Klaus Maskos

Mechanism of inhibition of the human matrix metalloproteinase stromelysin-1 by TIMP-1.

Matrix metalloproteinases (MMPs) are zinc endopeptidases that are required for the degradation of extracellular matrix components during normal embryo development, morphogenesis and tissue remodelling. Their proteolytic activities are precisely regulated by endogenous tissue inhibitors of metalloproteinases (TIMPs). Disruption of this balance results in diseases such as arthritis, atherosclerosis, tumour growth and metastasis. Here we report the crystal structure of an MMP-TIMP complex formed between the catalytic domain of human stromelysin-1 (MMP-3) and human TIMP-1. TIMP-1, a 184-residue protein, has the shape of an elongated, contiguous wedge. With its long edge, consisting of five different chain regions, it occupies the entire length of the active-site cleft of MMP-3. The central disulphide-linked segments Cys 1-Thr 2-Cys 3-Val 4 and Ser 68-Val 69 bind to either side of the catalytic zinc. Cys 1 bidentally coordinates this zinc, and the Thr-2 side chain extends into the large specificity pocket of MMP-3. This unusual architecture of the interface between MMP-3 and TIMP-1 suggests new possibilities for designing TIMP variants and synthetic MMP inhibitors with potential therapeutic applications.

Crystal structure of the complex formed by the membrane type 1-matrix metalloproteinase with the tissue inhibitor of metalloproteinases-2, the soluble progelatinase A receptor

The proteolytic activity of matrix metalloproteinases (MMPs) towards extracellular matrix components is held in check by the tissue inhibitors of metalloproteinases (TIMPs). The binary complex of TIMP‐2 and membrane‐type‐1 MMP (MT1‐MMP) forms a cell surface located ‘receptor’ involved in pro‐MMP‐2 activation. We have solved the 2.75 Å crystal structure of the complex between the catalytic domain of human MT1‐MMP (cdMT1‐MMP) and bovine TIMP‐2. In comparison with our previously determined MMP‐3–TIMP‐1 complex, both proteins are considerably tilted to one another and show new features. CdMT1‐MMP, apart from exhibiting the classical MMP fold, displays two large insertions remote from the active‐site cleft that might be important for interaction with macromolecular substrates. The TIMP‐2 polypeptide chain, as in TIMP‐1, folds into a continuous wedge; the A‐B edge loop is much more elongated and tilted, however, wrapping around the S‐loop and the β‐sheet rim of the MT1‐MMP. In addition, both C‐terminal edge loops make more interactions with the target enzyme. The C‐terminal acidic tail of TIMP‐2 is disordered but might adopt a defined structure upon binding to pro‐MMP‐2; the Ser2 side‐chain of TIMP‐2 extends into the voluminous S1′ specificity pocket of cdMT1‐MMP, with its Oγ pointing towards the carboxylate of the catalytic Glu240. The lower affinity of TIMP‐1 for MT1‐MMP compared with TIMP‐2 might be explained by a reduced number of favourable interactions.

Structural properties of matrix metalloproteinases

Abstract. Matrix metalloproteinases (MMPs) are involved in extracellular matrix degradation. Their proteolytic activity must be precisely regulated by their endogenous protein inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Disruption of this balance results in serious diseases such as arthritis, tumour growth and metastasis. Knowledge of the tertiary structures of the proteins involved is crucial for understanding their functional properties and interference with associated dysfunctions. Within the last few years, several three-dimensional MMP and MMP-TIMP structures became available, showing the domain organization, polypeptide fold and main specificity determinants. Complexes of the catalytic MMP domains with various synthetic inhibitors enabled the structure-based design and improvement of high-affinity ligands, which might be elaborated into drugs. A multitude of reviews surveying work done on all aspects of MMPs have appeared in recent years, but none of them has focused on the three-dimensional structures. This review was written to close the gap.

Insights into MMP-TIMP interactions.

