Biomedical subjects

C S Craik

Publications and source records attributed to C S Craik.

At least 55 records · Page 3Linked to original sources

PubMed1996

Three-dimensional structures of HIV-1 and SIV protease product complexes.

Strain is eliminated as a factor in hydrolysis of the scissile peptide bond by human immunodeficiency virus (HIV)-1 and simian immunodeficiency virus (SIV), based on the first eight complexes of products of hydrolysis with the enzymes. The carboxyl group generated at the scissile bond interacts with both catalytic aspartic acids. The structures directly suggest the interactions of the gemdiol intermediate with the active site. Based on the structures, the nucleophilic water is displaced stereospecifically by substrate binding toward one catalytic aspartic acid, while the scissile carbonyl becomes hydrogen bonded to the other catalytic aspartic acid in position for hydrolysis. Crystal structures for two N-terminal (P) products and two C-terminal (Q) products provide unambiguous density for the ligands at 2.2-2.6 A resolution and 17-21% R factors. The N-terminal product, Ac-S-L-N-F/, overlaps closely with the N-terminal sequences of peptidomimetic inhibitors bound to the protease. Comparison of the two C-terminal products, /F-L-E-K and /F(NO2)-E-A-Nle-S, indicates that the P2' residue is highly constrained, while the positioning of the P1' and P3' residues are sequence dependent.

Aspartic Acid↗

PubMed1996

Delocalizing trypsin specificity with metal activation.

Recognition for proteolysis by trypsin depends almost exclusively on tight binding of arginine or lysine side chains by the primary substrate specificity pocket. Although extended subsite interactions are important for catalysis, the majority of binding energy is localized in the P1 pocket. Analysis of the interactions of trypsin with the P1 residue of the bound inhibitors ecotin and bovine pancreatic trypsin inhibitor suggested that the mutation D189S would improve metal-assisted trypsin N143H, E151H specificity toward peptides that have a Tyr at P1 and a His at P2'. In the presence of transition metals, the catalytic efficiency of the triple mutant Tn N143H, E151H, D189S improved toward the tyrosine-containing peptide AGPYAHSS. Trypsin N143H, E151H, D189S exhibits a 25-fold increase in activity with nickel and a 150-fold increase in activity with zinc relative to trypsin N143H, E151H on this peptide. In addition, activity of trypsin N143H, E151H, D189S toward an arginine-containing peptide, YLVGPRGHFYDA, is enhanced by copper, nickel, and zinc. With this substrate, copper yields a 30-fold, nickel a 70-fold, and zinc a 350-fold increase in activity over background hydrolysis without metal. These results demonstrate that the engineering of multiple substrate binding subsites in trypsin can be used to delocalize protease specificity by increasing relative substrate binding contributions from alternate engineered subsites.

Amino Acid Sequence↗

PubMed1996

X-ray structures of a designed binding site in trypsin show metal-dependent geometry.

The three-dimensional structures of complexes of trypsin N143H, E151H bound to ecotin A86H are determined at 2.0 A resolution via X-ray crystallography in the absence and presence of the transition metals Zn2+, Ni2+, and Cu2+. The binding site for these transition metals was constructed by substitution of key amino acids with histidine at the trypsin-ecotin interface in the S2'/P2' pocket. Three histidine side chains, two on trypsin at positions 143 and 151 and one on ecotin at position 86, anchor the metals and provide extended catalytic recognition for substrates with His in the P2' pocket. Comparisons of the three-dimensional structures show the different geometries that result upon the binding of metal in the engineered tridentate site and suggest a structural basis for the kinetics of the metal-regulated catalysis. Of the three metals, the binding of zinc results in the most favorable binding geometry, not dissimilar to those observed in naturally occurring zinc binding proteins.

Bacterial Proteins↗

PubMed1996

Substrate recognition by recombinant serine collagenase 1 from Uca pugilator.

