Structural basis for the interaction between pectin methylesterase and a specific inhibitor protein

Pectin, one of the main components of the plant cell wall, is secreted in a highly methyl-esterified form and subsequently deesterified in muro by pectin methylesterases (PMEs). In many developmental processes, PMEs are regulated by either differential expression or posttranslational control by protein inhibitors (PMEIs). PMEIs are typically active against plant PMEs and ineffective against microbial enzymes. Here, we describe the three-dimensional structure of the complex between the most abundant PME isoform from tomato fruit (Lycopersicon esculentum) and PMEI from kiwi (Actinidia deliciosa) at 1.9-A resolution. The enzyme folds into a right-handed parallel beta-helical structure typical of pectic enzymes. The inhibitor is almost all helical, with four long alpha-helices aligned in an antiparallel manner in a classical up-and-down four-helical bundle. The two proteins form a stoichiometric 1:1 complex in which the inhibitor covers the shallow cleft of the enzyme where the putative active site is located. The four-helix bundle of the inhibitor packs roughly perpendicular to the main axis of the parallel beta-helix of PME, and three helices of the bundle interact with the enzyme. The interaction interface displays a polar character, typical of nonobligate complexes formed by soluble proteins. The structure of the complex gives an insight into the specificity of the inhibitor toward plant PMEs and the mechanism of regulation of these enzymes.     

Serine protease mechanism: structure of an inhibitory complex of alpha-lytic protease and a tightly bound peptide boronic acid

The structure of the complex formed between alpha-lytic protease, a serine protease secreted by Lysobacter enzymogenes, and N-tert-butyloxycarbonylalanylprolylvaline boronic acid (Ki = 0.35 nM) has been studied by X-ray crystallography to a resolution of 2.0 A. The active-site serine forms a covalent, nearly tetrahedral adduct with the boronic acid moiety of the inhibitor. The complex is stabilized by seven hydrogen bonds between the enzyme and inhibitor with additional stabilization arising from van der Waals interactions between enzyme and inhibitor side chains and the burying of 330 A2 of hydrophobic surface area. Hydrogen bonding between Asp-102 and His-57 remains intact in the enzyme-inhibitor complex, and His N epsilon 2 is well positioned to donate its hydrogen to the leaving group. Little change in the positions of protease residues was observed on complex formation (root mean square main chain deviation = 0.13 A), suggesting that in its native state the enzyme is complementary to tetrahedral reaction intermediates or to the nearly tetrahedral transition state for the reaction.     

An efficient synthesis of argifin: a natural product chitinase inhibitor with chemotherapeutic potential

The first synthesis of the cyclopentapeptide family 18 chitinase inhibitor argifin has been achieved by a combination of solid phase and solution chemistry. Synthetic argifin is a nanomolar inhibitor of chitinase B1 from Aspergillus fumigatus and the high-resolution X-ray structure of the synthesized material in complex with the same enzyme is identical to that previously obtained for the natural product.     

The refined 2.3 A crystal structure of human leukocyte elastase in a complex with a valine chloromethyl ketone inhibitor

The stoichiometric complex formed between human leukocyte elastase and a synthetic MeO-Suc-Ala-Ala-Pro-Val chloromethyl ketone inhibitor was co-crystallized and its X-ray structure determined, using Patterson search methods. Its structure has been crystallographically refined to a final R value of 0.145 (8.0 and 2.3 A). The enzyme structure is very similar to that recently observed in a complex formed with the ovomucoid third domain from turkey [(1986) EMBO J. 5,2453-2458]. The rms deviation of all alpha-carbon atoms is 0.32 A. The peptidic inhibitor is bound in a similar overall conformation as the ovomucoid binding segment. Covalent bonds are formed between Val-P1 of the inhibitor and His-57 NE2 and Ser-195 OG of the enzyme. The carbonyl carbon is tetrahedrally deformed to a hemiketal. The valine side chain is arranged in the S1 pocket in the g-conformation.     

