Engineering, characterization and phage display of hepatitis C virus NS3 protease and NS4A cofactor peptide as a single-chain protein

The polyprotein encoded by hepatitis C virus (HCV) genomic RNA is processed into functional polypeptides by both host- and virus-encoded proteases. The HCV-encoded NS3 protease and its cofactor peptide NS4A form a non-covalent complex, which participates in processing the viral polyprotein. This proteolytic activity is believed to be essential for virus proliferation and thus the NS3 protease is a prime target for developing anti-HCV pharmacological agents. Recent X-ray crystallography structural studies have revealed the nature of this non-covalent complex between NS3 protease and the 'active' central segment of NS4A, providing the opportunity to design a single-chain polypeptide. To this end, the DNA sequence encoding for the NS4A peptide (residues 21-34) was genetically fused via a short linker, capable of making a beta-turn, to the N-terminus of the NS3 protease domain. This engineered single-chain NS3-protease (scNS3) is fully active with kinetic parameters virtually identical with those of the NS3/ NS4A non-covalent complex. Moreover, the scNS3 protease can be displayed on filamentous phage and affinity selected using an immobilized specific inhibitor. The scNS3 expressed as a soluble protein and in a phage-display format facilitates enzyme engineering for further structural studies and in vitro selection of potential drug-resistant mutants. These are important steps towards developing effective anti-protease compounds.     

Rational design and functional expression of a constitutively active single-chain NS4A-NS3 proteinase

BACKGROUND: The proteinase domain of the hepatitis C virus NS3 protein is involved in the maturation of the viral polyprotein. A central hydrophobic domain of the NS4A protein is required as a cofactor for its proteolytic activity. The three-dimensional structure of the proteinase domain alone and complexed with an NS4A-derived peptide has been solved recently and revealed that the N terminus of the proteinase is in near proximity to the C terminus of the cofactor. To study the molecular basis of the enzyme activation by its cofactor and to overcome the difficulties of structural and functional investigation associated with a two-species complex, we rationally designed a link to bridge the two molecules in order to have a single polypeptide construct. RESULTS: The engineered construct led to the production of a stable, monomeric protein with proteolytic activity that is independent from the addition of a synthetic peptide representing the cofactor domain of the NS4A protein. The protein is active on both protein and synthetic peptide substrates. Spectroscopic and kinetic analysis of the recombinant NS4A-NS3 single-chain proteinase demonstrated features superimposable with the isolated NS3 proteinase domain complexed with the NS4A cofactor. CONCLUSIONS: We designed a very tight connection between the NS3 and NS4A polypeptide chains with the rationale that this would allow a more stable structure to be formed. The engineered single-chain enzyme was indistinguishable from the NS3 proteinase complexed with its NS4A cofactor in all enzymatic and physico-chemical properties investigated.     

Age-related changes in the inner zone of the adrenal cortex of the rat--a morphologic and biochemical study

Despite an urgent medical need, a broadly effective anti-viral therapy for the treatment of infections with hepatitis C viruses (HCVs) has yet to be developed. One of the approaches to anti-HCV drug discovery is the design and development of specific small molecule drugs to inhibit the proteolytic processing of the HCV polyprotein. This proteolytic processing is catalyzed by a chymotrypsin-like serine protease which is located in the N-terminal region of non-structural protein 3 (NS3). This protease domain forms a tight, non-covalent complex with NS4A, a 54 amino acid activator of NS3 protease. The C-terminal two-thirds of the NS3 protein contain a helicase and a nucleic acid-stimulated nucleoside triphosphatase (NTPase) activities which are probably involved in viral replication. This review will focus on the structure and function of the serine protease activity of NS3/4A and the development of inhibitors of this activity.     

