Matrix metalloproteinase (MMP) inhibitors are potential therapeutic agents for various diseases including cancer and osteoarthritis. Recent data from clinical trials with MMP inhibitors indicate that there is a great need for selective inhibitors. X-ray crystallography [1], [2], [3] has been used as a tool to help understand specific binding interactions of inhibitors to various MMPs. Large conformational changes have been noted when comparing the structures of the active MMP-3 catalytic domain and the one inhibited by a hydroxamic acid inhibitor. Both soaking and co-crystallization methods were used to generate the MMP-3/ inhibitor complex crystals for data collection. The same inhibitor has also been co-crystallized with MMP-1 and MMP-13. Comparisons of the structures of three inhibited enzymes, MMP-1, 3, and 13 show that MMP-3 and 13 are extremely similar. There are major differences in the binding pockets, especially in the S1’ pocket between MMP-1 and MMP-3/13. These structural studies have helped design more selective inhibitors that can be used as therapeutic agents with improved safety profile.

Aminomethyltransferase, a component of the glycine cleavage system termed T-protein, reversibly catalyzes the degradation of the aminomethyl moiety of glycine attached to the lipoate cofactor of H-protein, resulting in the production of ammonia, 5,10-methylenetetrahydrofolate, and dihydrolipoate-bearing H-protein in the presence of tetrahydrofolate (THF).Several mutations in the human T-protein gene are known to cause non-ketotic hyperglycinemia.Previously we determined the

Since their inception during the eighties, MMP inhibitors (MMPIs) have gone through several cycles of metamorphosis. The design of early MMPIs was based on the cleavage site of peptide substrates. The second generation contained a substituted succinate scaffold (e.g., marimastat) coupled to a modified amino acid residue. The lower molecular weight analogs with multiple substitution possibilities produced a series of MMP inhibitors with varying degrees of selectivity for various MMPs. The introduction of sulfonamides in the midnineties added a new dimension to this field. The simplicity of synthesis coupled with high potency (e.g., CGS-27023A, AG-3340) produced a number of clinical candidates. This review highlights some of the key features that contributed to the discovery of this novel series of MMP inhibitors.

Matrix metalloproteinases are believed to play an important role in pathological conditions such as osteoarthritis, rheumatoid arthritis and tumor invasion. Stromelysin is a zinc-dependent proteinase and a member of the matrix metalloproteinase family. We have solved the crystal structure of an active uninhibited form of truncated stromelysin and a complex with a hydroxamate-based inhibitor. The catalytic domain of the enzyme of residues 83-255 is an active fragment. Two crystallographically independent molecules, A and B, associate as a dimer in the crystals. There are three alpha-helices and one twisted, five-strand beta-sheet in each molecule, as well as one catalytic Zn, one structural Zn and three structural Ca ions. The active site of stromelysin is located in a large, hydrophobic cleft. In particular, the S1' specificity site is a deep and highly hydrophobic cavity. The structure of a hydroxamate-phosphinamide-type inhibitor-bound stromelysin complex, formed by diffusion soaking, has been solved as part of our structure-based design strategy. The most important feature we observed is an inhibitor-induced conformational change in the S1' cavity which is triggered by Tyr223. In the uninhibited enzyme structure, Tyr223 completely covers the S1' cavity, while in the complex, the P1' group of the inhibitor displaces the Tyr223 in order to fit into the S1' cavity. Furthermore, the displacement of Tyr223 induces a major conformational change of the entire loop from residue 222 to residue 231. This finding provides direct evidence that Tyr223 plays the role of gatekeeper of the S1' cavity. Another important intermolecular interaction occurs at the active sit of molecule A, in which the C-terminal tail (residues 251-255) from molecule B inserts. The C-terminal tail interacts extensively with the active site of molecule A, and the last residue (Thr255) coordinated to the catalytic zinc as the fourth ligand, much like a product inhibitor would. The inhibitor-induced conformational change and the intermolecular C-terminal-zinc coordination are significant in understanding the structure-activity relationships of the enzyme.

A new series of hydroxamic acid-based matrix metalloproteinase (MMP) inhibitors containing a unique phosphinamide motif derived from D-amino acid was designed, synthesized, and tested for enzyme inhibition. Compounds with an R configuration at phosphorus were found to be potent MMP inhibitors while molecules with the S configuration were almost inactive. Structure-activity relationship studies of the series led to the discovery of the potent inhibitor 16 with IC50 = 20.5 nM and 24.4 nM against fibroblast collagenase (MMP-1) and stromelysin (MMP-3), respectively. The binding mode of this novel phosphinamide-based series of MMP inhibitors was established based on X-ray crystallography of the complex of stromelysin and 16.

