Peter Reinemer

The X-ray crystal structure of the catalytic domain of human neutrophil collagenase inhibited by a substrate analogue reveals the essentials for catalysis and specificity.

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 A 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 five‐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.(ABSTRACT TRUNCATED AT 250 WORDS)

THE THREE-DIMENSIONAL STRUCTURE OF CLASS PI GLUTATHIONE S-TRANSFERASE IN COMPLEX WITH GLUTATHIONE SULFONATE AT 2.3 A RESOLUTION

The three‐dimensional structure of class pi glutathione S‐transferase from pig lung, a homodimeric enzyme, has been solved by multiple isomorphous replacement at 3 A resolution and preliminarily refined at 2.3 A resolution (R = 0.24). Each subunit (207 residues) is folded into two domains of different structure. Domain I (residues 1–74) consists of a central four‐stranded beta‐sheet flanked on one side by two alpha‐helices and on the other side, facing the solvent, by a bent, irregular helix structure. The topological pattern resembles the bacteriophage T4 thioredoxin fold, in spite of their dissimilar sequences. Domain II (residues 81–207) contains five alpha‐helices. The dimeric molecule is globular with dimensions of about 55 A ×52 A ×45 A. Between the subunits and along the local diad, is a large cavity which could possibly be involved in the transport of nonsubstrate ligands. The binding site of the competitive inhibitor, glutathione sulfonate, is located on domain I, and is part of a cleft formed between intrasubunit domains. Glutathione sulfonate is bound in an extended conformation through multiple interactions. Only three contact residues, namely Tyr7, Gln62 and Asp96 are conserved within the family of cytosolic glutathione S‐transferases. The exact location of the binding site(s) of the electrophilic substrate is not clear. Catalytic models are discussed on the basis of the molecular structure.

Three-dimensional structure of class π glutathione S-transferase from human placenta in complex with S-hexylglutathione at 2.8 Å resolution☆

The three-dimensional structure of human class pi glutathione S-transferase from placenta (hGSTP1-1), a homodimeric enzyme, has been solved by Patterson search methods and refined at 2.8 A resolution to a final crystallographic R-factor of 19.6% (8.0 to 2.8 A resolution). Subunit folding topology, subunit overall structure and subunit association closely resembles the structure of porcine class pi glutathione S-transferase. The binding site of a competitive inhibitor, S-hexylglutathione, is analyzed and the locations of the binding regions for glutathione (G-site) and electrophilic substrates (H-site) are determined. The specific interactions between protein and the inhibitors glutathione peptide are the same as those observed between glutathione sulfonate and the porcine isozyme. The H-site is located adjacent to the G-site, with the hexyl moiety lying above a segment (residues 8 to 10) connecting strand beta 1 and helix alpha A where it is in hydrophobic contact with Tyr7, Phe8, Val10, Val35 and Tyr106. Catalytic models are discussed on the basis of the molecular structure.

Crystal Structure of the Interleukin-4/Receptor α Chain Complex Reveals a Mosaic Binding Interface

Interleukin-4 (IL-4) is a principal regulatory cytokine during an immune response and a crucial determinant for allergy and asthma. IL-4 binds with high affinity and specificity to the ectodomain of the IL-4 receptor alpha chain (IL4-BP). Subsequently, this intermediate complex recruits the common gamma chain (gamma c), thereby initiating transmembrane signaling. The crystal structure of the intermediate complex between human IL-4 and IL4-BP was determined at 2.3 A resolution. It reveals a novel spatial orientation of the two proteins, a small but unexpected conformational change in the receptor-bound IL-4, and an interface with three separate clusters of trans-interacting residues. Novel insights on ligand binding in the cytokine receptor family and a paradigm for receptors of IL-2, IL-7, IL-9, and IL-15, which all utilize gamma c, are provided.

Structural implications for the role of the N terminus in the ‘superactivation’ of collagenases: A crystallographic study

For the collagenases PMNL‐CL and FIB‐CL, the presence of the N‐terminal Phe79 correlates with an increase in proteolytic activity. We have determined the X‐ray crystal structure of the recombinant Phe79‐Gly242 catalytic domain of human neutrophil collagenase (PMNL‐CL, MMP‐8) using the recently solved model of the Met80‐Gly242 form for phasing and subsequently refined it to a final crystalographic R‐factor of 18.0% at 2.5 Å 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 Pro86, which is disordered in the Met80‐Gly242 form, packs against a concave hydrophobic surface made by the C‐terminal helix. The N‐terminal Phe79 ammonium group makes a salt link with the side chain carboxylate group of the strictly conserved Asp232. Stabilization of the catalytic site might be conferred via strong hydrogen bonds made by the adjacent, likewise strictly conserved Asp233 with the characteristic ‘Met‐turn’, which forms the base of the active site residues.

