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We report DNA construction, baculovirus expression, and partial characterization of a minienzyme form of the human matrix metalloproteinase-9 (MMP-9). The MMP-9 minienzyme gene construct consisting of the pre, pro, and catalytic domains of the MMP-9 was introduced into Sf9 insect cells using a baculovirus expression system. The expression of the recombinant MMP-9 minienzyme was estimated to be approximately 0.8 mg/L of cell medium. The recombinant protein was purified using a single-step gelatin-Sepharose affinity column and yielded a highly stable and active minienzyme with gelatinolytic activity. Moreover, two interesting findings related to MMP-9 interactions with heparin and TIMP-1 resulted from our studies. First, the pro and catalytic domains of the human MMP-9 are not sufficient for heparin affinity. Second, in contrast to the prevailing consensus, TIMP-1 blockade of the enzymatic activity of MMP-9 does not require prior binding to the C-terminus of its MMP-9 protein substrate., (Copyright 2001 Elsevier Science.)

Human stuttering is a simple example of the repetition of sounds or symbols, sometimes associated with single letters, and may be used to illustrate the amazing repetition of amino acids (symbolized by a letter, e.g. W) in proteins. A survey of available databases with highly improbable strings of single amino acids is tabulated. This paper concludes with a challenge to the crystallographic community to probe the structural origins of the structure-function relationship in this neglected area. When nature stutters, we should pay attention.

The South American imported fire ant (Solenopsis invicta), without natural enemies in the United States, widely infests the southern United States, causing more than a half billion dollars in health and agriculture-related damage annually in Texas alone. Fire ants are resistant to most insecticides, so control will require a more fundamental understanding of their biochemistry and metabolism leading to the design of selective, ecologically safe insecticides. The 4th instar larvae play a crucial role in the nutrition of the colony by secreting proteinases (especially chymotrypsin) which digest food products for the entire colony. The first structure of an ant proteolytic enzyme, fire ant chymotrypsin, was determined to atomic resolution (1.7 A). A structural comparison of the ant and mammalian structures confirms the "universality" of the serine proteinase motif and reveals a difference at residues 147-148, which are proteolytically removed in the bovine enzyme but are firmly intact in the ant chymotrypsin, suggesting a different activation mechanism for the latter. Likewise, the absence of the covalently attached propeptide domain (1-15) further suggests an uncharacteristic activation mechanism. The presence of Gly189 in the S1 site is an atypical feature of this chymotrypsin and is comparable only to human leukocyte elastase, hornet chymotrypsin and fiddler crab collagenase. Binding studies confirm the chymotrypsin nature of this novel enzyme., (Copyright 2000 Academic Press.)

Drug design is a creative act of the same magnitude as composing, sculpting, or writing. The results can touch the lives of millions, but the creator is rarely one scientist and the rewards are distributed differently in the arts than in the sciences. The mechanisms of creativity are the same, i.e., incremental (plodding from darkness to dawn) or sudden (the "Eureka" effect) realization, but both are poorly understood. Creativity remains a human characteristic, but it is directly related to the tools available, especially computer software and hardware. While modelling software continues to mature, very little new has evolved in terms of hardware. Here, we discuss the history of molecular modelling and describe two novel modelling tools, a haptic device and a program, SCULPT, to generate solid molecular models at atomic resolution.

The matrix metalloproteinases are crucial in the physiological and pathological degradation of the mammalian extracellular matrix, including breast tumours, and osteoarthritic cartilage. These enzymes are classified according to their matrix substrate specificity. Collagenase-3 (MMP-13) is a member of this family and preferentially cleaves type II collagen, cartilage, fibronectin and aggrecan. Collagenase-3 is normally expressed in hypertrophic chondrocytes, periosteal cells, and osteoblasts during bone development. The structure of the catalytic domain of recombinant mouse collagenase-3, complexed to the hydroxamate inhibitor (RS-113456), is reported at 2.0 A resolution. Molecular replacement and weak phasing information from a single derivative determined the structure. Neither molecular replacement nor derivative methods had a sufficient radius of convergence to yield a refinable structure. The structure illuminates the atomic zinc ion interactions with functional groups in the active site, emphasizing zinc ligation and the very voluminous hydrophobic P1' group for the inhibitor potency. The structure provides insight into the specificity of this enzyme, facilitating design of specific inhibitors to target various diseases.