The three-dimensional structure of the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans has been refined at 1.75 Å resolution utilizing the DNA-based protein sequence. The final model incorporates 8034 atoms per molecule, including 552 molecules of solvent, and gives an R-factor of 0.163. The molecule is an H2L2 hetero-tetramer containing a non-crystallographic 2-fold axis of symmetry. The 373-residue H subunit is folded into seven repeats of a four-stranded antiparallel β-sheet motif, arranged in a propeller-like pattern about a pseudo-7-fold rotational axis of symmetry. Each L subunit contains 131 residues folded in a tight structure composed of five β-strands in two sheets and crosslinked by six disulfide bonds. In addition there is an intrasubunit covalent linkage between two tryptophan side-chains that form the unique redox center, tryptophan tryptophylquinone (TTQ). The active site contains the O-6 carbonyl of TTQ, the side-chains of Asp32L, Asp76L, Tyr119L and Thr122L, and two solvent molecules. A potential “gate” (Phe55H) separates the closed active-site cavity from a channel containing a group of highly ordered water molecules to bulk solvent. Phe55H and Tyr119L, and a number of neighboring oxygen atoms, may also provide a binding site for monovalent cations that are known to affect the reactivity and spectral properties of TTQ as well as the oxidative half reaction. The overall reaction has been dissected into a number of discrete steps that may require participation by several individual amino acid residues in the active site acting as general acids and bases.

The copper-containing amine oxidase from the yeast Hansenula polymorpha (YAO) has been crystallized and partially solved by molecular replacement. It catalyzes the oxidative deamination of primary amines by molecular oxygen to the corresponding aldehydes, ammonia and hydrogen peroxide. It contains a covalently bound redox cofactor, topa quinone, generated by post-translational modification of a single tyrosine side chain. The crystals of YAO are orthorhombic, with space-group symmetry P2(1)2(1)2(1) and unit-cell dimensions a = 138.8, b = 148.2, c = 234.0 A and diffract X-rays beyond 2.0 A resolution. Solution by molecular replacement using the E. coli amine oxidase structure [Parsons, Convery, Wilmot, Yadav, Blakeley, Corner, Philips, McPherson & Knowles (1995). Structure, 3, 1171-1184] as a search model reveals that there are three dimers in the asymmetric unit in a trigonal arrangement having 32 point-group symmetry. The solution agrees well with the self-rotation function of YAO. The non-crystallographic threefold axis lies parallel to a crystallographic twofold screw axis and each dimer has twofold symmetry. Phases from the refined model based on the molecular-replacement solution were used to solve one heavy-atom derivative. Model building from the unbiased isomorphous replacement phases is in progress.

The quinoprotein methanol dehydrogenase and cytochrome c-551i are two soluble acidic proteins that form a physiological complex in which electrons are transferred from pyrroloquinoline quinone to heme. The oxidation of methanol dehydrogenase by the cytochrome was studied as a function of ionic strength using stopped-flow spectroscopy. The dissociation constant (Kd) for complex formation decreased 2-fold with increasing ionic strength from 0.21 to 1.3 M and increased at higher ionic strengths. The rate constant for the electron transfer reaction (kET) increased 2-fold with increasing ionic strength from 0.21 to 1.3 M and decreased at higher ionic strengths. The variation of Kd and kET over this range of ionic strengths was described by Van Leeuwen theory, which takes into account monopole-dipole and dipole-dipole forces, in addition to the monopole-monopole force, to predict the interactions between large molecules. Analysis of the kinetic results in terms of these electrostatic interactions indicated the probable orientations for protein-protein binding and electron transfer. To explain the ionic strength dependence of the observed kET, a model is presented in which the true kET is reduced by a factor Kc, an equilibrium constant that describes some rearrangement of the proteins after a nonoptimal collision to produce the most efficient orientation for electron transfer. This model is consistent with the notion that the large reorganizational energy obtained from temperature-dependence studies of this electron transfer reaction [Harris, T. K., & Davidson, V. L. (1993) Biochemistry 32, 14145-14150] is due to such an intracomplex rearrangement.(ABSTRACT TRUNCATED AT 250 WORDS)

