Use of protein unfolding studies to determine the conformational and dimeric stabilities of HIV-1 and SIV proteases.

The free energies of dimer dissociation of the retroviral proteases (PRs) of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) were determined by measuring the effects of denaturants on the protein fluorescence upon the unfolding of the enzymes. HIV-1 PR was more stable to denaturation by chaotropes and extremes of pH and temperature than SIV PR, indicating that the former enzyme has greater conformational stability. The urea unfolding curves of both proteases were sigmoidal and single phase. The midpoints of the transition curves increased with increasing protein concentrations. These data were best described by and fitted to a two-state model in which folded dimers were in equilibrium with unfolded monomers. This denaturation model conforms to cases in which protein unfolding and dimer dissociation are concomitant processes in which folded monomers do not exist [Bowie, J. U., & Sauer, R. T. (1989) Biochemistry 28, 7140-7143]. Accordingly, the free energies of unfolding reflect the stabilities of the protease dimers, which for HIV-1 PR and SIV PR were, respectively, delta GuH2O = 14 +/- 1 kcal/mol (Ku = 39 pM) and 13 +/- 1 kcal/mol (Ku = 180 pM). The binding of a tight-binding, competitive inhibitor greatly stabilized HIV-1 PR toward urea-induced unfolding (delta GuH2O = 19.3 +/- 0.7 kcal/mol, Ku = 7.0 fM). There were also profound effects caused by adverse pH on the protein conformation for both HIV-1 PR and SIV PR, resulting in unfolding at pH values above and below the respective optimal ranges of 4.0-8.0 and 4.0-7.0     

Characterization of human immunodeficiency virus type 1 dimeric RNA from wild-type and protease-defective virions.

We have characterized the dimeric genomic RNA in particles of both wild-type and protease (PR)-deficient human immunodeficiency virus type 1 (HIV-1). We found that the dimeric RNA isolated from PR- mutant virions has a lower mobility in nondenaturing gel electrophoresis than that from wild-type virions. It also dissociates into monomers at a lower temperature than the wild-type dimer. Thus, the dimer in PR- particles is in a conformation different from that in wild-type particles. These results are quite similar to recent findings on Moloney murine leukemia virus and suggest that a postassembly, PR-dependent maturation event is a common feature in genomic RNAs of retroviruses. We also measured the thermal stability of the wild-type and PR- dimeric RNAs under different ionic conditions. Both forms of the dimer were stabilized by increasing Na+ concentrations. However, the melting temperatures of the two forms were not significantly affected by the identity of the monovalent cation present in the incubation buffer. This observation is in contrast with recent reports on dimers formed in vitro from short segments of HIV-1 sequence: the latter dimers are specifically stabilized by K+ ions. K+ stabilization of dimers formed in vitro has been taken as evidence for the presence of guanine quartet structures. The results suggest that guanine quartets are not involved in the structure linking full-length, authentic genomic RNA of HIV-1 into a dimeric structure.     

Activity in monomers of human cytomegalovirus protease.

Herpesvirus proteases require dimerization for activity, although crystallographic data indicate that each monomeric subunit possesses a well-separated and complete active site. This suggests that dimerization stabilizes the monomeric protease subunits in an active conformation. Chemical cross-linking with disuccinimidyl glutarate was used to capture human cytomegalovirus protease in its various conformations. The cross-linked protease retained activity under mildly chaotropic conditions (0.25 to 0.75 M urea) in contrast to non-cross-linked protease which lost activity. Identification of active protease species by incorporation of radioactive diisopropylfluorophosphate showed that in addition to cross-linked dimers, cross-linked protease monomers were responsible for a significant fraction of the total protease activity. These results are consistent with the hypothesis that herpesvirus protease activation occurs by stabilization of an active conformer in the dimer.     

Functional consequences of the Kaposi's sarcoma-associated herpesvirus protease structure: regulation of activity and dimerization by conserved structural elements

The structure of Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr), at 2.2 A resolution, reveals the active-site geometry and defines multiple possible target sites for drug design against a human cancer-producing virus. The catalytic triad of KSHV Pr, (Ser114, His46, and His157) and transition-state stabilization site are arranged as in other structurally characterized herpesviral proteases. The distal histidine-histidine hydrogen bond is solvent accessible, unlike the situation in other classes of serine proteases. As in all herpesviral proteases, the enzyme is active only as a weakly associated dimer (K(d) approximately 2 microM), and inactive as a monomer. Therefore, both the active site and dimer interface are potential targets for antiviral drug design. The dimer interface in KSHV Pr is compared with the interface of other herpesviral proteases. Two conserved arginines (Arg209), one from each monomer, are buried within the same region of the dimer interface. We propose that this conserved arginine may provide a destabilizing element contributing to the tuned micromolar dissociation of herpesviral protease dimers.     