Uca pugilator serine collagenase 1 was cloned and sequenced from a fiddler crab hepatopancreas cDNA library. A full-length sequence encodes a 270-amino acid pre-pro-enzyme highly identical in structure to the chymotrypsin family of serine proteases. The zymogen form of the enzyme was expressed in Saccharomyces cerevisiae as a fusion with the alpha-factor signal sequence under control of the alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase promoter. Upon activation with trypsin, the recombinant collagenase possesses collagenolytic properties identical to those of the enzyme isolated from the crab hepatopancreas. The collagenase substrate binding pocket recognizes a wide range of basic, hydrophobic, and neutral polar residues. beta-Branched and acidic amino acids are poor substrates. Acylation is rate-limiting for collagenase versus peptidyl amides, rather than deacylation, as for trypsin and chymotrypsin. Correlations relating substrate volume and hydrophobicity to catalysis were found for collagenase and compared to those for chymotrypsin and elastase. Relative enzyme efficiencies on single amino acid versus tetrapeptide amide substrates show that collagenase derives less catalytic efficiency from binding of the primary substrate residue than trypsin or chymotrypsin, but compensates in binding of the extended peptidyl residues. Serine collagenase 1 is a novel member of the chymotrypsin protease family, by virtue of its amino acid sequence and multifunctional active site.

Amino Acid Sequence↗

PubMed1996

Isolation and characterization of two cDNAs from Atlantic cod encoding two distinct psychrophilic elastases.

The cDNAs encoding two different Atlantic cod elastases have been isolated and sequenced. The predicted amino acid sequences revealed two preproelastases, consisting of a signal peptide, an activation peptide and a mature enzyme of 242 and 239 amino acids. Amino acid sequence identity between the two cod elastases was 60.1% and identity with mammalian elastases ranged from 50-64%. The two cod elastases contain all the major structural features common to serine proteases, such as the catalytic triad His57, Asp102 and Ser195. Both cod elastases have a high content of methionine, consistent with previous findings in psychrophilic fish enzymes.

Adaptation, Physiological↗

PubMed1996

Structure-based design of parasitic protease inhibitors.

To streamline the preclinical phase of pharmaceutical development, we have explored the utility of structural data on the molecular target and synergy between computational and medicinal chemistry. We have concentrated on parasitic infectious diseases with a particular emphasis on the development of specific noncovalent inhibitors of proteases that play a key role in the parasites' life cycles. Frequently, the structure of the enzyme target of pharmaceutical interest is not available. In this setting we have modeled the structure of the relevant enzyme by virtue of its sequence similarity with proteins of known structure. For example, we have constructed a homology-based model of falcipain, the trophozoite cysteine protease, and used the computational ligand identification algorithm DOCK to identify in compuo enzyme inhibitors including oxalic bis(2-hydroxy-1-naphthyl-methylene)hydrazide (1) [Ring, C. S.; Sun, E.; McKerow, J. H.; Lee, G.; Rosenthal, P. J., Kuntz, I. D.; Cohen, F. E., Proc. Natl Acad. Sci. U.S.A. 1993, 90, 3583]. Compound 1 inhibits falcipain (IC50 6 microM) and the organism in vitro as judged by hypoxanthine uptake (IC50 7 microM). Following this lead, to date, we have identified potent bis arylacylhydrazides (IC50 150 nM) and chalcones (IC50 200 nM) that are active against both chloroquine-sensitive and chloroquine-resistant strains of malaria. In a second example, cruzain, the crystallographically determined structure of a papain-like cysteine protease, resolved to 2.35 A, was available. Aided by DOCK, we have identified a family of bis-arylacylhydrazides that are potent inhibitors of cruzain (IC50 600 microM). These compounds represent useful leads for pharmaceutical development over strict enzyme inhibition criteria in a structure-based design program.

Aldehydes↗

PubMed1996

A new class of HIV-1 protease inhibitor: the crystallographic structure, inhibition and chemical synthesis of an aminimide peptide isostere.