Multiple enzymatic activities associated with recombinant NS3 protein of hepatitis C virus

The hepatitis C virus (HCV) nonstructural 3 protein (NS3) contains at least two domains associated with multiple enzymatic activities; a serine protease activity resides in the N-terminal one-third of the protein, whereas RNA helicase activity and RNA-stimulated nucleoside triphosphatase activity are associated with the C-terminal portion. To study the possible mutual influence of these enzymatic activities, a full-length NS3 polypeptide of 67 kDa was expressed as a nonfusion protein in Escherichia coli, purified to homogeneity, and shown to retain all three enzymatic activities. The protease activity of the full-length NS3 was strongly dependent on the activation by a synthetic peptide spanning the central hydrophobic core of the NS4A cofactor. Once complexed with the NS4A-derived peptide, the full-length NS3 protein and the isolated N-terminal protease domain cleaved synthetic peptide substrates with comparable efficiency. We show that, as in the case of the isolated protease domain, the protease activity of full-length NS3 undergoes inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A-NS4B and NS5A-NS5B. We have also characterized and quantified the NS3 ATPase, RNA helicase, and RNA-binding activities under optimized reaction conditions. Compared with the isolated N-terminal and C-terminal domains, recombinant full-length NS3 did not show significant differences in the three enzymatic activities analyzed in independent in vitro assays. We have further explored the possible interdependence of the NS3 N-terminal and C-terminal domains by analyzing the effect of polynucleotides on the modulation of all NS3 enzymatic functions. Our results demonstrated that the observed inhibition of the NS3 proteolytic activity by single-stranded RNA is mediated by direct interaction with the protease domain rather than with the helicase RNA-binding domain.     

Serine protease of hepatitis C virus expressed in insect cells as the NS3/4A complex

Hepatitis C virus (HCV) protease NS3 and its protein activator NS4A participate in the processing of the viral polyprotein into its constituent nonstructural proteins. The NS3/4A complex is thus an attractive target for antiviral therapy against HCV. We expressed the full-length NS3 and NS4A in insect cells as a soluble fusion protein with an N-terminal polyhistidine tag and purified the two proteins to homogeneity. Cleavage at the junction between HisNS3 and NS4A occurs during expression, producing a noncovalent complex between HisNS3 and NS4A with a subnanomolar dissociation constant. We purified the HisNS3/4A complex by detergent extraction of cell lysate and by metal chelate chromatography. We removed the His tag by thrombin cleavage and then further purified the complex by gel filtration. The purified NS3/4A complex is active in a protease assay using a synthetic peptide substrate derived from the NS5A-NS5B junction, with kcat/K(m) of 3700 (+/- 600) M-1 s-1, an order of magnitude above those previously reported for NS3 expressed by other strategies. This high protease activity implies that the full-length sequences of NS3 and NS4A are required for optimal activity of the NS3 protease domain. We examined the dependence of the NS3/4A protease activity on buffer conditions, temperature, and the presence of detergents. We find that, under most conditions, NS3 protease activity is dependent on the aggregation state of the NS3/4A complex. The monodisperse, soluble form of the NS3/4A complex is associated with the highest protease activity.     

Contribution of domain interface residues to the stability of antibody CH3 domain homodimers

Dimers of CH3 domains from human IgG1 were used to study the effect of mutations constructed at a domain-domain interface upon domain dissociation and unfolding, "complex stability". Alanine replacement mutants were constructed on one side of the interface for each of the sixteen interdomain contact residues by using a single-chain CH3 dimer in which the carboxyl terminus of one domain was joined to the amino terminus of the second domain via a (G4S)4 linker. Single-chain variants were expressed in Escherichia coli grown in a fermentor and recovered in yields of 6-90 mg L-1 by immobilized metal affinity chromatography. Guanidine hydrochloride-induced denaturation was used to follow domain dissociation and unfolding. Surprisingly, the linker did not perturb the complex stability for either the wild type or two destabilizing mutants. The CH3 domain dissociation and unfolding energetics are dominated by six contact residues where corresponding alanine mutations each destabilize the complex by >2.0 kcal mol-1. Five of these residues (T366, L368, F405, Y407, and K409) form a patch at the center of the interface and are located on the two internal antiparallel beta-strands. These energetically key residues are surrounded by 10 residues on the two external beta-strands whose contribution to complex stability is small (three have a Delta DeltaG of 1.1-1.3 kcal mol-1) or very small (seven have a Delta DeltaG of 相似文献   