Crotalus adamanteus snake venom adamalysin II is the structural prototype of the adamalysin or ADAM family comprising proteolytic domains of snake venom metalloproteinases, multimodular mammalian reproductive tract proteins, and tumor necrosis factor alpha convertase, TACE, involved in the release of the inflammatory cytokine, TNFalpha. The structure of adamalysin II in noncovalent complex with two small-molecule right-hand side peptidomimetic inhibitors (Pol 647 and Pol 656) has been solved using X-ray diffraction data up to 2.6 and 2.8 A resolution. The inhibitors bind to the S'-side of the proteinase, inserting between two protein segments, establishing a mixed parallel-antiparallel three-stranded beta-sheet and coordinate the central zinc ion in a bidentate manner via their two C-terminal oxygen atoms. The proteinase-inhibitor complexes are described in detail and are compared with other known structures. An adamalysin-based model of the active site of TACE reveals that these small molecules would probably fit into the active site cleft of this latter metalloproteinase, providing a starting model for the rational design of TACE inhibitors.

Matrix metalloproteinase enzymes have been implicated in degenerative processes like tumor cell invasion, metastasis, and arthritis. Specific metalloproteinase inhibitors have been used to block tumor cell proliferation. We have examined the interaction of batimastat (BB-94) with a metalloproteinase [atrolysin C (Ht-d), EC 3.4.24.42] active site at 2.0-angstroms resolution (R = 16.8%). The title structure exhibits an unexpected binding geometry, with the thiophene ring deeply inserted into the primary specificity site. This unprecedented binding geometry dramatizes the significance of the cavernous primary specificity site, pointing the way for the design of a new generation of potential antitumor drugs.

The structure of the metalloproteinase and hemorrhagic toxin atrolysin C form d (EC 3.4.24.42), from the venom of the western diamondback rattlesnake Crotalus atrox, has been determined to atomic resolution by x-ray crystallographic methods. This study illuminates the nature of inhibitor binding with natural (< Glu-Asn-Trp, where < Glu is pyroglutamic acid) and synthetic (SCH 47890) ligands. The primary specificity pocket is exceptionally deep; the nature of inhibitor and productive substrate binding is discussed. Insights gained from the study of these complexes facilitate the design of potential drugs to treat diseases where matrix metalloproteinases have been implicated, e.g., arthritis and tumor metastasis.

1. Adamalysin II, alias proteinase II, a 24-kDa zinc-endopeptidase from the snake venom of Crotalus adamanteus, is a member of a large family of metalloproteinases isolated as small proteinases or proteolytic domains of mosaic hemorrhagic proteins from various snake venoms. Homologous domains have been recently detected in multimodular mammalian reproductive tract proteins and in mammalian gene products, somatic rearrangements of which seem to be linked to primary breast cancers. 2. The 2.0 A X-ray crystal structure of adamalysin II reveals an ellipsoidal molecule with a shallow active-site cleft separating a relatively irregularly folded sub-domain from the main molecular body composed of a 5-stranded beta-sheet and four alpha-helices. Opposite to this active-site cleft is an integrated calcium ion liganded by carbonyl and strongly conserved carboxylate/carboxamide residues. The folding of the peptide fragment containing the zinc-binding motif HExxHxxGxxH bears only a distant resemblance to thermolysin; it is identical to that found in astacin, in collagenases, and in serralysins, with the three histidines (His142, His146, His152) and a water molecule (linked to the glutamic acid Glu143) likewise constituting the zinc ligand; similar to collagenases, but in contrast to astacin, adamalysin II lacks a fifth (tyrosine) zinc ligand, leaving its zinc-ion tetrahedrally coordinated. Furthermore, adamalysin II shares an identical active-site basement formed by a common Met-turn. 3. Due to their virtually identical active-site environment and similar folding topology, the snake venom metalloproteinases (hitherto called adamalysins) and the three other proteinases might be grouped into a common superfamily called metzincins with distinct differences from the thermolysin family.