The essential role of HIV-1 protease (HIV-1 PR) in the viral life cycle makes it an attractive target for the development of substrate-based inhibitors that may find efficacy as anti-AIDS drugs. However, resistance has arisen to potent peptidomimetic drugs necessitating the further development of novel chemical backbones for diversity based chemistry focused on probing the active site for inhibitor interactions and binding modes that evade protease resistance. AQ148 is a potent inhibitor of HIV-1 PR and represents a new class of transition state analogues incorporating an aminimide peptide isostere. A 3-D crystallographic structure of AQ148, a tetrapeptide isostere, has been determined in complex with its target HIV-1 PR to a resolution of 2.5 A and used to evaluate the specific structural determinants of AQ148 potency and to correlate structure-activity relationships within the class of related compounds. AQ148 is a competitive inhibitor of HIV-1 PR with a Ki value of 137 nM. Twenty-nine derivatives have been synthesized and chemical modifications have been made at the P1, P2, P1', and P2' sites. The atomic resolution structure of AQ148 bound to HIV-1 PR reveals both an inhibitor binding mode that closely resembles that of other peptidomimetic inhibitors and specific protein/inhibitor interactions that correlate with structure-activity relationships. The structure provides the basis for the design, synthesis and evaluation of the next generation of hydroxyethyl aminimide inhibitors. The aminimide peptide isostere is a scaffold with favorable biological properties well suited to both the combinatorial methods of peptidomimesis and the rational design of potent and specific substrate-based analogues.

Carbamates↗

PubMed1996

Phage display of proteases and macromolecular inhibitors.

We describe methods for displaying the protease trypsin and the macromolecular protease inhibitor ecotin on the surface of filamentous phage. Our strategy for selecting variant ecotins against target proteases is also described. We believe that the two proteins that have been displayed serve as ideal models for studying molecular recognition in detail. The ability to search efficiently through a large number of variant proteins for desired properties using phage display technology and the in vitro selection methods described opens a new avenue for studying protein-ligand interactions, as well as creating proteins with novel functions.

Bacterial Proteins↗

PubMed1996

Intracellular expression of human immunodeficiency virus type 1 (HIV-1) protease variants inhibits replication of wild-type and protease inhibitor-resistant HIV-1 strains in human T-cell lines.

The enzymatic activity of the human immunodeficiency type 1 (HIV-1) protease (PR) is crucial to render HIV-1 virions mature and infectious. Hence, genetic intervention strategies based on trans-dominant (td) variants of the HIV-1 PR might be an alternative to current pharmacological and gene therapy regimens for AIDS. CD4-positive human CEM-SS T-cell lines were generated which constitutively expressed HIV-1 td PR variants. HIV-1 infection experiments demonstrated severely reduced HIV-1 replication in these td PR CEM-SS cell lines compared with control T cells expressing wild-type PR. Furthermore, replication of an HIV-1 isolate bearing a PR inhibitor-resistant PR was blocked, showing that genetic intervention strategies based on td PRs can be effective against HIV-1 isolates containing PR inhibitor-resistant mutants.

CD4-Positive T-Lymphocytes↗

PubMed1995

Trans-dominant inhibitory human immunodeficiency virus type 1 protease monomers prevent protease activation and virion maturation.