Expression of the Japanese encephalitis virus NS3 and NS2b proteins as glutathione S-transferase fusions

Flaviviruses generate their structural and nonstructural proteins by proteolytic processing of a single large polyprotein precursor. These proteolytic events are brought about both by host cell signalase and a virally encoded protease. The virally encoded proteolytic activity has been shown to reside within the nonstructural protein 3 (NS3) and requires the product of the nonstructural 2b (NS2b) gene. In order to obtain sufficient quantities of pure NS2b and NS3 proteins for kinetic analysis, we have expressed both these proteins in recombinant systems as fusions to glutathione S-transferase (GST). The fusion constructs were driven by the strong bacteriophage T7 promoter. Transfection of these constructs into the African green monkey kidney cell line CV-1 previously infected with a recombinant vaccinia virus expressing the T7 RNA polymerase resulted in synthesis of the fusion proteins. Both the fusion proteins could be purified to homogeneity in a single step using a glutathione agarose affinity matrix.     

Product inhibition of the hepatitis C virus NS3 protease

The nonstructural protein NS3 of the hepatitis C virus (HCV) harbors a serine protease domain that is responsible for most of the processing events of the nonstructural region of the polyprotein. Its inhibition is presently regarded as a promising strategy for coping with the disease caused by HCV. In this work, we show that the NS3 protease undergoes inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B cleavage sites, whereas no inhibition is observed with a cleavage product of the intramolecular NS3-NS4A junction. The Ki values of the hexamer inhibitory products [Ki(NS4A) = 0.6 microM, Ki(NS5A) = 1.4 microM, and Ki(NS4B) = 180 microM] are lower than the Km values of the respective substrate peptides [Km(NS4A-NS4B) = 10 microM, Km(NS5A-NS5B) = 3.8 microM, and Km(NS4B-NS5A) > 1000 microM]. Mutagenesis experiments have identified Lys136 as an important determinant for product binding. The phenomenon of product inhibition can be exploited to optimize peptide inhibitors of NS3 protease activity that may be useful in drug development.     

RNA-dependent RNA polymerase activity of the soluble recombinant hepatitis C virus NS5B protein truncated at the C-terminal region

The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRP), which is the central catalytic enzyme of HCV replicase. We established a new method to purify soluble HCV NS5B in the glutathione S-transferase-fused form NS5Bt from Escherichia coli which lacks the C-terminal 21 amino acid residues encompassing a putative anchoring domain (anino acids 2990-3010). The recombinant soluble protein exhibited RdRP activity in vitro which was dependent upon the template and primer, but it did not exhibit the terminal transferase activity that has been reported to be associated with the recombinant NS5B protein from insect cells. The RdRP activity of purified glutathione S-transferase-NS5Bt and thrombin-cleavaged non-fused NS5Bt shares most of the properties. Substitution mutations of NS5Bt at the GDD motif, which is highly conserved among viral RdRPs, and at the clustered basic residues (amino acids 2919-2924 and 2693-2699) abolished the RdRP activity. The C-terminal region of NS5B, which is dispensable for the RdRP activity, dramatically affected the subcellular localization of NS5B retaining it in perinuclear sites in transiently overexpressed mammalian cells. These results may provide some clues to dissecting the molecular mechanism of the HCV replication and also act as a basis for developing new anti-viral drugs.     