ABSTRACT: The proteolytic activity of the matrix metalloproteinases (MMPs) involved in extracellular matrix degradation must be precisely regulated by their endogenous protein inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Disruption of this balance can result in serious diseases such as arthritis and tumor growth and metastasis. Knowledge of the tertiary structures of the proteins involved in such processes is crucial for understanding their functional properties and to interfere with associated dysfunctions. Within the last few years, several three‐dimensional structures have been determined showing the domain organization, the polypeptide fold, and the main specificity determinants of the MMPs. Complexes of the catalytic MMP domains with various synthetic inhibitors enabled the structure‐based design and improvement of high‐affinity ligands, which might be elaborated into drugs. Very recently, structural information also became available for some TIMP structures and MMP‐TIMP complexes, and these new data elucidated important structural features that govern the enzyme‐inhibitor interaction.

Structural basis of the matrix metalloproteinases and their physiological inhibitors, the tissue inhibitors of metalloproteinases

Abstract The matrix metalloproteinases (MMPs) constitute a family of multidomain zinc endopeptidases with a metzincin-like catalytic domain, which are involved in extracellular matrix degradation but also in a number of other important biological processes. Under healthy conditions, their proteolytic activity is precisely regulated by their main endogenous protein inhibitors, the tissue inhibitors of metalloproteinases. Disruption of this balance results in pathophysiological processes such as arthritis, tumor growth and metastasis, rendering the MMPs attractive targets for inhibition therapy. Knowledge of their tertiary structures is crucial for a full understanding of their functional properties and for rational drug design. Since the first appearance of atomic MMP structures in 1994, a large amount of structural information has become available on the catalytic domains of MMPs and their substrate specificity, interaction with synthetic inhibitors and the TIMPs, the domain organization, and on complex formation with other proteins. This review will outline our current structural knowledge of the MMPs and the TIMPs.

A novel strategy for inhibition of α-amylases: yellow meal worm α-amylase in complex with the Ragi bifunctional inhibitor at 2.5 å resolution

Abstract Background: α -Amylases catalyze the hydrolysis of α -D-(1,4)-glucan linkages in starch and related compounds. There is a wide range of industrial and medical applications for these enzymes and their inhibitors. The Ragi bifunctional α -amylase/trypsin inhibitor (RBI) is the prototype of the cereal inhibitor superfamily and is the only member of this family that inhibits both trypsin and α -amylases. The mode of inhibition of α -amylases by these cereal inhibitors has so far been unknown. Results: The crystal structure of yellow meal worm α -amylase (TMA) in complex with RBI was determined at 2.5 a resolution. RBI almost completely fills the substrate-binding site of TMA. Specifically, the free N terminus and the first residue (Ser1) of RBI interact with all three acidic residues of the active site of TMA (Asp 185, Glu222 and Asp287). The complex is further stabilized by extensive interactions between the enzyme and inhibitor. Although there is no significant structural reorientation in TMA upon inhibitor binding, the N-terminal segment of RBI, which is highly flexible in the free inhibitor, adopts a 3 10 -helical conformation in the complex. RBIs trypsin-binding loop is located opposite the α -amylase-binding site, allowing simultaneous binding of α -amylase and trypsin. Conclusions: The binding of RBI to TMA constitutes a new inhibition mechanism for α -amylases and should be general for all α -amylase inhibitors of the cereal inhibitor superfamily. Because RBI inhibits two important digestive enzymes of animals, it constitutes an efficient plant defense protein and may be used to protect crop plants from predatory insects.

Inhibition of influenza virus replication via small molecules that induce the formation of higher-order nucleoprotein oligomers

Influenza nucleoprotein (NP) plays multiple roles in the virus life cycle, including an essential function in viral replication as an integral component of the ribonucleoprotein complex, associating with viral RNA and polymerase within the viral core. The multifunctional nature of NP makes it an attractive target for antiviral intervention, and inhibitors targeting this protein have recently been reported. In a parallel effort, we discovered a structurally similar series of influenza replication inhibitors and show that they interfere with NP-dependent processes via formation of higher-order NP oligomers. Support for this unique mechanism is provided by site-directed mutagenesis studies, biophysical characterization of the oligomeric ligand:NP complex, and an X-ray cocrystal structure of an NP dimer of trimers (or hexamer) comprising three NP_A:NP_B dimeric subunits. Each NP_A:NP_B dimeric subunit contains two ligands that bridge two composite, protein-spanning binding sites in an antiparallel orientation to form a stable quaternary complex. Optimization of the initial screening hit produced an analog that protects mice from influenza-induced weight loss and mortality by reducing viral titers to undetectable levels throughout the course of treatment.