Production of infectious human immunodeficiency virus (HIV) requires proper polyprotein processing by the dimeric viral protease. The trans-dominant inhibitory activity of a defective protease monomer with the active site Asp-25 changed to Asn was measured by transient transfection. A proviral plasmid that included the drug-selectable Escherichia coli gpt gene was used to deliver the wild-type (wt) or mutant proteases to cultured cells. Coexpression of the wt proviral DNA (HIV-gpt) with increasing amounts of the mutant proviral DNA (HIV-gpt D25N) results in a concomitant decrease in proteolytic activity monitored by in vivo viral polyprotein processing. The viral particles resulting from inactivation of the protease were mostly immature, consisting predominantly of unprocessed p55gag and p160gag-pol polyproteins. In the presence of HIV-1 gp160 env, the number of secreted noninfectious particles correlated with the presence of increasing amounts of the defective protease. Greater than 97% reduction in infectivity was observed at a 1:6 ratio of wt to defective protease DNA. This provides an estimate of the level of inhibition required for effectively preventing virion processing. Stable expression of the defective protease in monkey cells reduced the yield of infectious particles from these cells by 90% upon transfection with the wt proviral DNA. These results show that defective subunits of the viral protease exert a trans-dominant inhibitory effect resulting from the formation of catalytically compromised heterodimers in vivo, ultimately yielding noninfectious viral particles.

Animals↗

PubMed1995

Regulation of clathrin assembly and trimerization defined using recombinant triskelion hubs.

Clathrin polymerization into a polyhedral vesicle coat drives receptor sorting at cellular membranes during endocytosis and organelle biogenesis. To study clathrin self-assembly, we expressed the C-terminal third of the clathrin heavy chain in bacteria. The recombinant fragment trimerized, bound clathrin light chains, and morphologically resembled the hub domain of the triskelion-shaped clathrin molecule. Self-assembly of recombinant hubs demonstrated a regulatory role for clathrin light chains and for the distal portions of triskelion legs in clathrin coat formation. Deletion mutagenesis of the hub localized a domain mediating light chain binding and clathrin self-assembly and mapped a transferable trimerization domain. These studies define molecular interactions controlling clathrin self-assembly and establish a recombinant system for future analysis.

Animals↗

PubMed1995

Trypsin specificity increased through substrate-assisted catalysis.

Histidine 57 of the catalytic triad of trypsin was replaced with alanine to determine whether the resulting variant would be capable of substrate-assisted catalysis [Carter, P., & Wells, J. A. (1987) Science 237, 394-9]. A 2.5-fold increase in kcat/Km was observed on tri- or tetrapeptide substrates containing p-nitroanilide leaving groups and histidine at P2. In contrast, hydrolysis of peptide substrates extending from P6 to P6' is improved 70-300-fold by histidine in the P2 or P1' position. This preference creates new protease specificities for sequences HR decreases, R decreases H, HK decreases, and K decreases H. The ability of histidine from either the P2 or the P1' position of substrate to participate in catalysis emphasizes the considerable variability of proteolytically active orientations which can be assumed by the catalytic triad. Trypsin H57A is able to hydrolyze fully folded ornithine decarboxylase with complete specificity at a site containing the sequence HRH. Trypsin H57A was compared to enteropeptidase in its ability to cleave a propeptide from trypsinogen. Trypsin H57A cleaved the propeptide of a variant trypsinogen containing an introduced FPVDDDHR cleavage site only 100-fold slower than enteropeptidase cleaved trypsinogen. The selective cleavage of folded proteins suggests that trypsin H57A can be used for specific peptide and protein cleavage. The extension of substrate-assisted catalysis to the chymotrypsin family of proteolytic enzymes indicates that it may be possible to apply this strategy to a wide range of serine proteases and thereby develop various unique specificities for peptide and protein hydrolysis.

Amino Acid Sequence↗

PubMed1995

Isolation of a high affinity inhibitor of urokinase-type plasminogen activator by phage display of ecotin.