Synthesis and characterization of a pancreatic trypsin inhibitor homologue and a model inhibitor.

The synthesis and characterization of protein proteinase inhibitor homologues with variations in the amino acid composition in the vicinity of the reactive site should aid the understanding of the mechanism by which inhibition of enzymatic activity occurs. A homologue inhibitor in which the reactive-site residue Ala-16 of basic pancreatic trypsin inhibitor (Kunitz) (BPTI) is replaced by Phe has been synthesized to study the effect of this replacement on the dissociation constants of the enzyme-inhibitor complexes. The replacement of Ala-16 by Phe causes a dramatic increase in the K1 value of the trypsin-BPTI complex while that of the chymotrypsin-BPTI complex remains essentially the same. This cannot be explained simply in terms of increased steric crowding. The Phe replacement probably causes a small change in the local conformation of the reactive site of the inhibitor which leads to a large decrease in the stability of the very tight trypsin-BPTI complex. This conformation change apparently can be tolerated in the less tightly bound chymotrypsin-BPTI complex. On the basis of the known structure of BPTI, a cyclic heptadecapeptide containing one disulfide bond was synthesized as a model inhibitor in order to determine if a smaller peptide can be designed to act as a highly efficient inhibitor for trypsin. This heptadecapeptide which contains all of the amino acid residues of BPTI taking part in the interaction of the proteinase inhibitor with trypsin binds 3 X 10(7) time more weakly to the enzyme than native BPTI does. It thus appears that even though only a small part of the inhibitor molecule enters directly into interaction with the enzyme, the remaining portions of the molecule which hold the structure of the inhibitor rigid are essential for the strong interaction.     

The crystal structure of human dipeptidyl peptidase I (cathepsin C) in complex with the inhibitor Gly-Phe-CHN2

hDDPI (human dipeptidyl peptidase I) is a lysosomal cysteine protease involved in zymogen activation of granule-associated proteases, including granzymes A and B from cytotoxic T-lymphocytes and natural killer cells, cathepsin G and neutrophil elastase, and mast cell tryptase and chymase. In the present paper, we provide the first crystal structure of an hDPPI-inhibitor complex. The inhibitor Gly-Phe-CHN2 (Gly-Phe-diazomethane) was co-crystallized with hDPPI and the structure was determined at 2.0 A (1 A=0.1 nm) resolution. The structure of the native enzyme was also determined to 2.05 A resolution to resolve apparent discrepancies between the complex structure and the previously published structure of the native enzyme. The new structure of the native enzyme is, within the experimental error, identical with the structure of the enzyme-inhibitor complex presented here. The inhibitor interacts with three subunits of hDPPI, and is covalently bound to Cys234 at the active site. The interaction between the totally conserved Asp1 of hDPPI and the ammonium group of the inhibitor forms an essential interaction that mimics enzyme-substrate interactions. The structure of the inhibitor complex provides an explanation of the substrate specificity of hDPPI, and gives a background for the design of new inhibitors.     

Two polymorphs of a covalent complex between papain and a diazomethylketone inhibitor.

The three-dimensional structure of two polymorphs of a ZLFG-CH2-papain covalent complex has been determined by X-ray crystallography. The structures indicate that: (i) the methylene carbon atom of the inhibitor is covalently bound to the Sgamma atom of Cys25 of papain; (ii) the hydrophobic S2 pocket formed by Pro68, Val133, Val157, and Asp158 is occupied by the inhibitor's phenylalanyl P2 side chain; (iii) extensive hydrogen bonding and hydrophobic interactions are responsible for the interaction of the inhibitor with the enzyme. Comparison with similar structures suggests that in covalent complexes preservation of main chain-main chain interactions between the enzyme and the inhibitor may have higher priority than the P-S interactions.     