DsbA and DsbC-catalyzed oxidative folding of proteins with complex disulfide bridge patterns in vitro and in vivo

Oxidative protein folding in the periplasm of Escherichia coli is catalyzed by the thiol-disulfide oxidoreductases DsbA and DsbC. We investigated the catalytic efficiency of these enzymes during folding of proteins with a very complex disulfide pattern in vivo and in vitro, using the Ragi bifunctional inhibitor (RBI) as model substrate. RBI is a 13.1 kDa protein with five overlapping disulfide bonds. We show that reduced RBI can be refolded quantitatively in glutathione redox buffers in vitro and spontaneously adopts the single correct conformation out of 750 possible species with five disulfide bonds. Under oxidizing redox conditions, however, RBI folding is hampered by accumulation of a large number of intermediates with non-native disulfide bonds, while a surprisingly low number of intermediates accumulates under optimal or reducing redox conditions. DsbC catalyzes folding of RBI under all redox conditions in vitro, but is particularly efficient in rearranging buried, non-native disulfide bonds formed under oxidizing conditions. In contrast, the influence of DsbA on the refolding reaction is essentially restricted to reducing redox conditions where disulfide formation is rate limiting. The effects of DsbA and DsbC on folding of RBI in E.coli are very similar to those observed in vitro. Whereas overexpression of DsbA has no effect on the amount of correctly folded RBI, co-expression of DsbC enhanced the efficiency of RBI folding in the periplasm of E.coli about 14-fold. Addition of reduced glutathione to the growth medium together with DsbC overexpression further increased the folding yield of RBI in vivo to 26-fold. This shows that DsbC is the bacterial enzyme of choice for improving the periplasmic folding yields of proteins with very complex disulfide bond patterns.

Structural basis of matrix metalloproteinases and tissue inhibitors of metalloproteinases

The matrix metalloproteinases (MMPs) constitute a family of secreted/cell-surface-anchored multidomain zinc endopeptidases, all of which exhibit a catalytic domain of a common metzincin-like topology, and which are involved in degradation of the extracellular matrix but also in a number of other biologic processes. Normally, the proteolytic activity of the MMPs is precisely regulated by their main endogenous protein inhibitors, in particular the tissue inhibitors of metalloproteinases (TIMPs). Disruption of this balance results in serious diseases such as arthritis, tumor growth, and tumor metastasis, rendering the MMPs attractive targets for inhibition therapy. Knowledge of their tertiary structures is crucial for a full understanding of their functional properties and their associations with dysfunctions. Since the reports of the first atomic structures of MMPs and TIMPs in 1994, considerable structural information has become available about both of these families of substances. Many of the MMP structures have been determined as complexes with synthetic inhibitors, facilitating knowledge-based drug design. This review focuses on the currently available 3D structural information about MMPs and TIMPs.

The Structure of CDK8/CycC Implicates Specificity in the CDK/Cyclin Family and Reveals Interaction with a Deep Pocket Binder

Cyclin-dependent kinase (CDK) 8 associates with cyclin C (CycC) and belongs to the CDK module of the Mediator of transcription, together with MED12 and MED13. CDK8 is involved in the regulation of mRNA transcription and was identified as a potent oncogene in colon cancerogenesis. We have solved the 2.2-Å crystal structure of CDK8/CycC in complex with sorafenib, an anti-cancer drug of clinical relevance. The CDK8 structure reveals a unique CycC recognition helix that explains the specificity of the CDK8/CycC pair and discrimination among the highly promiscuous binding in the CDK/cyclin family. In contrast to all CDKs, the CDK8 activation loop appears not to be phosphorylated. Based on the structure, we discuss an alternate mode of CDK8 activation to the general CDK activation by T-loop phosphorylation. Sorafenib binds to the catalytic cleft of CDK8. It displays a deep pocket binding mode and is the first small molecule to induce a DFG-out conformation in the CDK family, which is actually DMG-out in CDK8.