Ecotin, a serine protease inhibitor found in the periplasm of Escherichia coli, is unique in its ability and mechanism of inhibiting serine proteases of a broad range of substrate specificity. However, although the catalytic domain of human urokinase-type plasminogen activator (uPA) has 40% identity to bovine trypsin and the substrate specificities of these two proteases are virtually identical, ecotin inhibits uPA almost 10,000-fold less efficiently than trypsin. Ecotin was expressed on the surface of filamentous bacteriophage (ecotin phage) to allow the isolation of more potent inhibitors of uPA from a library of ecotin variants. The 142-amino acid inhibitor was fused to the C-terminal domain of the M13 minor coat protein, pIII, through a Gly-Gly-Gly linker and assembled into phage particles. The ecotin phage were shown to react with anti-ecotin antibodies, revealing a stoichiometry of approximately one ecotin per bacteriophage. The ecotin displayed on the surface of phage inhibited trypsin with an equilibrium dissociation constant of 6.7 nM, in close approximation to that of free ecotin, indicating that phage-associated ecotin is correctly folded and functionally active. Reactive-site amino acids 84 and 85 of ecotin were then randomized and a library of 400 unique ecotin phage was created. Three hundred thousand members of the library were screened with immobilized uPA and subjected to three rounds of binding and in vitro selection. DNA sequence analysis of the selected ecotin phage showed that ecotin M84R/M85R predominated while ecotin M84R, M84K, and M84R/M85K were present at a lower frequency. The four ecotin variants were overexpressed and purified and their affinities toward uPA were determined. Each of the selected ecotin variants exhibited increased affinity for uPA when compared to wild-type ecotin with ecotin M84R/M85R showing a 2800-fold increase in binding affinity.

Amino Acid Sequence↗

PubMed1995

The crystal structure of cruzain: a therapeutic target for Chagas' disease.

Trypanosoma cruzi, a protozoan parasite, is the etiologic agent of American trypanosomiasis or Chagas' disease. Chagas' disease afflicts more than 24 million individuals in South and Central America producing a debilitating life-long disease. It is the leading cause of heart failure in many Latin American countries. Currently, there is no satisfactory treatment for this parasitic infection. Cruzain (also known as cruzipain, gp 57/51), the major cysteine protease present in T. cruzi, is critical for the development and survival of the parasite within the host cells, making this enzyme a target for potential trypanocidal drugs. Here we report the X-ray crystal structure of cruzain complexed with the potent inhibitor Z-Phe-Ala-fluoromethyl ketone. The structure was determined at 2.35 A (Rcryst = 0.15) by molecular replacement using a modified papain as the search model. The refined structure is compared to papain. Features which distinguish cruzain from papain are discussed since they may aid in the design of specificity inhibitors. Fluorescence microscopy shows that a biotinylated form of the bound inhibitor does not effectively reach host proteases in their lysosomal compartment, but is selectively taken up by the parasite. The inhibitor greatly reduces parasitemia in a cell culture system, without adverse effects to mammalian cells. This biological selectivity can be exploited, in conjunction with unique active site features revealed by the crystal structure, to develop chemotherapy for Chagas' disease.

Animals↗

PubMed1995

Engineered metal regulation of trypsin specificity.

Histidine substrate specificity has been engineered into trypsin by creating metal binding sites for Ni2+ and Zn2+ ions. The sites bridge the substrate and enzyme on the leaving-group side of the scissile bond. Application of simple steric and geometric criteria to a crystallographically derived enzyme-substrate model suggested that histidine specificity at the P2' position might be achieved by a tridentate site involving amino acid residues 143 and 151 of trypsin. Trypsin N143H/E151H hydrolyzes a P2'-His-containing peptide (AGPYAHSS) exclusively in the presence of nickel or zinc with a high level of catalytic efficiency. Since cleavage following the tyrosine residue is normally highly disfavored by trypsin, this result demonstrates that a metal cofactor can be used to modulate specificity in a designed fashion. The same geometric criteria applied in the primary S1 binding pocket suggested that the single-site mutation D189H might effect metal-dependent His specificity in trypsin. However, kinetic and crystallographic analysis of this variant showed that the design was unsuccessful because His189 rotates away from substrate causing a large perturbation in adjacent surface loops. This observation suggests that the reason specificity modification at the trypsin S1 site requires extensive mutagenesis is because the pocket cannot deform locally to accommodate alternate P1 side chains. By taking advantage of the extended subsites, an alternate substrate specificity has been engineered into trypsin.