Crystallographic analysis of a complex between human immunodeficiency virus type 1 protease and acetyl-pepstatin at 2.0-A resolution

The mode of binding of acetyl-pepstatin to the protease from the human immunodeficiency virus type 1 (HIV-1) has been determined by x-ray diffraction analysis. Crystals of an acetyl-pepstatin-HIV-1 protease complex were obtained in space group P2(1)2(1)2 (unit cell dimensions a = 58.39 A, b = 86.70 A, c = 46.27 A) by precipitation with sodium chloride. The structure was phased by molecular replacement methods, and a model for the structure was refined using diffraction data to 2.0 A resolution (R = 0.176 for 12901 reflections with I greater than sigma (I); deviation of bond distances from ideal values = 0.018 A; 172 solvent molecules included). The structure of the protein in the complex has been compared with the structure of the enzyme without the ligand. A core of 44 amino acids in each monomer, including residues in the active site and residues at the dimer interface, remains unchanged on binding of the inhibitor (root mean square deviation of alpha carbon positions = 0.39 A). The remaining 55 residues in each monomer undergo substantial rearrangement, with the most dramatic changes occurring at residues 44-57 (these residues comprise the so-called flaps of the enzyme). The flaps interact with one another and with the inhibitor so as to largely preserve the 2-fold symmetry of the protein. The inhibitor is bound in two approximately symmetric orientations. In both orientations the peptidyl backbone of the inhibitor is extended; a network of hydrogen bonds is formed between the inhibitor and the main body of the protein as well as between the inhibitor and the flaps. Hydrophobic side chains of residues in the body of the protein form partial binding sites for the side chains of the inhibitor; hydrophobic side chains of residues in the flaps complete these binding sites.     

Structure-based inhibitor design for an enzyme that binds different steroids: a potent inhibitor for human type 5 17beta-hydroxysteroid dehydrogenase

Human type 5 17beta-hydroxysteroid dehydrogenase plays a crucial role in local androgen formation in prostate tissue. Several chemicals were synthesized and tested for their ability to inhibit this enzyme, and a series of estradiol derivatives bearing a lactone on the D-ring were found to inhibit its activity efficiently. The crystal structure of the type 5 enzyme in complex with NADP and such a novel inhibitor, EM1404, was determined to a resolution of 1.30 A. Significantly more hydrogen bonding and hydrophobic interactions were defined between EM1404 and the enzyme than in the substrate ternary complex. The lactone ring of EM1404 accounts for important interactions with the enzyme, whereas the amide group at the opposite end of the inhibitor contributes to the stability of three protein loops involved in the construction of the substrate binding site. EM1404 has a strong competitive inhibition, with a Ki of 6.9+/-1.4 nM, demonstrating 40 times higher affinity than that of the best inhibitor previously reported. This is observed despite the fact that the inhibitor occupies only part of the binding cavity. Attempts to soak the inhibitor into crystals of the binary complex with NADP were unsuccessful, yielding a structure with a polyethylene glycol fragment occupying the substrate binding site. The relative crystal packing is discussed. Combined studies of small molecule inhibitor synthesis, x-ray crystallography, enzyme inhibition, and molecular modeling make it possible to analyze the plasticity of the substrate binding site of the enzyme, which is essential for developing more potent and specific inhibitors for hormone-dependent cancer therapy.     

Interaction of human leukocyte elastase with a N-aryl azetidinone suicide substrate: Conformational analyses based on the mechanism of action of serine proteinases