A series of new acyl, urea, and carbonate derivatives of 7-amino-4-chloro-3-methoxyisocoumarin were synthesized and evaluated as irreversible inhibitors of human neutrophil elastase (HNE) and porcine pancreatic elastase (PPE). Inhibition of HNE is directly related to the hydrophobicity of the substituent on the 7-amino group. The N-Tos-Phe derivative (19) is the best HNE inhibitor with a second-order rate constant kobs/[I] = 200,000 M-1 s-1. The closest analogue in this series, the 3,3-diphenylpropionyl derivative 5, had a kobs/[I] = 130,000 M-1 s-1 with HNE. In contrast to the Tos-Phe derivative 19, phenylacetyl derivative 2 and carbonates 22 and 25 gave extremely stable enzyme-inhibitor complexes with deacylation half-lives longer than 48 h with both elastases. N-Phenylurea derivative 25 was the best inhibitor for PPE with a second-order rate constant kobs/[I] = 7300 M-1 s-1. The crystal structure of a complex of PPE with N-tosyl-Phe derivative 19 was determined at 1.85-A resolution and refined to a final R factor of 16.9%. The isocoumarin forms an acyl enzyme with Ser-195, while His-57 is near the inhibitor, but not covalently linked. The Tos-Phe makes a few hydrophobic contacts with the S' subsites of PPE, but appears to be interacting primarily with itself in the PPE structure. This region of HNE is more hydrophobic and modeling indicates that the inhibitor would probably make additional contacts with the enzyme.

Eighteen climbers actively ascended Mount Rainier (elevation 4,392 m) twice during a randomized, double-blind, concurrent, placebo-controlled, crossover trial comparing the use of acetazolamide, 250 mg, dexamethasone, 4 mg, and placebo every 8 hours as prophylaxis for acute mountain sickness. Each subject was randomly assigned to receive placebo during one ascent and one of the active medications during the other ascent. Assessment of acute mountain sickness was performed using the Environmental Symptoms Questionnaire and a clinical interview. At the summit or high point attained above base camp, the use of dexamethasone significantly reduced the incidence of acute mountain sickness and the severity of symptoms. Cerebral and respiratory symptom severity scores for subjects receiving dexamethasone (0.26 +/- 0.16 and 0.20 +/- 0.19, respectively) were significantly lower than similar scores for both acetazolamide (0.80 +/- 0.80 and 1.20 +/- 1.05; P = 0.25) and placebo (1.11 +/- 1.02 and 1.45 +/- 1.27; P = .025). Neither the use of dexamethasone nor that of acetazolamide measurably affected other physical or mental aspects. Compared with placebo, dexamethasone appears to be effective for prophylaxis of symptoms associated with acute mountain sickness accompanying rapid ascent. The precise role of dexamethasone for the prophylaxis of acute mountain sickness is not known, but it can be considered for persons without contraindications who are intolerant of acetazolamide, for whom acetazolamide is ineffective, or who must make forced, rapid ascent to high altitude for a short period of time with a guaranteed retreat route.