Amino Acid Sequence↗

PubMed1995

Structural basis of substrate specificity in the serine proteases.

Structure-based mutational analysis of serine protease specificity has produced a large database of information useful in addressing biological function and in establishing a basis for targeted design efforts. Critical issues examined include the function of water molecules in providing strength and specificity of binding, the extent to which binding subsites are interdependent, and the roles of polypeptide chain flexibility and distal structural elements in contributing to specificity profiles. The studies also provide a foundation for exploring why specificity modification can be either straightforward or complex, depending on the particular system.

Amino Acid Sequence↗

PubMed1995

Conversion of human 15-lipoxygenase to an efficient 12-lipoxygenase: the side-chain geometry of amino acids 417 and 418 determine positional specificity.

Positional specificity determinants of human 15-lipoxygenase were examined by site-directed mutagenesis and by kinetic analysis of the wild-type and variant enzymes. By comparing conserved differences among sequences of 12- and 15-lipoxygenases, a small region responsible for functional differences between 12- and 15-lipoxygenases has been identified. Furthermore, the replacement of only two amino acids in 15-lipoxygenase (at 417 and 418 in the primary sequence) by those found in certain 12-lipoxygenases results in an enzyme that has activity similar to 12-lipoxygenase. An examination of the activity of nine variants of lipoxygenase demonstrated that the amino acid side-chain bulk and geometry of residues 417 and 418 are the key components of the positional specificity determinant of 15-lipoxygenase. Overexpression of a variant (containing valines at positions 417 and 418) that performs predominantly 12-lipoxygenation was achieved in a baculo-virus-insect cell culture system. This variant was purified to > 90% homogeneity and its kinetics were compared with the wild-type 15-lipoxygenase. The variant enzyme has no change in its apparent KM for arachidonic acid and a minor (3-fold) change in its Vmax. For linoleic acid, the variant has no change in its KM and a 10-fold reduction in its Vmax, as expected for an enzyme performing predominantly 12-lipoxygenation. The results are consistent with a model in which two amino acids of 15-lipoxygenase (isoleucine 417 and methionine 418) constitute a structural element which contributes to the regiospecificity of the enzyme. Replacement of these amino acids with those found in certain 12-lipoxygenases results in an enzyme which can bind arachidonic acid in a catalytic register that prefers 12-lipoxygenation.

Arachidonate 12-Lipoxygenase↗

PubMed1995

Defining the level of human immunodeficiency virus type 1 (HIV-1) protease activity required for HIV-1 particle maturation and infectivity.

The human immunodeficiency virus type 1 (HIV-1) protease is the enzyme required for processing of the Gag and Gag-Pol polyproteins to yield mature, infectious virions. Although the complete absence of proteolytic activity prevents maturation, the level of activity sufficient for maturation and subsequent infectivity has not been determined. Amino acid substitutions that reduce catalytic activity without affecting substrate recognition have been engineered into the active site of the HIV-1 protease. The catalytic efficiency (kcat) of the HIV-1 protease is decreased 4-fold when threonine 26 is replaced by serine (T26S) and approximately 50-fold when alanine 28 is replaced by serine (A28S). Genes containing these mutations were cloned into a proviral vector for analysis of their effects on virion maturation and infectivity. The results show that virions containing the T26S protease variant, in which only 25% of the protease is active, are very similar to wild-type virions, although slight reductions in infectivity are observed. Virions containing the A28S protease variant are not infectious, even though a limited amount of polyprotein processing does occur. There appears to be a linear correlation between the level of protease activity and particle infectivity. Our observations suggest that a threshold of protease activity exists between a 4-fold and 50-fold reduction, below which processing is insufficient to yield infectious particles. Our data also suggest that a reduction of protease activity by 50-fold or greater is sufficient to prevent the formation of infectious particles.

Animals↗