The three-dimensional interaction of the enzyme-activated (suicide) inhibitor AA 231-1 [N(2-chloromethyl)-3,3-difluoro-azetidin-2-one] with human leukocyte elastase has been studied using computer graphics and molecular mechanics. Systematic conformational analyses and energy minimizations have been performed for the inhibitor AA 231-1 and its presumed complexes formed during the enzymatic process of inactivation, i.e., the Michaelis complex, the acyl-enzyme, and the inactivated enzyme with the covalently bound inhibitor. The β-lactam ring characteristics of modeled AA 231-1 were in agreement with crystallo-graphic data of related structures. Lowest energy conformatinos were found when the angle between the planes of the β-lactam ring and that of its phenyl substituent was about −60 or 60°. To study the interaction with the enzyne, the enzyme-inhibitor complexes were constructed by docking the inhibitor in the active site using enzyme coordinates from an X-ray crystallographic structure. The whole enzyme structure was used for conformational analyses and energy mechanics. Favorable conformations for the Michaelis complex have been obtained in which the carbonyl oxygen of the inhibitor was located in the oxyanion hole and the hydroxyl of Ser195 was in position to interact with the β-lactam carbonyl carbon on the α face of AA 231-1. Simulations of the approach of the benzylic carbon by the nucleophilic amino acid His40 or His57 through an S_N2 displacement on the halomethyl group of AA 231-1 were performed. The results agreed with the alkylation of the imidazole nitroge Nϵ2 of His57 leading to the inactivated enzyme (bis-adduct form).     

Crystallographic evidence of a transglycosylation reaction: ternary complexes of a psychrophilic alpha-amylase

The psychrophilic Pseudoalteromonas haloplanctis alpha-amylase is shown to form ternary complexes with two alpha-amylase inhibitors present in the active site region, namely, a molecule of Tris and a trisaccharide inhibitor or heptasaccharide inhibitor, respectively. The crystal structures of these complexes have been determined by X-ray crystallography to 1.80 and 1.74 A resolution, respectively. In both cases, the prebound inhibitor Tris is expelled from the active site by the incoming oligosaccharide inhibitor substrate analogue, but stays linked to it, forming well-defined ternary complexes with the enzyme. These results illustrate competition in the crystalline state between two inhibitors, an oligosaccharide substrate analogue and a Tris molecule, bound at the same time in the active site region. Taken together, these structures show that the enzyme performs transglycosylation in the complex with the pseudotetrasaccharide acarbose (confirmed by a mutant structure), leading to a well-defined heptasaccharide, considered as a more potent inhibitor. Furthermore, the substrate-induced ordering of water molecules within a channel highlights a possible pathway used for hydrolysis of starch and related poly- and oligosaccharides.     

Crystal structure of GDP-mannose dehydrogenase: a key enzyme of alginate biosynthesis in P. aeruginosa

The enzyme GMD from Pseudomonas aeruginosa catalyzes the committed step in the synthesis of the exopolysaccharide alginate. Alginate is a major component of P. aeruginosa biofilms that protect the bacteria from the host immune response and antibiotic therapy. The 1.55 A crystal structure of GMD in ternary complex with its cofactor NAD(H) and product GDP-mannuronic acid reveals that the enzyme forms a domain-swapped dimer with two polypeptide chains contributing to each active site. The extensive dimer interface provides multiple opportunities for intersubunit communication. Comparison of the GMD structure with that of UDP-glucose dehydrogenase reveals the structural basis of sugar binding specificity that distinguishes these two related enzyme families. The high-resolution structure of GMD provides detailed information on the active site of the enzyme and a template for structure-based inhibitor design.     

Structure of isocitrate lyase, a persistence factor of Mycobacterium tuberculosis

Isocitrate lyase (ICL) plays a pivotal role in the persistence of Mycobacterium tuberculosis in mice by sustaining intracellular infection in inflammatory macrophages. The enzyme allows net carbon gain by diverting acetyl-CoA from beta-oxidation of fatty acids into the glyoxylate shunt pathway. Given its potential as a drug target against persistent infections, we solved its structure without ligand and in complex with two inhibitors. Covalent modification of an active site residue, Cys 191, by the inhibitor 3-bromopyruvate traps the enzyme in a catalytic conformation with the active site completely inaccessible to solvent. The structure of a C191S mutant of the enzyme with the inhibitor 3-nitropropionate provides further insight into the reaction mechanism.     