Complex formation between the hepatitis C virus serine protease and a synthetic NS4A cofactor peptide

The NS3 protein of the hepatitis C virus contains a serine protease that, upon binding to its cofactor, NS4A, is responsible for maturational cleavages that occur in the nonstructural region of the viral polyprotein. We have studied in vitro complex formation between the NS3 protease domain expressed in Escherichia coli and a synthetic peptide spanning the minimal domain of the NS4A cofactor. Complex dissociation constants in the low micromolar range were measured using different techniques such as activity titration, fluorescence titration, and pre-equilibrium analysis of complex formation. Cofactor binding was strictly dependent on the glycerol content of buffer solutions and was not significantly influenced by substrate saturation of the enzyme. NS4A peptide binding to NS3 was accompanied by changes in the circular dichroism spectrum in the region between 270 and 290 nm, as well as by an enhancement of tryptophan fluorescence. Conversely, no changes in the far UV region of the circular dichroism spectrum were detectable. These data are indicative of induced tertiary structure changes and suggest that the secondary structure content of the uncomplexed enzyme does not differ significantly from that of the NS3-cofactor complex. Pre-equilibrium measurements of complex formation showed very low values for k(on), suggesting conformational transitions to be rate limiting for the association reaction.     

Peptide-based inhibitors of the hepatitis C virus serine protease

Hexapeptide DDIVPC-OH is a competitive inhibitor of the hepatitis C virus (HCV) NS3 protease complexed with NS4A cofactor peptide. This hexapeptide corresponds to the N-terminal cleavage product of an HCV dodecapeptide substrate derived from the NS5A/5B cleavage site. Structure-activity studies on Ac-DDIVPC-OH revealed that side chains of the P4, P3 and P1 residues contribute the most to binding and that the introduction of a D-amino acid at the P5 position improves potency considerably. Furthermore, there is a strong preference for cysteine at the P1 position and conservative replacements, such as serine, are not well tolerated.     

Novel hepatitis C virus protease inhibitors: thiazolidine derivatives

This study evaluated the inhibitory effects of thiazolidine derivatives on hepatitis C virus (HCV) protease and other human serine proteases. The inhibition efficacy was tested with a reversed-phase high-performance liquid chromatography (HPLC) assay system using a NS3-NS4A fusion protein as the HCV protease and a synthetic peptide substrate that mimics the NS5A-5B junction. Nine thiazolidine derivatives showed more than 50% inhibition at 50 microg/ml. The most potent derivative was RD4-6250, with 50% inhibition at a concentration of 2.3 microg/ml; this concentration was lower than those of other protease inhibitors reported previously. The most selective derivative was RD4-6205, with 50% inhibition at a concentration of 6.4 microg/ml, a lower concentration than those on other serine proteases (chymotrypsin, trypsin, plasmin, and elastase). These results suggest that the RD4-6205 skeleton is an important structure for inhibitory activity on the HCV protease NS3-NS4A.     

The chlamydial EUO gene encodes a histone H1-specific protease

Chlamydia trachomatis is an obligate intracellular pathogen, long recognized as an agent of blinding eye disease and more recently as a common sexually transmitted infection. Recently, two eukaryotic histone H1-like proteins, designated Hc1 and Hc2, have been identified in Chlamydia. Expression of Hc1 in recombinant Escherichia coli produces chromatin condensation similar to nucleoid condensation observed late in the parasite's own life cycle. In contrast, chromatin decondensation, observed during the early life cycle, accompanies down-regulation and nondetection of Hc1 and Hc2 among internalized organisms. We reasoned that the early upstream open reading frame (EUO) gene product might play a role in Hc1 degradation and nucleoid decondensation since it is expressed very early in the chlamydial life cycle. To explore this possibility, we fused the EUO coding region between amino acids 4 and 177 from C. trachomatis serovar Lz with glutathione S-transferase (GST) and examined the effects of fusion protein on Hc1 in vitro. The purified fusion protein was able to digest Hc1 completely within 1 h at 37 degrees C. However, GST alone exhibited no Hc1-specific proteolytic activity. The chlamydial EUO-GST gene product also cleaves very-lysine-rich calf thymus histone H1 and chicken erythrocyte histone H5 but displays no measurable activity towards core histones H2A, H2B, H3, and H4 or chlamydial RNA polymerase alpha-subunit. This proteolytic activity appears sensitive to the serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF) and aspartic protease inhibitor pepstatin but resistant to high temperature and other broad-spectrum protease inhibitors. The proteolytic activity specified by the EUO-GST fusion product selectively digested the C-terminal portion of chlamydial Hc1, the domain involved in DNA binding, while leaving the N terminus intact. At a molar equivalent ratio of 1:1 between Hc1 and DNA, the EUO gene product cleaves Hc1 complexed to DNA and this cleavage appears sufficient to initiate dissociation of DNA-Hc1 complexes. However, at a higher molar equivalent ratio of Hc1/DNA (10:1), there is partial protection conferred upon Hc1 to an extent that prevents dissociation of DNA-Hc1 complexes.     