The complex of porcine pancreatic elastase (PPE) with 7-amino-3-(2-bromoethoxy)-4-chloroisocoumarin, a potent mechanism-based inhibitor, was crystallized and the crystal structure determined at 1.9-A resolution with a final R factor of 17.1%. The unbiased difference Fourier electron density map showed continuous density from O gamma of Ser 195 to the benzoyl carbonyl carbon atom and from N epsilon 2 of His 57 to the carbon atom at the 4-position of the isocoumarin ring in the inhibitor. This suggested unambiguously that the inhibitor was doubly covalently bound to the enzyme. It represents the first structural evidence for irreversible binding of an isocoumarin inhibitor to PPE through both Ser 195 and His 57 in the active site. The PPE-inhibitor complex is only partially activated in solution by hydroxylamine and confirms the existence of the doubly covalently bound complex along with the acyl enzyme. The benzoyl carbonyl oxygen atom of the inhibitor is not situated in the oxyanion hole formed by the amide (greater than NH) groups of Gly 193 and Ser 195. The complex is stabilized by the hydrogen-bonding interactions in the active site (from the N epsilon 2 of Gln 192 to the bromine atom in the inhibitor and the amino group at the 7-position of the isocoumarin ring to the carbonyl oxygen of Thr 41) and by van der Waals interactions. The inhibition rates of several 7-substituted 4-chloro-3-(bromoalkoxy)isocoumarins toward PPE were measured.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular dynamic simulations (30ps) of the Michaelis complex of hexapeptide (Thr-Pro-nVal-Leu-Tyr-Thr) bound to porcine pancreatic elastase (PPE) hydrated by about 2000 water molecules have been performed using the AMBER 3.0 program package. Dynamical properties of the conformation of the active site have been examined. A comparison with previously reported simulations of native PPE shows that after the substrate is bound, the catalytically crucial H-bond between O gamma-H group of (Ser 195) and nitrogen N epsilon (His 57) is more readily formed. These results show, however, that the H-bond does not adopt the most favorable conformation. The O gamma-H group of Ser 195 has a statistical preference for an attractive interaction with the O = C carbonyl (Ser 214) rather than the nitrogen N epsilon (His 57).

Two molecular dynamics simulations (100 and 50 ps) of native porcine pancreatic elastase i.e., without bound substrate and with the active site hydrated by a dome of water (630 molecules) have been performed. Dynamical properties of the catalytic tetrad have been examined. While relative conformations of the Asp 102, His 57, and Ser 214 are rather stable in time, the side chain of Ser 195 undergoes several conformational changes. No preferences are observed for the formation of a hydrogen bond between the O gamma-H group (Ser 195) and nitrogen N, (His 57). A cluster of ordered water molecules effectively competes with the H-O gamma group (Ser 195) and thereby prevents the formation of this H bond, which is generally agreed to be crucial for catalysis.

The variety of results of crystallographic studies of the serine proteases complexed with isocoumarin inhibitors presents a challenging problem to modeling methods and molecular energetics. Therefore, the thermodynamic cycle-perturbation technique has been used to study a model system of elastase and two peptidic inhibitors. Using the program AMBER, the technique correctly predicts changes of the binding constants for the trifluoroacetyl dipeptide inhibitors in comparison with available experimental (kinetic and crystallographic) data. However, the absolute values obtained are shown to be sensitive to the specific electrostatic interaction potential parameters used in the simulations. The reader and user are cautioned that thermodynamic cycle-perturbation results may be too optimistic by underestimating the accuracy of free energy values. This is especially a matter of concern for those cases where a direct comparison with experimental values is not possible, viz., (1) the stimulation of binding of novel compounds, (2) structurally uncertain binding sites, or (3) structurally different binding modes. With our best 4-31G* ESP (electrostatic potential) charges we were able to reproduce experimentally determined free energy differences (delta delta A) with an accuracy of about 1.5 kcal/mol. Dynamically induced structural changes in the binding site of elastase, and particularly changes in hydrogen-bond patterns of the binding site, are also reported.

Massive amounts of coordinate data result from molecular dynamics calculations. The animation program MDKINO is a simple but powerful tool for previewing or reviewing the results. In recent simulations of elastase, we have examined hydrogen bonding patterns, conformational changes involving shifts in ring positions and rotations of amino acid side chains, electric fields in interatomic space, and electric forces acting on chosen nuclei. Animation is also useful for checking on the stability of calculations in progress. Simple programming techniques achieve acceptable levels of animation with readily available hardware (PS330 or PS390 display with a serial interface to a laboratory VAX). In about half an hour, it is possible to make and watch a color stereo "movie" of a selected subsystem of a simulation (up to 1,000 frames of about 100 atoms each).