Structural study of porcine pancreatic elastase complexed with 7-amino-3-(2-bromoethoxy)-4-chloroisocoumarin as a nonreactivatable doubly covalent enzyme-inhibitor complex

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)     

Structural evidence for a ligand coordination switch in liver alcohol dehydrogenase

The use of substrate analogues as inhibitors provides a way to understand and manipulate enzyme function. Here we report two 1 A resolution crystal structures of liver alcohol dehydrogenase in complex with NADH and two inhibitors: dimethyl sulfoxide and isobutyramide. Both structures present a dynamic state of inhibition. In the dimethyl sulfoxide complex structure, the inhibitor is caught in transition on its way to the active site using a flash-freezing protocol and a cadmium-substituted enzyme. One inhibitor molecule is partly located in the first and partly in the second coordination sphere of the active site metal. A hydroxide ion bound to the active site metal lies close to the pyridine ring of NADH, which is puckered in a twisted boat conformation. The cadmium ion is coordinated by both the hydroxide ion and the inhibitor molecule, providing structural evidence of a coordination switch at the active site metal ion. The structure of the isobutyramide complex reveals the partial formation of an adduct between the isobutyramide inhibitor and NADH. It provides evidence of the contribution of a shift from the keto to the enol tautomer during aldehyde reduction. The different positions of the inhibitors further refine the knowledge of the dynamics of the enzyme mechanism and explain how the crowded active site can facilitate the presence of a substrate and a metal-bound hydroxide ion.     

Recognition between a bacterial ribonuclease, barnase, and its natural inhibitor, barstar

BACKGROUND: Protein-protein recognition is fundamental to most biological processes. The information we have so far on the interfaces between proteins comes largely from several protease-inhibitor and antigen-antibody complexes. Barnase, a bacterial ribonuclease, and barstar, its natural inhibitor, form a tight complex which provides a good model for the study and design of protein-protein non-covalent interactions. RESULTS: Here we report the structure of a complex between barnase and a fully functional mutant of barstar determined by X-ray analysis. Barstar is composed of three parallel alpha-helices stacked against a three-stranded parallel, beta-sheet, and sterically blocks the active site of the enzyme with an alpha-helix and adjacent loop. The buried surface in the interface between the two molecules totals 1630 A2. The barnase-barstar complex is predominantly stabilized by charge interactions involving positive charges in the active site of the enzyme. Asp39 of barstar binds to the phosphate-binding site of barnase, mimicking enzyme-substrate interactions. CONCLUSION: The phosphate-binding site of the enzyme is the anchor point for inhibitor binding. We propose that this is also likely to be the case for other ribonuclease inhibitors.     

X-ray analysis of a complex of Escherichia coli uracil DNA glycosylase (EcUDG) with a proteinaceous inhibitor. The structure elucidation of a prokaryotic UDG.

Uracil-DNA glycosylase (UDG), a key highly conserved DNA repair enzyme involved in uracil excision repair, was discovered in Escherichia coli . The Bacillus subtilis bacteriophage, PBS-1 and PBS-2, which contain dUMP residues in their DNA, express a UDG inhibitor protein, Ugi which binds to UDG very tightly to form a physiologically irreversible complex. The X-ray analysis of the E. coli UDG ( Ec UDG)-Ugi complex at 3.2 A resolution, leads to the first structure elucidation of a bacterial UDG molecule. This structure is similar to the enzymes from human and viral sources. A comparison of the available structures involving UDG permits the delineation of the constant and the variable regions of the molecule. Structural comparison and mutational analysis also indicate that the mode of action of the enzyme from these sources are the same. The crystal structure shows a remarkable spatial conservation of the active site residues involved in DNA binding in spite of significant differences in the structure of the enzyme-inhibitor complex, in comparison with those from the mammalian and viral sources. Ec UDG could serve as a prototype for UDGs from pathogenic prokaryotes, and provide a framework for possible drug development against such pathogens with emphasis on features of the molecule that differ from those in the human enzyme.     