Proteolytic processing of the replicase ORF1a protein of equine arteritis virus

To study the proteolytic processing of the equine arteritis virus (EAV) replicase open reading frame 1a (ORF1a) protein, specific antisera were raised in rabbits, with six synthetic peptides and a bacterial fusion protein as antigens. The processing of the EAV ORF1a product in infected cells was analyzed with Western blot (immunoblot) and immunoprecipitation techniques. Additional information was obtained from transient expression of ORF1a cDNA constructs. The 187-kDa ORF1a protein was found to be subject to at least five proteolytic cleavages. The processing scheme, which covers the entire ORF1a protein, results in cleavage products of approximately 29, 61, 22, 31, 41, and 3 kDa, which were named nonstructural proteins (nsps) 1 through 6, respectively. Pulse-chase experiments revealed that the cleavages at the nsp1/2 and nsp2/3 junctions are the most rapid processing steps. The remaining nsp3456 precursor is first cleaved at the nsp4/5 site. Final processing of the nsp34 and nsp56 intermediates is extremely slow. As predicted from previous in vitro translation experiments (E. J. Snijder, A. L. M. Wassenaar, and W. J. M. Spaan, J. Virol. 66:7040-7048, 1992), a cysteine protease domain in nsp1 was shown to be responsible for the nsp1/2 cleavage. The other processing steps are carried out by the putative EAV serine protease in nsp4 and by a third protease, which remains to be identified.     

A novel ligand-binding site in the zeta-form 14-3-3 protein recognizing the platelet glycoprotein Ibalpha and distinct from the c-Raf-binding site

We reported previously that the zeta-form 14-3-3 protein (14-3-3zeta) binds to a platelet adhesion receptor, glycoprotein (GP) Ib-IX, and this binding is dependent on the SGHSL sequence at the C terminus of GPIbalpha. In this study, we have identified a binding site in the helix I region of 14-3-3zeta (residues 202-231) required for binding to GPIb-IX complex and to the cytoplasmic domain of GPIbalpha. We also show that phosphorylation-dependent binding of c-Raf to 14-3-3zeta requires helix G (residues 163-187) but not helix I. Thus, the GPIbalpha-binding site is distinct from the binding sites for RSXpSXP motif-dependent ligands. Furthermore, we show that wild type 14-3-3zeta has a higher affinity for GPIb-IX complex than recombinant GPIbalpha cytoplasmic domain. Deletion of helices A and B (residues 1-32) disrupts 14-3-3zeta dimerization and decreases its affinity for GPIb-IX. Disruption of 14-3-3zeta dimerization, however, does not reduce 14-3-3zeta binding to recombinant GPIbalpha cytoplasmic domain. This suggests a dual site recognition mechanism in which a 14-3-3zeta dimer interacts with both GPIbalpha and GPIbbeta (known to contain a phosphorylation-dependent binding site), resulting in high affinity binding.     