Functional role of proteolytic cleavage at arginine-275 of human tissue plasminogen activator as assessed by site-directed mutagenesis

Activation of the zymogen form of a serine protease is associated with a conformational change that follows proteolysis at a specific site. Tissue-type plasminogen activator (t-PA) is homologous to mammalian serine proteases and contains an apparent activation cleavage site at arginine-275. To clarify the functional consequences of cleavage at arginine-275 of t-PA, site-specific mutagenesis was performed to convert arginine-275 to a glutamic acid. The mutant enzyme (designated Arg-275----Glu t-PA) could be converted to the two-chain form by Staphylococcus aureus V8 protease but not by plasmin. The one-chain form was 8 times less active against the tripeptide substrate H-D-isoleucyl-L-prolyl-L-arginine-p-nitroanilide (S-2288), and the ability of the enzyme to activate plasminogen in the absence of fibrinogen was reduced 20-50 times compared to the two-chain form. In contrast, one-chain Arg-275----Glu t-PA has equal activity to the two-chain form when assayed in the presence of physiological levels of fibrinogen and plasminogen. Fibrin bound significantly more of the one-chain form of t-PA than the two-chain form for both the wild-type and mutated enzymes. One- and two-chain forms of the wild-type and mutated plasminogen activators slowly formed complexes with plasma protease inhibitors, although the one-chain forms showed decreased complex formation with alpha 2-macroglobulin. The one-chain form of t-PA therefore is fully functional under physiologic conditions and has an increased fibrin binding compared to the two-chain form.     

The dimer interfaces of protease and extra-protease domains influence the activation of protease and the specificity of GagPol cleavage

Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation.     

Characterization of an active single polypeptide form of the human immunodeficiency virus type 1 protease

The pepsin-like aspartyl proteases consist of a single polypeptide chain with topologically similar amino- and carboxyl-terminal domains, each of which contributes 1 aspartic acid residue to the active site. This structure has been proposed to have evolved by gene duplication and fusion from a dimeric enzyme composed of two identical polypeptide chains, such as the aspartyl protease (PRT) of human immunodeficiency virus type 1 (HIV-1). To determine if a single polypeptide form of the HIV-1 protease would be enzymatically active, two protease coding regions were linked to form a dimeric gene (pFGGP). Expression of this gene in Escherichia coli yielded a protein with the expected molecular mass of 22 kDa. The in vitro kinetic parameters of PRT and FGGP (where FGGP is the single polypeptide form of the HIV-1 protease with 2 glycine residues connecting the two subunits) for three peptide substrates are similar. Construction and analysis of a CheY-GAG-FGGP fusion protein demonstrated that FGGP is capable of precursor processing in vivo. Mutation of one or both of the active site aspartates to either asparagine or glutamate rendered the enzyme inactive, demonstrating that both active site aspartate residues are required for enzymatic activity.     

Insight into the structural similarity between HIV protease and secreted aspartic protease-2 and binding mode analysis of HIV-Candida albicans inhibitors

The analysis of the structural similarity between Candida albicans Sap2 and HIV-1 aspartic proteases by molecular modeling gave insight into the common requirements for inhibition of both targets. Structure superimposition of Sap2 and HIV-1 protease confirmed the similarity between their active sites and flap regions. HIV-1 protease inhibitors herein investigated can fit the active site of Sap2, adopting very similar ligand-backbone conformations. In particular, key anchoring sites consisting of Gly85 in Sap2 and Ile50 in HIV-1 protease, both belonging to their corresponding flap regions, were found as elements of a similar binding-mode interaction. The knowledge of the molecular basis for binding to both Sap2 and HIV-1 proteases may ultimately lead to the development of single inhibitor acting on both targets.     