Structure of a type II thymidine kinase with bound dTTP

The structure of human cytosolic thymidine kinase in complex with its feedback inhibitor 2'-deoxythymidine-5'-triphosphate was determined. This structure is the first representative of the type II thymidine kinases found in several pathogens. The structure deviates strongly from the known structures of type I thymidine kinases such as the Herpes simplex enzyme. It contains a zinc-binding domain with four cysteines complexing a structural zinc ion. Interestingly, the backbone atoms of the type II enzyme bind thymine via hydrogen-bonds, in contrast to type I, where side chains are involved. This results in a specificity difference exploited for antiviral therapy. The presented structure will foster the development of new drugs and prodrugs for numerous therapeutic applications.     

The 2.0 A structure of malarial purine phosphoribosyltransferase in complex with a transition-state analogue inhibitor.

Malaria is a leading cause of worldwide mortality from infectious disease. Plasmodium falciparum proliferation in human erythrocytes requires purine salvage by hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase). The enzyme is a target for the development of novel antimalarials. Design and synthesis of transition-state analogue inhibitors permitted cocrystallization with the malarial enzyme and refinement of the complex to 2.0 A resolution. Catalytic site contacts in the malarial enzyme are similar to those of human hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) despite distinct substrate specificity. The crystal structure of malarial HGXPRTase with bound inhibitor, pyrophosphate, and two Mg(2+) ions reveals features unique to the transition-state analogue complex. Substrate-assisted catalysis occurs by ribooxocarbenium stabilization from the O5' lone pair and a pyrophosphate oxygen. A dissociative reaction coordinate path is implicated in which the primary reaction coordinate motion is the ribosyl C1' in motion between relatively immobile purine base and (Mg)(2)-pyrophosphate. Several short hydrogen bonds form in the complex of the enzyme and inhibitor. The proton NMR spectrum of the transition-state analogue complex of malarial HGXPRTase contains two downfield signals at 14.3 and 15.3 ppm. Despite the structural similarity to the human enzyme, the NMR spectra of the complexes reveal differences in hydrogen bonding between the transition-state analogue complexes of the human and malarial HG(X)PRTases. The X-ray crystal structures and NMR spectra reveal chemical and structural features that suggest a strategy for the design of malaria-specific transition-state inhibitors.     

Binding properties of an intrinsic ATPase inhibitor and occurrence in yeast mitochondria of a protein factor which stabilizes and facilitates the binding of the inhibitor to F1F0-ATPase

The content of an intrinsic ATPase inhibitor in mitochondria was determined by a radioimmunoassay procedure which showed the molar ratio of the inhibitor to ATPase to be 1:1. The ratio in submitochondrial particles, where half of the enzyme was activated, was the same as that of mitochondria, indicating that the inhibitor protein has affinity for the mitochondrial membrane as well as for F1-ATPase. The inhibitor protein could be removed from the mitochondrial membrane by incubation with 0.5 M Na2SO4 and concomitantly the enzyme was fully activated. The enzyme fully activated by the salt treatment was inactivated again by the externally added ATPase inhibitor in the presence of ATP and Mg2+. The enzyme-inhibitor complex (inactive) on the mitochondrial membrane was more stable than the solubilized enzyme-inhibitor complex but gradually dissociated in the absence of ATP and Mg2+. However, in mitochondria, the enzyme activity was inhibited even in the absence of the cofactors. A protein factor stabilizing the enzyme-inhibitor complex on the mitochondrial membrane was isolated from yeast mitochondria. This factor stabilized the inhibitor complex of membrane-bound ATPase while having no effect on that of purified F1-ATPase. It also efficiently facilitated the binding of the inhibitor to membrane-bound ATPase to form the complex, which reversibly dissociated at slightly alkaline pH.