Effects of prior exercise on exercise-induced arterial hypoxemia in young women

Insulin-like growth factor binding proteins (IGFBP) proteases have been proposed to be involved in changes of serum IGFBP pattern during pregnancy. IGFBP-4 and -5 are degraded specifically by proteases in pregnancy serum in vitro, whereas IGFBP-3 proteolytic activity was also detected in nonpregnancy serum. To identify and characterize IGFBP proteases, human pregnancy serum was fractionated by size exclusion chromatography revealing IGFBP-4 protease activities in fractions coeluting with proteins of approximately 600-kDa and 50- to 100-kDa molecular mass. In both fractions, a predominant 50-kDa gelatinase was found, suggesting that parts of the gelatinase activity might aggregate or are complexed with other proteins forming a higher molecular complex. Hydroxyapatite chromatography and chromatofocusing of the 50- to 100-kDa serum fraction showed that the IGFBP-4 protease and the 50-kDa gelatinase activity were copurified. When the 50-kDa gelatinase-containing band was excised from the polyacrylamide gel, it exhibited IGFBP-4 proteolytic activity, resulting in the formation of 17- and 10-kDa fragments. [125I] IGFBP substrate zymography combined with fragment blotting showed that the 1,10-phenanthroline-sensitive 50-kDa protease activity purified by chromatofocusing also cleaved IGFBP-3 and -5. Other proteases detected in pregnancy serum fractions with Mr estimates of 79-, 30-, and 22-kDa degraded IGFBP-3 and -5 but not IGFBP-4. [125I] IGFBP-5 substrate zymography revealed that the 30-kDa IGFBP protease was inhibited by serine protease inhibitors. Whereas 1,10-phenanthroline inhibited the IGFBP proteolytic activity in the solution assay, serine protease inhibitors failed to affect proteolysis, indicating the predominant contribution of the metalloproteinase to IGFBP proteolysis. Tissue inhibitors of matrix metalloproteinases-1 and -2 revealed weak or no inhibition of IGFBP-4 and -5 proteolytic activity, whereas a hydroxamic acid-based inhibitor, potentially inhibiting disintegrin metalloproteases, completely prevented the proteolysis of IGFBPs. Whereas no specific immunoreactivity of the 50-kDa protein with antimatrix metalloproteinase-1, -2, -3, -9, or -13 antibodies was observed, antidisintegrin domain-specific antibodies bound to the 50-kDa gelatinase. These studies provide the first direct biochemical evidence that human pregnancy serum contains a 50-kDa IGFBP protease with properties of a soluble disintegrin metalloproteinase that appears to be potentially involved in regulating IGF bioavailability for placental and fetal growth.     

Three-dimensional structure of an Fab-peptide complex: structural basis of HIV-1 protease inhibition by a monoclonal antibody

F11.2.32, a monoclonal antibody raised against HIV-1 protease (Kd = 5 nM), which inhibits proteolytic activity of the enzyme (K(inh) = 35(+/-3)nM), has been studied by crystallographic methods. The three-dimensional structure of the complex between the Fab fragment and a synthetic peptide, spanning residues 36 to 46 of the protease, has been determined at 2.2 A resolution, and that of the Fab in the free state has been determined at 2.6 A resolution. The refined model of the complex reveals ten well-ordered residues of the peptide (P36 to P45) bound in a hydrophobic cavity at the centre of the antigen-binding site. The peptide adopts a beta hairpin-like structure in which residues P38 to P42 form a type II beta-turn conformation. An intermolecular antiparallel beta-sheet is formed between the peptide and the CDR3-H loop of the antibody; additional polar interactions occur between main-chain atoms of the peptide and hydroxyl groups from tyrosine residues protruding from CDR1-L and CDR3-H. Three water molecules, located at the antigen-antibody interface, mediate polar interactions between the peptide and the most buried hypervariable loops, CDR3-L and CDR1-H. A comparison between the free and complexed Fab fragments shows that significant conformational changes occur in the long hypervariable regions, CDR1-L and CDR3-H, upon binding the peptide. The conformation of the bound peptide, which shows no overall structural similarity to the corresponding segment in HIV-1 protease, suggests that F11.2.32 might inhibit proteolysis by distorting the native structure of the enzyme.     