Class π glutathione S‐transferase from pig lung

A cytosolic glutathione S-transferase from pig lung was purified 210-fold to apparent homogeneity. The enzyme was classified as a class pi isoenzyme on the basis of its physical and chemical properties. It is homodimeric with a subunit Mr of 23,500, has a pI of 7.2, and shows a high specific activity towards ethacrynic acid. The glutathione analogues, S-hexylglutathione and glutathione sulfonate, were strong reversible inhibitors. The enzymes primary structure, established entirely by protein chemical methods, consists of 203 amino acids and is highly similar (82-84% residue identity) to the rat and human class pi isoenzymes. Furthermore, there was no evidence of microheterogeneity or post-translational modifications. Each subunit contains a highly reactive cysteine residue, the modification of which leads to enzyme inactivation. None of the cysteine residues in the pig enzyme appear to form intramolecular disulfide bonds. Singel crystals of the glutathione-S-transferase-glutathione-sulfonate complex were obtained by the hanging-drop method of vapour diffusion from poly(ethylene glycol) 4000 solutions. The crystals belong to the orthorhombic space group P212121 with unit cell dimensions of a = 10.125 nm, b = 8.253 nm and c = 5.428 nm and diffract to better than 0.22 nm.

Equilibrium unfolding of class π glutathione S-transferase

The equilibrium unfolding transition of class pi glutathione S-transferase, a homodimeric protein, from porcine lung was monitored by spectroscopic methods (fluorescence emission and ultraviolet absorption), and by enzyme activity changes. Solvent (guanidine hydrochloride and urea)-induced denaturation is well described by a two-state model involving significant populations of only the folded dimer and unfolded monomer. Neither a folded, active monomeric form nor stable unfolding intermediates were detected. The conformational stability, delta Gu (H2O), of the native dimer was estimated to be about 25.3 +/- 2 kcal/mol at 20 degrees C and pH6.5.

Mutational substitution of residues implicated by crystal structure in binding the substrate glutathione to human glutathione S-transferase π

Site-directed substitution mutations were introduced into a cDNA expression vector (pUC120 pi) that encoded a human glutathione S-transferase pi isozyme to non-conservatively replace four residues (Tyr7, Arg13, Gln62 and Asp96). Our earlier X-ray crystallographic analysis implicated these residues in binding and/or chemically activating the substrate glutathione. Each substitution mutation decreased the specific activity of the enzyme to less than 2% of the wild-type. Glutathione-binding was also reduced; however, the Tyr7----Phe mutant still retained 27% of the wild-type capacity to bind glutathione, underlining the primary role that this residue is likely to play in chemically activating the glutathione molecule during catalysis.

A Versatile High-Throughput Screen for Inhibitors of Lipid Kinase Activity: Development of an Immobilized Phospholipid Plate Assay for Phosphoinositide 3-Kinase γ

The family of phosphoinositide 3-kinases (PI3K) regulates fundamental cellular responses such as proliferation, apoptosis, motility, and adhesion. In particular, the PI3K γ isoform plays a critical role in the control of cell migration. Despite the attractiveness of PI3-kinases as drug targets, drug discovery efforts have been hampered by the lack of appropriate lipid kinase assay formats suitable for high-throughput screening. The authors report the development of a simple and robust 384-well plate assay that is based on33 P-phosphate transfer from radiolabeled [γ33 P]ATP to phosphatidylinositol immobilized on Maxisorp™ plates. The established assay format for PI3K γ was easily adapted to the automated screening platform and was successfully employed for high-throughput screening. Enzymatic and inhibition characteristics of recombinant human PI3K γ determined with the plate assay are in very good agreement with previously reported values determined in other assay formats. Maximal catalytic activity of PI3K γ was observed at pH 7.0. The apparent Km value for ATP using a 1:1 mixture of phosphatidylinositol and phosphatidylserine was determined to be 7.3μM (6.0-8.6 μM, 95% confidence interval [CI]). IC50 values for known PI3-kinase inhibitors were determined to be 1.45 nM (1.17-1.80 nM, 95% CI) for wortmannin and estimated from partial inhibition data to be 1400, 2830, and 21,400 nM for quercetin, LY294002, and staurosporine, respectively. This novel assay approach allows for screening of inhibitors of lipid kinases in high-throughput mode and thereby may facilitate the identification of novel inhibitory structures for drug development.

Macrocyclic statine-based inhibitors of BACE-1

Minimal sequence requirements for binding of substrate‐derived statine peptides to the aspartyl enzyme were established on the basis of the X‐ray cocrystal structure of the hydroxyethylene‐octapeptide OM00‐3 in complexation with BACE‐1. With this information to hand, macrocyclic compounds that conformationally restrict and preorganize the peptide backbone for an entropically favoured binding to the enzyme active site cleft were designed. By means of a side chain‐to‐side chain ring closure between two aspartyl residues in the P2 and P3′ positions through phenylene‐1,3‐dimethanamine, a 23‐membered ring structure was obtained; this structure retained an extended conformation of the peptide backbone, including the transition state analogue statine for tight interactions with the two aspartyl residues of the active centre. The conformational preorganization of the inhibitor molecule was verified by NMR structural analysis and was then confirmed by the crystal structure of the BACE‐1/inhibitor complex. Detailed insights into the binding mode of this macrocyclic inhibitor explained its moderate binding affinity in cell‐free assays (Ki=2.5 μM) and yielded precious information for possible structural optimization in view of the lack of steric clashes of the macrocycle with the flap domain of the enzyme.