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Biomedical subjects

C S Craik

Publications and source records attributed to C S Craik.

At least 19 recordsLinked to original sources
PubMed2000

Yeast two-hybrid assay for examining human immunodeficiency virus protease heterodimer formation with dominant-negative inhibitors and multidrug-resistant variants.

The yeast two-hybrid assay was used to study the dimerization of engineered and naturally occurring variants of human immunodeficiency virus (HIV) protease (PR) monomers. Defective monomers that were previously shown to exhibit a dominant-negative (D-N) effect in cultured mammalian cells were tested for their ability to interact in the two-hybrid assay. Similarly, monomers with dimer-interface substitutions and monomers harboring in vivo selected mutations that confer multidrug resistance (mdr) in an AIDS patient were tested for interaction in yeast. Dimer formation between wt monomers with catalytic aspartates was not detected in yeast, whereas the dimerization of PR monomers harboring the acid active site substitution D25N was readily demonstrated. The use of inactive monomers harboring the D25N substitution as a genetic background for studying additional HIV PR mutations allowed for the probing of interactions between monomers with mdr-associated mutations with those based on the HIV-1 HXB2R sequence. The HTLVIII/HIV-1 HXB2R clone has been the basis for a large number of HIV-related plasmids, primers, antibodies, and other specific reagents throughout the HIV research community. The results of our assay suggest that HXB2R-based D-N PR inhibitors associate with variant monomers based on the recently obtained nucleotide sequence from an AIDS patient with a multidrug-resistant virus. These results further encourage the use of D-N PR inhibitors as antiviral agents which may complement existing small-molecule combination therapies.

Acquired Immunodeficiency Syndrome↗
PubMed1999

Reverse biochemistry: use of macromolecular protease inhibitors to dissect complex biological processes and identify a membrane-type serine protease in epithelial cancer and normal tissue.

Serine proteases of the chymotrypsin fold are of great interest because they provide detailed understanding of their enzymatic properties and their proposed role in a number of physiological and pathological processes. We have been developing the macromolecular inhibitor ecotin to be a "fold-specific" inhibitor that is selective for members of the chymotrypsin-fold class of proteases. Inhibition of protease activity through the use of wild-type and engineered ecotins results in inhibition of rat prostate differentiation and retardation of the growth of human PC-3 prostatic cancer tumors. In an effort to identify the proteases that may be involved in these processes, reverse transcription-PCR with PC-3 poly(A)+ mRNA was performed by using degenerate oligonucleotide primers. These primers were designed by using conserved protein sequences unique to chymotrypsin-fold serine proteases. Five proteases were identified: urokinase-type plasminogen activator, factor XII, protein C, trypsinogen IV, and a protease that we refer to as membrane-type serine protease 1 (MT-SP1). The cloning and characterization of the MT-SP1 cDNA shows that it encodes a mosaic protein that contains a transmembrane signal anchor, two CUB domains, four LDLR repeats, and a serine protease domain. Northern blotting shows broad expression of MT-SP1 in a variety of epithelial tissues with high levels of expression in the human gastrointestinal tract and the prostate. A His-tagged fusion of the MT-SP1 protease domain was expressed in Escherichia coli, purified, and autoactivated. Ecotin and variant ecotins are subnanomolar inhibitors of the MT-SP1 activated protease domain, suggesting a possible role for MT-SP1 in prostate differentiation and the growth of prostatic carcinomas.

Amino Acid Sequence↗
PubMed1999

Auto-inactivation by cleavage within the dimer interface of Kaposi's sarcoma-associated herpesvirus protease.

An autolysis site of functional and structural significance has been mapped within the dimer interface of Kaposi's sarcoma-associated herpesvirus protease. Cleavage 27 residues from the C terminus of the 230 amino acid residue, 25 kDa protein was observed to cause a loss of dimerization and proteolytic activity, even though no active site moieties were lost. Gel-filtration chromatography and analytical ultracentrifugation were used to analyze the changes in oligomerization upon autolysis. The selective auto-disruption of this essential protein-protein interface by proteolytic cleavage resulted in a 60 % loss in mean residue ellipticity by circular dichroism as well as a 20 % weaker, 10 nm red-shifted intrinsic protein fluorescence emission spectrum. These apparent conformational changes induced a strict inhibition of enzymatic activity. An engineered substitution at the P1' position of this cleavage site attenuated autolysis by the enzyme and restored wild-type dimerization. In addition to retaining full proteolytic activity in a continuous fluorescence-based enzyme assay, this protease variant allowed the determination of the enzyme's dimerization dissociation constant of 1.7 (+/-0.9) microM. The structural perturbations observed in this enzyme may play a role in viral maturation, and offer general insight into the allosteric relationship between the dimer interface and active site of herpesviral proteases. The functional coupling between oligomerization and activity presented here may allow for a better understanding of such phenomena, and the design of an enzyme variant stabilized to autolysis should further the structural and mechanistic characterization of this viral protease.