Intramolecular regulation of the sequence-specific mRNA interferase activity of MazF fused to a MazE fragment with a linker cleavable by specific proteases

The genomes of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) consist of single-stranded RNA encoding polyproteins, which are processed to individual functional proteins by virus-encoded specific proteases. These proteases have been used as targets for drug development. Here, instead of targeting these proteases to inhibit viral infection, we utilized the protease activity to activate a toxic protein to prevent viral infection. We engineered the MazE-MazF antitoxin-toxin system of Escherichia coli to fuse a C-terminal 41-residue fragment of antitoxin MazE to the N-terminal end of toxin MazF with a linker having a specific protease cleavage site for either HIV PR (HIV-1 protease), NS3 protease (HCV protease), or factor Xa. These fusion proteins formed a stable dimer (instead of the MazF(2)-MazE(2)-MazF(2) heterohexamer in nature) to inactivate the ACA (sequence)-specific mRNA interferase activity of MazF. When the fusion proteins were incubated with the corresponding proteases, the MazE fragment was cleaved from the fusion proteins, releasing active MazF, which then acted as an ACA-specific mRNA interferase cleaving single-stranded MS2 phage RNA. The intramolecular regulation of MazF toxicity by proteases as demonstrated may provide a novel approach for preventive and therapeutic treatments of infection by HIV-1, HCV, and other single-stranded RNA viruses.     

Structural properties of FliH,an ATPase regulatory component of the Salmonella type III flagellar export apparatus

FliH is a regulatory component for FliI, the ATPase that is responsible for driving flagellar protein export in Salmonella. FliH consists of 235 amino acid residues, has a quite elongated shape, exists as a homodimer and together with FliI forms a heterotrimer. Here, we have investigated the structural properties of the FliH homodimer in further detail. Like intact His-tagged FliH homodimer, fragment His-FliH(N2) (consisting of the first 102 amino acid residues of FliH), exhibited anomalous elution behavior in gel filtration chromatography; the same was true of His-FliH(C1) (consisting of amino acid residues 119-235), but to a much lesser degree. Thus the elongated shape of FliH appears to derive primarily from its N-terminal region. A deletion version of N-His-FliH, lacking amino acid residues 101-140, does not dimerize and so we were able to establish the gel filtration properties of an almost full-size monomeric form; it also exhibited anomalous elution behavior. We performed trypsin proteolysis of the FliH homodimer and subjected the cleavage products to gel filtration chromatography. FliH was degraded by trypsin and a contaminating protease into two stable fragments: FliH(Prt1) (missing both the first ten and the last 12 amino acid residues), and FliH(Prt2) (missing both the first ten and the last 63 amino acid residues); however, substantial amounts remained undigested even after 24 hours. Under native conditions, both FliH(Prt1) and FliH(Prt2) co-eluted with undigested His-FliH from the gel filtration column, indicating that the fragments exist as a hybrid dimer with intact FliH. These results suggest that the two subunits within the dimer differ in their proteolytic susceptibility. No heterotrimer was observed by gel filtration chromatography when His-FliI was mixed with either His-FliH/FliH(Prt1) or His-FliH/FliH(Prt2) hybrid dimers. A hybrid dimer of FliH and His-FliHDelta1 (lacking the first ten amino acid residues) retained the ability to form a complex with His-FliI. In contrast, hybrid dimers consisting of FliH and either His-FliH(W223ochre) or His-FliH(V172ochre) failed to complex to His-FliI, demonstrating that the C-terminal region of both FliH monomers within the FliH dimer are required for heterotrimer formation.     

One functional switch mediates reversible and irreversible inactivation of a herpesvirus protease

Distinct mechanisms have evolved to regulate the function of proteolytic enzymes. Viral proteases in particular have developed novel regulatory mechanisms, presumably due to their comparatively rapid life cycles and responses to constant evolutionary pressure. Herpesviruses are a family of human pathogens that require a viral protease with a concentration-dependent zymogen activation involving folding of two alpha-helices and activation of the catalytic machinery, which results in formation of infectious virions. Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) is unique among the herpesvirus proteases in possessing an autolysis site in the dimer interface, which removes the carboxyl-terminal 27 amino acids comprising an alpha-helix adjacent to the active site. Truncation results in the irreversible loss of dimerization and concomitant inactivation. We characterized the conformational and functional differences between the active dimer, inactive monomer, and inactive truncated protease to determine the different protease regulatory mechanisms that control the KSHV lytic cycle. Circular dichroism revealed a loss of 31% alpha-helicity upon dimer dissociation. Comparison of the full-length and truncated monomers by NMR showed differences in 21% of the protein structure, mainly located adjacent to the dimer interface, with little perturbation of the overall protein upon truncation. Fluorescence polarization and active site labeling, with a transition state mimetic, characterized the functional effects of these conformational changes. Substrate turnover is abolished in both the full-length and truncated monomers; however, substrate binding remained intact. Disruption of the helix 6 interaction with the active site oxyanion loop is therefore used in two independent regulatory mechanisms of proteolytic activity.     