The crystal structures of porcine pancreatic elastase complexed to two similar benzoxazinone inhibitors are reported to 2.09 A and 1.76 A resolution, and refined to conventional R factors of 0.153 and 0.172.

Predictive methods have been extended to generate a proposed tertiary structure of rat elastase II on the basis of primary amino acid sequence and structural homologies within the family of mammalian serine proteinases. Force field refinement calculations were used to relax the structure. Structurally conserved molecules of solvation were introduced and the structure was again refined by means of force-field calculations. The resulting structure suggests probable substrate cleavage preferences. An independent statistical analysis of the crystallographically refined structures of serine proteinases (0.01-0.13 A, RMS) shows a close similarity to the final predicted model of rat pancreatic elastase II (0.03-0.14 A, RMS).

The Protein Data Bank is a computer-based archival file for macromolecular structures. The Bank stores in a uniform format atomic co-ordinates and partial bond connectivities, as derived from crystallographic studies. Text included in each data entry gives pertinent information for the structure at hand (e.g. species from which the molecule has been obtained, resolution of diffraction data, literature citations and specifications of secondary structure). In addition to atomic co-ordinates and connectivities, the Protein Data Bank stores structure factors and phases, although these latter data are not placed in any uniform format. Input of data to the Bank and general maintenance functions are carried out at Brookhaven National Laboratory. All data stored in the Bank are available on magnetic tape for public distribution, from Brookhaven (to laboratories in the Americas), Tokyo (Japan), and Cambridge (Europe and worldwide). A master file is maintained at Brookhaven and duplicate copies are stored in Cambridge and Tokyo. In the future, it is hoped to expand the scope of the Protein Data Bank to make available co-ordinates for standard structural types (e.g. alpha-helix, RNA double-stranded helix) and representative computer programs of utility in the study and interpretation of macromolecular structures.

X-ray crystallographic data to 2.57 A resolution (1 A = 0.1 nm) have been measured for the complex of a peptidyl trifluoromethylketone inhibitor with porcine pancreatic elastase (PPE); R = 0.14. The inhibitor forms a stable complex with the enzyme by means of a covalent attachment to active site Ser195O gamma, resulting in a hemiketal moiety with tetrahedral geometry. The tripeptide protion binds as an antiparallel beta-sheet, with four hydrogen bonds augmenting the active-site covalent linkage, Ki = 9.5 microM. His57 exhibits a bifurcated H-bond to both Ser195O gamma and an F atom of the inhibitor. This study is one of a series which explores the binding geometry of a variety of small substrates and inhibitors to PPE. This peptidyl-PPE complex affords insight into the binding geometry of a novel trifluoromethylketone moiety to a serine proteinase.

The hexapeptide substrate Thr-Pro-nVal-NMeLeu-Tyr-Thr reacts with porcine pancreatic elastase sufficiently slowly that accelerated crystallographic data collection procedures and two-dimensional transferred nuclear Overhauser enhancement measurements could be used to study the geometry of binding. Both studies report a time-averaged population of the Michaelis complex state, prior to proteolysis. This result provides an important data point along the reaction coordinate pathway for serine proteases. Crystallographic data to 1.80-A resolution were used in the structure analysis with refinement to an R-factor of 0.19.