Amino Acid Sequence↗
PubMed1998

Definition and redesign of the extended substrate specificity of granzyme B.

Granzyme B is a protease involved in the induction of rapid target cell death by cytotoxic lymphocytes. Definition of the substrate specificity of granzyme B allows for the identification of in vivo substrates in this process. By using the combinatorial methods of synthetic substrate libraries and substrate-phage display, an optimal substrate for granzyme B that spans over six subsites was determined to be Ile-Glu-Xaa-(Asp downward arrowXaa)-Gly, with cleavage of the Asp downward arrowXaa peptide bond. Granzyme B proteolysis was shown to be highly dependent on the length and sequence of the substrate, supporting the role of granzyme B as a regulatory protease. Arginine 192 was identified as a determinant of P3-Glu and P1-Asp substrate specificity. Mutagenesis of arginine 192 to glutamate reversed the preference for negatively charged amino acids at P3 to positively charged amino acids. The preferred substrate sequence matches the activation sites of caspase 3 and caspase 7 and thus is consistent with the role of granzyme B in activation of these proteases during apoptosis. The caspase substrate poly(ADP)-ribose polymerase is cleaved by granzyme B in a cell-free assay at two sites that resemble the granzyme B specificity determined by the combinatorial methods. Many caspase substrates contain granzyme B cleavage sites and are proposed as potential granzyme B targets, suggesting a redundant function with certain caspases.

Animals↗
PubMed1998

Ecotin: a serine protease inhibitor with two distinct and interacting binding sites.

The interaction between ecotin and target proteases with trypsin-like specificity has been systematically dissected to understand the structural basis of ecotin's broad inhibitory specificity and the role of the secondary binding site. Site-directed and region-specific mutagenesis were preformed at ecotin's primary site P1 residue (84), the C-terminal dimer interface (133 to 142), and two surface loops of the secondary binding site (67 to 70, 108 to 113). Substitutions at the P1 position resulted in less than fivefold difference in the potency of ecotin binding to rat trypsin, suggesting that the extended binding site is important in binding. A ten amino acid C-terminal truncation variant showed threefold weaker self-association but remained a dimer. The interactions of the secondary binding site of ecotin with bovine trypsin, rat trypsin and human urokinase-type plasminogen activator (uPA) were investigated with alanine substitutions in ecotin at Trp67, Gly68, Tyr69, Asp70, Arg108, Asn110, Lys112 and Leu113, which formed contacts between the inhibitor and protease. By combining these mutations at the secondary binding site with mutations in the primary binding site the molecular recognition between ecotin and its target serine proteases was probed. The contrast in the Ki values of the various ecotin variants towards bovine trypsin, rat trypsin and human uPA established the role of ecotin's secondary binding site in recognizing these homologous serine proteases. Ecotin binds to proteases with a chymotrypsin fold through a combination of primary and secondary site surface loops and is amenable to redesign of its potency and specificity for this class of enzymes.

Animals↗
PubMed1998

Engineering bidentate macromolecular inhibitors for trypsin and urokinase-type plasminogen activator.

Ecotin, a dimeric serine protease inhibitor from Escherichia coli, is a novel platform for inhibitor design. An approach using the three-dimensional structure of the ecotin-trypsin complex to guide combinatiorial design efforts was taken to create potent bidentate ecotin inhibitors for trypsin and human urokinase-type plasminogen activator (uPA). The ecotin surface loop that was redesigned is composed of residues 67 to 70 (60 s loop), and binds to the target protease at a region 25 A from the enzyme active site. Two ecotin phage display libraries were constructed to exploit the binding interactions at the 60 s loop. The ecotin 60X4 library, in which residues 67 to 70 of ecotin were randomized, was panned against rat and bovine trypsin in parallel for four rounds. Panning against bovine trypsin resulted in enrichment of ecotin phage but did not yield a consensus sequence. Panning against rat trypsin resulted in enrichment as well as the ecotin consensus sequence WGFP at positions 67 to 70. The variant ecotin encoded by this sequence inhibited rat trypsin at 80 pM, a 12-fold improvement over ecotin wild-type (WT). A second generation library, ecotin M84R+60X4 including an additional methionine to arginine substitution at position 84 in the primary binding site of ecotin, was generated for panning against uPA and rat trypsin. Panning against rat trypsin resulted in enrichment but no consensus sequence. Panning against uPA resulted in enrichment as well as the different ecotin consensus sequence WGYR at positions 67 to 70. Ecotin M84R+D70R bound to uPA at 50 pM, a 56,000-fold increase in binding compared to ecotin WT. Furthermore, ecotin M84R+D70R achieved a 13,680-fold preference of specificity towards uPA versus rat trypsin. The fact that the 60 s loop of ecotin plays different roles in binding to trypsin and uPA suggests this site can be used to introduce specificity and potency for other members of the serine proteases with a chymotrypsin fold.