The crystal structure of amicyanin, a cupredoxin isolated from Paracoccus denitrificans, has been determined by molecular replacement. The structure has been refined at 2.0 angstrom resolution using energy-restrained least-squares procedures to a crystallographic residual of 15.7%. The copper-free protein, apoamicyanin, has also been refined to 1.8 angstrom resolution with residual 15.5%.The protein is found to have a beta-sandwich topology with nine beta-strands forming two mixed beta-sheets. The secondary structure is very similar to that observed in the other classes of cupredoxins, such as plastocyanin and azurin. Amicyanin has approximately 20 residues at the N-terminus that have no equivalents in the other proteins; a portion of these residues forms the first beta-strand of the structure.The copper atom is located in a pocket between the beta-sheets and is found to have four coordinating ligands: two histidine nitrogens, one cysteine sulfur, and, at a longer distance, one methionine sulfur. The geometry of the copper coordination is very similar to that in the plant plastocyanins. Three of the four copper ligands are located in the loop between beta-strands eight and nine. This loop is shorter than that in the other cupredoxins, having only two residues each between the cysteine and histidine and the histidine and methionine ligands. The amicyanin and apoamicyanin structures are very similar; in particular, there is little difference in the positions of the coordinating ligands with or without copper.One of the copper ligands, a histidine, lies close to the protein surface and is surrounded on that surface by seven hydrophobic residues. This hydrophobic patch is thought to be important as an electron transfer site.

A ternary electron transfer protein complex has been crystallized and a preliminary structure investigation has been carried out. The complex is composed of a quinoprotein, methylamine dehydrogenase (MADH), a blue copper protein, amicyanin, and a c ‐type cytochrome ( c 551i ). All three proteins were isolated from Paracoccus denitrificans. The crystals of the complex are orthorhombic, space group C222 1 with cell dimensions a = 148.81 Å, b = 68.85 Å, and c = 187.18 Å. Two types of isomorphous crystals were prepared: one using native amicyanin and the other copper‐free apo‐amicyanin. The diffraction data were collected at 2.75 Å resolution from the former and at 2.4 Å resolution from the latter. The location of the MADH portion was determined by molecular replacement. The copper site of the amicyanin molecule was located in an isomorphous difference Fourier while the iron site of the cytochrome was found in an anomalous difference Fourier. The MADH from P. denitrificans (PD‐MADH) is an H 2 L 2 hetero‐tetramer with the H subunit containing 373 residues and the L subunit 131 residues, the latter containing a novel redox cofactor, tryptophan tryptophylquinone (TTQ). The amicyanin of P. denitrificans contains 105 residues and the cytochrome c 551i contains 155 residues. The ternary complex consists of one MADH tetramer with two molecules of amicyanin and two of c 551i , forming a hetero‐octamer; the octamer is located on a crystallographic diad. The relative positions of the three redox centers — i.e., the TTQ of MADH, the copper of amicyanin, and the heme group of c 551i — are presented.

The three‐dimensional structure of the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans (PD‐MADH) has been determined at 2.8 Å resolution by the molecular replacement method combined with map averaging procedures, using data collected from an area detector. The structure of methylamine dehydrogenase from Thio‐bacillus versutus, which contains an “X‐ray” sequence, was used as the starting search model. MADH consists of 2 heavy (H) and 2 light (L) subunits related by a molecular 2‐fold axis. The H subunit is folded into seven four‐stranded β‐segments, forming a disk‐shaped structure, arranged with pseudo‐7‐fold symmetry. A 31‐residue elongated tail exists at the N‐terminus of the H subunit in MADH from T. versutus but is partially digested in this crystal form of MADH from P. denitrificans, leaving the H subunit about 18 residues shorter. Each L subunit contains 127 residues arranged into 10 β‐strands connected by turns. The active site of the enzyme is located in the L subunit and is accessible via a hydrophobic channel between the H and L subunits. The redox cofactor of MADH, tryptophan tryptophylquinone is highly unusual. It is formed from two co‐valently linked tryptophan side chains at positions 57 and 107 of the L subunit, one of which contains an orthoquinone. © 1992 Wiley‐Liss, Inc.

The crystal structure of the complex between the quinoprotein methylamine dehydrogenase (MADH) and the type I blue copper protein amicyanin, both from Paracoccus denitrificans, has been determined at 2.5-A resolution using molecular replacement. The search model was MADH from Thiobacillus versutus. The amicyanin could be located in an averaged electron density difference map and the model improved by refinement and model building procedures. Nine beta-strands are observed within the amicyanin molecule. The copper atom is located between three antiparallel strands and is about 2.5 A below the protein surface. The major intermolecular interactions occur between amicyanin and the light subunit of MADH where the interface is largely hydrophobic. The copper atom of amicyanin and the redox cofactor of MADH are about 9.4 A apart. One of the copper ligands, His 95, lies between the two redox centers and may facilitate electron transfer between them.