Biological activity of different forms of bacteriophage lambda DNA]

Hershey circles and linear tandem aggregated forms of DNA have been obtained in vitro and treated with polynucleotide ligase to form phosphodiester bond. Using zone centrifugation in glycerol gradient covalently closed circles and linear dimers have been purified and their biological activity investigated. It was found that closed circular molecules lost most, if not all, of their activity in CaCl2-dependent system. In order to investigate the biological activity of tandem dimer molecules, hybrid dimers consisting of DNA's from lambda C1857 and lambda 1434 have been obtained. In plaque assay with the appropriate non-permissive strains of E. coli the efficiency of infectivity of hybrid dimers was measured. Biological activity of dimer molecules sealed with ligase was about 5% of the activity of linear monomers. Ig has been suggested that tandem dimers of lambda DNA joined by phosphodiester bond are able to penetrate into the CaCl2-treated host cells and both components of dimers are active during subsequent multiplication.     

Solution structure of the mature HIV-1 protease monomer: insight into the tertiary fold and stability of a precursor

We present the first solution structure of the HIV-1 protease monomer spanning the region Phe1-Ala95 (PR1-95). Except for the terminal regions (residues 1-10 and 91-95) that are disordered, the tertiary fold of the remainder of the protease is essentially identical to that of the individual subunit of the dimer. In the monomer, the side chains of buried residues stabilizing the active site interface in the dimer, such as Asp25, Asp29, and Arg87, are now exposed to solvent. The flap dynamics in the monomer are similar to that of the free protease dimer. We also show that the protease domain of an optimized precursor flanked by 56 amino acids of the N-terminal transframe region is predominantly monomeric, exhibiting a tertiary fold that is quite similar to that of PR1-95 structure. This explains the very low catalytic activity observed for the protease prior to its maturation at its N terminus as compared with the mature protease, which is an active stable dimer under identical conditions. Adding as few as 2 amino acids to the N terminus of the mature protease significantly increases its dissociation into monomers. Knowledge of the protease monomer structure and critical features of its dimerization may aid in the screening and design of compounds that target the protease prior to its maturation from the Gag-Pol precursor.     

A range of catalytic efficiencies with avian retroviral protease subunits genetically linked to form single polypeptide chains.

Molecular modeling based on the crystal structure of the Rous sarcoma virus (RSV) protease dimer has been used to link the two identical subunits of this enzyme into a functional, single polypeptide chain resembling the nonviral aspartic proteases. Six different linkages were selected to test the importance of different interactions between the amino acids at the amino and carboxyl termini of the two subunits. These linkages were introduced into molecular clones of fused protease genes and the linked protease dimers were expressed in Escherichia coli and purified. Catalytically active proteins were obtained from the inclusion body fraction after renaturation. The linked protease dimers exhibited a 10-20-fold range in catalytic efficiencies (Vmax/Km) on peptide substrates. Both flexibility and ionic interactions in the linkage region affect catalytic efficiency. Some of the linked protease dimers were 2-3-fold more active than the nonlinked enzyme purified from bacteria, although substrate specificities were unchanged. Similar relative efficiencies were observed using a polyprotein precursor as substrate. Mutation of one catalytic Asp in the most active linked protease dimer inactivated the enzyme, demonstrating that these proteins function as single polypeptide chains rather than as multimers.     

Disruption of the HIV-1 protease dimer with interface peptides: structural studies using NMR spectroscopy combined with [2-(13)C]-Trp selective labeling

