly-Lys*-Lys-Ala-Arg-NH2 (*, position of the cage group) for matrix metalloproteinases was synthesized and characterized. The synthesis implies the modification of a carbamidated lysine side-chain amine with a photocleavable 2-nitrobenzyl group. Mass spectrometry upon UV irradiation demonstrated the complete photolytic cleavage of the protecting group. Time-resolved laser-flash photolysis at 355 nm in combination with transient absorption spectroscopy determined the biphasic decomposition with a = 171 ± 3 ms (79%) and b = 2.9 ± 0.2ms (21%) at pH 6.0 of the photo induced release of 2-nitrobenzyl group. The recombinantly expressed catalytic domain of human membrane type I matrix metalloproteinase (MT1-MMP or MMP-14) was used to determine the hydrolysis efficiency for the caged peptide before and after photolysis. It turned out that the cage group sufficiently shields the peptide from peptidase activity, which can be thus controlled by UV light

Crystallization and preliminary X ray crystallographic analysis of the catalytic domain of membrane type 1 matrix metalloproteinase

Membrane type 1 matrix metalloproteinase (MT1-MMP) belongs to the large family of zinc-dependent endopeptidases termed MMPs that are located in the extracellular matrix. MT1-MMP was crystallized at 277 K using the vapour-diffusion method with PEG as a precipitating agent. Data sets for MT1-MMP were collected to 2.24 Å resolution at 100 K. The crystals belonged to space group P4(3)2(1)2, with unit-cell parameters a = 62.99, c = 122.60 Å. The crystal contained one molecule per asymmetric unit, with a Matthews coefficient (V (M)) of 2.90 Å(3) Da(−1); the solvent content is estimated to be 57.6%

Solvent water interactions within the active site of the membrane type I matrix metalloproteinase

Matrix metalloproteinases (MMP) are an important family of proteases which catalyze the degradation of extracellular matrix components. While the mechanism of peptide cleavage is well established, the process of enzyme regeneration, which represents the rate limiting step of the catalytic cycle, remains unresolved. This step involves the loss of the newly formed N-terminus (amine) and C-terminus (carboxylate) protein fragments from the site of catalysis coupled with the inclusion of one or more solvent waters. Here we report a novel crystal structure of membrane type I MMP (MT1-MMP or MMP-14), which includes a small peptide bound at the catalytic Zn site via its C-terminus. This structure models the initial product state formed immediately after peptide cleavage but before the final proton transfer to the bound amine; the amine is not present in our system and as such proton transfer cannot occur. Modeling of the protein, including earlier structural data of Bertini and coworkers [I. Bertini, et al., Angew. Chem., Int. Ed., 2006, 45, 7952–7955], suggests that the C-terminus of the peptide is positioned to form an H-bond network to the amine site, which is mediated by a single oxygen of the functionally important Glu240 residue, facilitating efficient proton transfer. Additional quantum chemical calculations complemented with magneto-optical and magnetic resonance spectroscopies clarify the role of two additional, non-catalytic first coordination sphere waters identified in the crystal structure. One of these auxiliary waters acts to stabilize key intermediates of the reaction, while the second is proposed to facilitate C-fragment release, triggered by protonation of the amine. Together these results complete the enzymatic cycle of MMPs and provide new design criteria for inhibitors with improved efficacy