Activation of thrombin-activable fibrinolysis inhibitor requires epidermal growth factor-like domain 3 of thrombomodulin and is inhibited competitively by protein C

Thrombomodulin is a cofactor protein on vascular endothelial cells that inhibits the procoagulant functions of thrombin and enhances thrombin-catalyzed activation of anticoagulant protein C. Thrombomodulin also accelerates the proteolytic activation of a plasma procarboxypeptidase referred to as thrombin-activable fibrinolysis inhibitor (TAFI). In this study, we describe structures on recombinant membrane-bound thrombomodulin that are required for human TAFI activation. Deletion of the N-terminal lectin-like domain and epidermal growth factor (EGF)-like domains 1 and 2 had no effect on TAFI or protein C activation, whereas deletions including EGF-like domain 3 selectively abolished thrombomodulin cofactor activity for TAFI activation. Provided that thrombomodulin EGF-like domain 3 was present, TAFI competitively inhibited protein C activation catalyzed by the thrombin-thrombomodulin complex. A thrombomodulin construct lacking EGF-like domain 3 functioned normally as a cofactor for protein C activation but was insensitive to inhibition by TAFI. Thus, the anticoagulant and antifibrinolytic cofactor activities of thrombomodulin have distinct structural requirements: protein C binding to the thrombin-thrombomodulin complex requires EGF-like domain 4, whereas TAFI binding also requires EGF-like domain 3.     

Cleavage activity of hepatitis C virus serine proteinase

To study the character of the hepatitis C virus (HCV) encoding serine proteinase and to search for inhibitors, a practical in vitro assay system using the purified enzyme and synthetic peptide substrates was established. The enzyme used was expressed in Escherichia coli as a fusion form with protein tags and purified to apparent homogeneity by single-step affinity chromatography. The purified enzyme exhibited proteolytic activity with pH optima of around eight, and the addition of NS4A fragments increased the activity as well as the thermal stability of the enzyme. The activity was inhibited by EDTA and some divalent ions, i.e., copper and zinc, though calcium, magnesium, and manganese were stimulative both in the presence and absence of the NS4A fragment. None of the common protease inhibitors, including serine protease inhibitors, effectively inhibited the activity. Based on the kinetic parameters of the cleavage reaction of the synthetic 20 mer peptides corresponding to the three cleavage sites, NS4A/4B, NS4B/5A, and NS5A/5B, the peptide with the NS5A/5B junction was found to be the most efficient substrate. Analysis of the minimal peptide substrate of NS5A/5B indicated that 5 to 7 amino acids on both sides of the junction were required for efficient cleavage. These findings are expected to contribute to the search for a proteinase inhibitor.     

The domain structure of human receptor-associated protein. Protease sensitivity and guanidine HCl denaturation

The 39-kDa receptor-associated protein (RAP), a specialized chaperone for endocytic receptors of the low density lipoprotein receptor gene family, is a triplicate repeat sequence (residues 1-100, 101-200, and 201-323, respectively), with the three repeats having different functional roles. The goal of the present study was to use a combination of protease sensitivity and guanidine denaturation analyses to investigate whether human RAP correspondingly contained multiple structural domains. Protease sensitivity analysis using six proteolytic enzymes of varying specificity showed that RAP has two protease-resistant regions contained within repeat 1 (residues 15-94) and repeat 3 (residues 223-323). Guanidine denaturation analysis showed that RAP has two phases in its denaturation, an early denaturation transition at 0.6 M guanidine HCl, and a broad second transition between 1.0 and 3.0 M guanidine HCl. Analysis of the denaturation of the individual repeats showed that, despite the similarity in sequence and protease sensitivity between repeats 1 and 3, repeat 1 was a stable structure, with a sharp transition midpoint at 2.4 M guanidine HCl, while repeat 3 was relatively unstable, with a transition midpoint at 0.6 M guanidine HCl. Repeat 2 had a denaturation profile almost identical to that of repeat 3. Denaturation analysis of the contiguous repeats 1 and 2 (residues 1-210) indicated that repeats 1 and 2 probably interact to form one structural domain represented by the broad transition, while repeat 3 constitutes a separate domain represented by the early transition. A two-domain model of RAP three-dimensional structure is proposed that integrates both structural and functional information, in which a helical segment from repeat 2 interacts with the known three-helix bundle of repeat 1 to form a four-helix bundle structural domain, while repeat 3 forms the other structural domain.