Animals↗
PubMed1998

Determining protein-protein interactions by oxidative cross-linking of a glycine-glycine-histidine fusion protein.

The Ni(II) complex of the tripeptide NH2-glycine-glycine-histidine-COOH (GGH) mediates efficient protein-protein cross-linking in the presence of oxidants such as oxone and monoperoxyphthalic acid (MMPP). Here we demonstrate that GGH fused to the amino terminus of a protein can still support cross-linking. The tripeptide was expressed at the amino terminus of ecotin, a dimeric macromolecular serine protease inhibitor found in the periplasm of Escherichia coli. In the presence of Ni(OAc)2 and MMPP, GGH-ecotin is cross-linked to give a species that has an apparent molecular mass of a GGH-ecotin dimer with no observable protein degradation. The cross-linking reaction occurs between two ecotin proteins in a dimer complex. Furthermore, GGH-ecotin can be cross-linked to a serine protease target, trypsin, and the reaction is specific for proteins that interact with ecotin. The cross-linking reaction has been carried out on small peptides, and the reaction products have been analyzed by matrix-assisted laser desorption/ionization mass spectrometry. The target of the reaction is tyrosine, and the product is bityrosyl cross-links. The yield of the cross-linking is on the order of 15%. However, the reaction efficiency can be increased 4-fold by a single amino acid substitution in the carboxy terminus of ecotin that places an engineered tyrosine within 5 A of a naturally occurring tyrosine. This cross-linking methodology allows for the protein cross-linking reagent to be encoded for at the DNA level, thus circumventing the need for posttranslational modification.

Acetates↗
PubMed1998

Domain flexibility in retroviral proteases: structural implications for drug resistant mutations.

Rigid body rotation of five domains and movements within their interfacial joints provide a rational context for understanding why HIV protease mutations that arise in drug resistant strains are often spatially removed from the drug or substrate binding sites. Domain motions associated with substrate binding in the retroviral HIV-1 and SIV proteases are identified and characterized. These motions are in addition to closure of the flaps and result from rotations of approximately 6-7 degrees at primarily hydrophobic interfaces. A crystal structure of unliganded SIV protease (incorporating the point mutation Ser 4 His to stabilize the protease against autolysis) was determined to 2.0 A resolution in a new space group, P3221. The structure is in the most "open" conformation of any retroviral protease so far examined, with six residues of the flaps disordered. Comparison of this and unliganded HIV structures, with their respective liganded structures by difference distance matrixes identifies five domains of the protease dimer that move as rigid bodies against one another: one terminal domain encompassing the N- and C-terminal beta sheet of the dimer, two core domains containing the catalytic aspartic acids, and two flap domains. The two core domains rotate toward each other on substrate binding, reshaping the binding pocket. We therefore show that, for enzymes, mutations at interdomain interfaces that favor the unliganded form of the target active site will increase the off-rate of the inhibitor, allowing the substrate greater access for catalysis. This offers a mechanism of resistance to competitive inhibitors, especially when the forward enzymatic reaction rate exceeds the rate of substrate dissociation.

Aspartic Acid Endopeptidases↗
PubMed1998

Engineering enzyme specificity.

Protein engineering is the application of knowledge to design and alter protein function and structure. Although powerful methods, from specific to random, have been developed for the redesign of protein architecture, their successful application is dependent on the information known about the protein. This database of information is providing a foundation for establishing rules that govern enzyme-substrate interactions.

Enzymes↗
PubMed1997

P-4 and RNKP-7, new granzyme-like serine proteases expressed in activated rat lymphocytes.

Serine proteases (granzymes) in killer lymphocytes are required for lymphocyte cytotoxic granules to lyse target cells. Herein we report the development of a 3-step PCR cloning technique to amplify novel granzyme genes and two new rat granzymes are described. Degenerate oligonucleotide primers were designed based on sequence motifs selectively expressed in granzymes. These motifs flank "delta" regions that are unique for each granzyme. Total RNA of RNK-16 cells or activated splenocytes was amplified by reverse transcriptase-PCR to obtain cDNA fragments of several new granzymes. Gene-specific primers based on these delta regions were then used for 3'-RACE to obtain clones with the 3' gene ends. Reverse (antisense) delta-based or active site serine primers were used with a granzyme 5'-UTR primer to obtain clones extending to the 5' ends. Using this technique, two new cDNAs, RNKP-4 and RNKP-7, which encode granzymes of 248 and 241 amino acids, respectively, were cloned from activated lymphocytes. RNKP-4 is likely the rat equivalent of mouse granzyme C. RNKP-7 is most closely related to granzymes F and G. Modeling of the predicted proteins suggests large/polar P1 (Gln/Asn) specificity for RNKP-4 and large/hydrophobic P1 (e.g., Phe) specificity for RNKP-7. These specific protease activities were found in cytotoxic RNK-16 lymphocyte granules indicating that the two new genes may be translated and stored as active granzymes.