We have analyzed the molecular interaction of 28 sulfonamide inhibitors with human carbonic anhydrase C (HCAC) using an interactive computer graphic display. Small aromatic sulfonamides gain most of their inhibitory power towards HCAC from the interaction of hydrogen bond acceptors at the para or meta positions with hydrophilic residues of the enzyme. Additional coordinated water molecules stabilize the complexes of heterocyclic sulfonamides (i.e., thiophenes, 1,3-thiazoles, and 1,3,4-thiadiazoles) with HCAC. We propose two different modes of binding of the heterocyclic ring with respect to the active site cleft: the heterocyclic sulfur atom of a 3,4-unsubstituted thiophene approaches the oxygen atom of a coordinated water molecule (sulfur "out"), whereas in 3,4-unsubstituted 1,3,4-thiadiazoles, the sulfur is in contact with a hydrophobic part of the receptor site (sulfur "in"). This proposal is consistent with crystallographic evidence. Sulfonamides with two aromatic or heterocyclic rings also interact with a hydrophobic pocket of the enzyme located greater than 10 A away from the active site metal Zn2+. We also discuss the possibility that the relative inactivity of thiazide diuretics is due to the steric interaction of the ortho chlorine atom with the enzyme receptor cavity.

Four isomeric forms of the Asn-102 PPE (D102N mutant according to the emerging protocol, [Knowles, Science, 236 (1987) 1252-1258]) have been investigated using energy minimization (EM) and molecular dynamics (MD) techniques. MD simulation data for 175 ps are reported for each form (in total 700 ps for about 2500 atoms). The His-57 N epsilon-protonated forms are calculated to be more stable than the N delta-protonated ones. The active site region of the most stable form is very similar to that found in the D102N rat trypsin enzyme [Craik et al., Science, 237 (1987) 909-913]. Conformations of the active sites and their hydrogen bond patterns are presented for each of these forms and are compared with the structure of the native enzyme active site. The pH dependent activity of the D102N derivative is discussed.

The crystal structure of porcine pancreatic elastase (PPE) complexed to carbobenzoxy-alanylisoleucine-boronic acid (ZAIB) is reported to 2.09-A resolution and refined to an R factor of 0.15. This is the first reported structural analysis of PPE with an isoleucine residu in the primary specificity pocket. The results include (1) marked displacement of the inhibitor out of the active site leading to (2) a close (2.2 A) direct contact between B (boron atom of the inhibitor) and N epsilon of His-57 and also (3) covalent bonding (1.5 A) to O gamma of Ser-195. A scheme for the mechanism of inhibition of PPE by ZAIB is proposed. A comparison with a peptidyl difluoromethyl ketone-PPE complex (Ki = 9.5 microns) is made to explain the strong inhibition of PPE by ZAIB (Ki = 0.3 micron). These results lead us to characterize this structure as a time- and space-averaged reaction intermediate, providing fresh insight into the cramped dimensions available in enzymatic catalyses.

A nuclear magnetic resonance study of the conformation of the tetrapeptide acetyl-Pro-Ala-Pro-Tyr-NH2 bound to porcine pancreatic elastase is presented. From two-dimensional transferred nuclear Overhauser enhancement measurements, a set of 23 approximate distance restraints between pairs of bound ligand protons, indicative of an extended type structure, is derived. The structure of the bound tetrapeptide is then refined from two different starting structures (an extended beta-strand and a polyproline helix) by restrained molecular dynamics, in which the interproton distances are incorporated into the total energy of the system in the form of effective potentials. Convergence to essentially the same average restrained dynamics structures is achieved. The refined structures are then modelled into the active site of elastase by interactive molecular graphics. The determination of the anchor point of the bound tetrapeptide on the enzyme was aided by a simultaneous crystallographic study which, despite the fact that only electron density for a Pro-X dipeptide fragment was visible, enabled both the approximate position and orientation of binding to be determined. It is found that the tetrapeptide is bound in the S' binding site in the reverse orientation found in other serine protease-inhibitor complexes and is stabilized both by hydrogen-bonding and by van der Waals' interactions.

A single crystal of porcine pancreatic elastase was mounted in a thin-walled capillary and allowed to react with acetyl-Ala-Pro-Ala-paranitroanalide. Diffraction data to 1.65 A resolution were measured and the isomorphous structure was solved from the difference Fourier map. The structure contains two surprises. Two molecules of the product: acetyl-Ala-Pro-Ala molecule are bound in the extended binding site. Both molecules are bound backwards with respect to the established mode of peptide binding.