HIV-1 protease (HIV-1 PR), which is encoded by retroviruses, is required for the processing of gag and pol polyprotein precursors, hence it is essential for the production of infectious viral particles. In vitro inhibition of the enzyme results in the production of progeny virions that are immature and noninfectious, suggesting its potential as a therapeutic target for AIDS. Although a number of potent protease inhibitor drugs are now available, the onset of resistance to these agents due to mutations in HIV-1 PR has created an urgent need for new means of HIV-1 PR inhibition. Whereas enzymes are usually inactivated by blocking of the active site, the structure of dimeric HIV-1 PR allows an alternative inhibitory mechanism. Since the active site is formed by two half-enzymes, which are connected by a four-stranded antiparallel beta-sheet involving the N- and C- termini of both monomers, enzyme activity can be abolished by reagents targeting the dimer interface in a region relatively free of mutations would interfere with formation or stability of the functional HIV-1 PR dimer. This strategy has been explored by several groups who targeted the four-stranded antiparallel beta-sheet that contributes close to 75% of the dimerization energy. Interface peptides corresponding to native monomer N- or C-termini of several of their mimetics demonstrated, mainly on the basis of kinetic analyses, to act as dimerization inhibitors. However, to the best of our knowledge, neither X-ray crystallography nor NMR structural studies of the enzyme-inhibitor complex have been performed to date. In this article we report a structural study of the dimerization inhibition of HIV-1 PR by NMR using selective Trp side chain labeling.     

Crystal structure of a cross-reaction complex between an anti-HIV-1 protease antibody and an HIV-2 protease peptide

The monoclonal antibody 1696, elicited by HIV-1 protease, inhibits the activity of both HIV-1 and HIV-2 proteases with inhibition constants in the low nanomolar range. The antibody cross-reacts with peptides derived from the N-terminal region of both proteases. The crystal structure of the recombinant single-chain Fv fragment of 1696 complexed with an N-terminal peptide from the HIV-2 protease has been determined at 1.88A resolution. Interactions of the peptide with scFv1696 are compared with the previously reported structure of scFv1696 in complex with the corresponding peptide from HIV-1 protease. The origin of cross-reactivity of mAb1696 with HIV proteases is discussed.     

Only one protomer is active in the dimer of SARS 3C-like proteinase

The severe acute respiratory syndrome coronavirus 3C-like protease has been proposed to be a key target for structurally based drug design against SARS. The enzyme exists as a mixture of dimer and monomer, and only the dimer was considered to be active. In this report, we have investigated, using molecular dynamics simulation and mutational studies, the problems as to why only the dimer is active and whether both of the two protomers in the dimer are active. The molecular dynamics simulations show that the monomers are always inactive, that the two protomers in the dimer are asymmetric, and that only one protomer is active at a time. The enzyme activity of the hybrid severe acute respiratory syndrome coronavirus 3C-like protease of the wild-type protein and the inactive mutant proves that the dimerization is important for enzyme activity and only one active protomer in the dimer is enough for the catalysis. Our simulations also show that the right conformation for catalysis in one protomer can be induced upon dimer formation. These results suggest that the enzyme may follow the association, activation, catalysis, and dissociation mechanism for activity control.     

Synthetic "interface" peptides alter dimeric assembly of the HIV 1 and 2 proteases.

Retroviral proteases are obligate homodimers and play an essential role in the viral life cycle. Dissociation of dimers or prevention of their assembly may inactivate these enzymes and prevent viral maturation. A salient structural feature of these enzymes is an extended interface composed of interdigitating N- and C-terminal residues of both monomers, which form a four-stranded beta-sheet. Peptides mimicking one beta-strand (residues 95-99), or two beta-strands (residues 1-5 plus 95-99 or 95-99 plus 95-99) from the human immunodeficiency virus 1 (HIV1) interface were shown to inhibit the HIV1 and 2 proteases (PRs) with IC50's in the low micromolar range. These interface peptides show cognate enzyme preference and do not inhibit pepsin, renin, or the Rous sarcoma virus PR, indicating a degree of specificity for the HIV PRs. A tethered HIV1 PR dimer was not inhibited to the same extent as the wild-type enzymes by any of the interface peptides, suggesting that these peptides can only interact effectively with the interface of the two-subunit HIV PR. Measurements of relative dissociation constants by limit dilution of the enzyme show that the one-strand peptide causes a shift in the observed Kd for the HIV1 PR. Both one- and two-strand peptides alter the monomer/dimer equilibrium of both HIV1 and HIV2 PRs. This was shown by the reduced cross-linking of the HIV2 PR by disuccinimidyl suberate in the presence of the interface peptides. Refolding of the HIV1 and HIV2 PRs with the interface peptides shows that only the two-strand peptides prevent the assembly of active PR dimers. Although both one- and two-strand peptides seem to affect dimer dissociation, only the two-strand peptides appear to block assembly. The latter may prove to be more effective backbones for the design of inhibitors directed toward retroviral PR dimerization in vivo.