Amino Acid Sequence↗
PubMed1997

Crystal structure of an ecotin-collagenase complex suggests a model for recognition and cleavage of the collagen triple helix.

The crystal structure of fiddler crab collagenase complexed with the dimeric serine protease inhibitor ecotin at 2.5 A resolution reveals an extended cleft providing binding sites for at least 11 contiguous substrate residues. Comparison of the positions of nine intermolecular main chain hydrogen bonding interactions in the cleft, with the known sequences at the cleavage site of type I collagen, suggests that the protease binding loop of ecotin adopts a conformation mimicking that of the cleaved strand of collagen. A well-defined groove extending across the binding surface of the enzyme readily accommodates the two other polypeptide chains of the triple-helical substrate. These observations permit construction of a detailed molecular model for collagen recognition and cleavage by this invertebrate serine protease. Ecotin undergoes a pronounced internal structural rearrangement which permits binding in the observed conformation. The capacity for such rearrangement appears to be a key determinant of its ability to inhibit a wide range of serine proteases.

Animals↗
PubMed1997

Structural basis for the broad substrate specificity of fiddler crab collagenolytic serine protease 1.

Crab collagenolytic serine protease 1 efficiently cleaves peptide bonds directly C-terminal to basic, polar, and hydrophobic amino acids. The crystal structure of this enzyme complexed to the protein inhibitor ecotin at 2.5 A resolution reveals a large primary binding pocket punctuated on one wall by the side chain of aspartate-226. Removal or relocation of this negatively charged group by site-directed mutagenesis generates variant enzymes which retain very high activities toward selected substrates. Full retention of activity toward hydrophobic substrates in collagenase D226G is accompanied by a 10-100-fold reduction in k(cat)/Km toward basic residues. In contrast, restoration of the negative charge in a trypsin-like position in collagenase D226G/G189D regenerates nearly full activity toward basic substrates while introducing a 5-fold decrease in k(cat)/Km toward hydrophobic amino acids. These results imply that the collagenase S1 pocket has multiple distinct binding sites for different amino acid side chains, a suggestion supported by molecular modeling studies based on the crystal structure. The ease of specificity modification in the primary binding site of this serine protease parallels similar observations with the bacterial enzymes alpha-lytic protease and subtilisin, and stands in sharp distinction to the extensive mutagenesis required to alter specificity in trypsin.

Animals↗
PubMed1997

Structural determinants of specificity in the cysteine protease cruzain.

The structure of cruzain, an essential protease from the parasite Trypanosoma cruzi, was determined by X-ray crystallography bound to two different covalent inhibitors. The cruzain S2 specificity pocket is able to productively bind both arginine and phenylalanine residues. The structures of cruzain bound to benzoyl-Arg-Ala-fluoromethyl ketone and benzoyl-Tyr-Ala-fluoromethyl ketone at 2.2 and 2.1 A, respectively, show a pH-dependent specificity switch. Glu 205 adjusts to restructure the S2 specificity pocket, conferring right binding to both hydrophobic and basic residues. Kinetic analysis of activated peptide substrates shows that substrates placing hydrophobic residues in the specificity pocket are cleaved at a broader pH range than hydrophilic substrates. These results demonstrate how cruzain binds both basic and hydrophobic residues and could be important for in vivo regulation of cruzain activity.

Animals↗
PubMed1997

Alkylation of a catalytic aspartate group of the SIV protease by an epoxide inhibitor.

Specific irreversible inhibition of the SIV protease by FMOC-protected piperidine epoxide 1 involves alkylation of the protein. Tryptic digestion of the alkylated protein and mass spectrometric analysis of the peptides identify an active site aspartic acid (Asp-25) as the single residue that is alkylated. Computer modeling of 1 bound in the crystal structure of the SIV protease using DOCK 3.5 indicates that 1 has appropriate access to the active site. It is able to align in an orientation that allows a proton to be transferred to the epoxide from one of the catalytic aspartic acid groups in conjunction with nucleophilic attack on the epoxide of the carboxylate moiety of the second catalytic aspartic acid residue. Hydrophobic interactions are not optimal for this process due, in part, to the rigidity of the inhibitor ring system and the planar conformation of the amide. The combination of modeling with protein alkylation can provide insights into structural modifications of the inhibitor that may lead to improved inhibitory activity.

Alkylation↗