ENDOR structural characterization of a catalytically competent acylenzyme reaction intermediate of wild-type TEM-1 beta-lactamase confirms glutamate-166 as the base catalyst

The catalytically competent active-site structure of a true acylenzyme reaction intermediate of TEM-1 beta-lactamase formed with the kinetically specific spin-labeled substrate 6-N-(2,2,5,5-tetramethyl-1-oxypyrrolinyl-3-carboxyl)-penicillanic acid isolated under cryoenzymologic conditions has been determined by angle-selected electron nuclear double resonance (ENDOR) spectroscopy. Cryoenzymologic experiments with use of the chromophoric substrate 6-N-[3-(2-furanyl)-propen-2-oyl]-penicillanic acid showed that the acylenzyme reaction intermediate could be stabilized in the -35 to -75 degrees C range with a half-life suitably long to allow freeze-quenching of the reaction species for ENDOR studies while a noncovalent Michaelis complex could be optically identified at temperatures only below -70 degrees C. The wild-type, Glu166Asn, Glu240Cys, and Met272Cys mutant forms of the mature enzyme were overexpressed in perdeuterated minimal medium to allow detection and assignment of proton resonances specific for the substrate and chemically modified amino acid residues in the active site. From analysis of the dependence of the ENDOR spectra on the setting of the static laboratory magnetic field H0, the dipolar contributions to the principal hyperfine coupling components were estimated to calculate the separations between the unpaired electron of the nitroxyl group and isotopically identified nuclei. These electron-nucleus distances were applied as constraints to assign the conformation of the substrate in the active site and of amino acid side chains by molecular modeling. Of special interest was that the ENDOR spectra revealed a water molecule sequestered in the active site of the acylenzyme of the wild-type protein that was not detected in the deacylation impaired Glu166Asn mutant. On the basis of the X-ray structure of the enzyme, the ENDOR distance constraints placed this water molecule within hydrogen-bonding distance to the carboxylate side chain of glutamate-166 as if it were poised for nucleophilic attack of the scissile ester bond. The ENDOR results provide experimental evidence of glutamate-166 in its functional role as the general base catalyst in the wild-type enzyme for hydrolytic breakdown of the acylenzyme reaction intermediate of TEM-1 beta-lactamase.     

alpha-lytic protease precursor: characterization of a structured folding intermediate

The bacterial alpha-lytic protease (alphaLP) is synthesized as a precursor containing a large N-terminal pro region (Pro) transiently required for correct folding of the protease [Silen, J. L., and Agard, D. A. (1989) Nature 341, 462-464]. Upon folding, the precursor is autocatalyticly cleaved to yield a tight-binding inhibitory complex of the pro region and the fully folded protease (Pro/alphaLP). An in vitro purification and refolding protocol has been developed for production of the disulfide-bonded precursor. A combination of spectroscopic approaches have been used to compare the structure and stability of the precursor with either the Pro/alphaLP complex or isolated Pro. The precursor and complex have significant similarities in secondary structure but some differences in tertiary structure, as well as a dramatic difference in stability. Correlations with isolated Pro suggest that the pro region part of the precursor is fully folded and acts to stabilize and structure the alphaLP region. Precursor folding is shown to be biphasic with the fast phase matching the rate of pro region folding. Further, the rate-limiting step in oxidative folding is formation of the disulfide bonds and autocatalytic processing occurs rapidly thereafter. These studies suggests a model in which the pro region folds first and catalyzes folding of the protease domain, forming the active site and finally causing autocatalytic cleavage of the bond separating pro region and protease. This last processing step is critical as it allows the protease N-terminus to rearrange, providing the majority of net stabilization of the product Pro/alphaLP complex.     

Kinetic and thermodynamic characterization of HIV-1 protease inhibitors

Interaction kinetic and thermodynamic analyses provide information beyond that obtained in general inhibition studies, and may contribute to the design of improved inhibitors and increased understanding of molecular interactions. Thus, a biosensor-based method was used to characterize the interactions between HIV-1 protease and seven inhibitors, revealing distinguishing kinetic and thermodynamic characteristics for the inhibitors. Lopinavir had fast association and the highest affinity of the tested compounds, and the interaction kinetics were less temperature-dependent as compared with the other inhibitors. Amprenavir, indinavir and ritonavir showed non-linear temperature dependencies of the kinetics. The free energy, enthalpy and entropy (DeltaG, DeltaH, DeltaS) were determined, and the energetics of complex association (DeltaG(on), DeltaH(on), DeltaS(on)) and dissociation (DeltaG(off), DeltaH(off), DeltaS(off)) were resolved. In general, the energetics for the studied inhibitors was in the same range, with the negative free energy change (DeltaG < 0) due primarily to increased entropy (DeltaS > 0). Thus, the driving force of the interaction was increased degrees of freedom in the system (entropy) rather than the formation of bonds between the enzyme and inhibitor (enthalpy). Although the DeltaG(on) and DeltaG(off) were in the same range for all inhibitors, the enthalpy and entropy terms contributed differently to association and dissociation, distinguishing these phases energetically. Dissociation was accompanied by positive enthalpy (DeltaH(off) > 0) and negative entropy (DeltaS(off) < 0) changes, whereas association for all inhibitors except lopinavir had positive entropy changes (DeltaS(on) > 0), demonstrating unique energetic characteristics for lopinavir. This study indicates that this type of data will be useful for the characterization of target-ligand interactions and the development of new inhibitors of HIV-1 protease.     

Generation and characterization of a human immunodeficiency virus type 1 (HIV-1) mutant resistant to an HIV-1 protease inhibitor.

A synthetic peptide, RPI 312, that specifically inhibits the protease of the human immunodeficiency virus type 1 (HIV-1) showed a potent inhibition on virus production, maturation, and infectivity. Treatment with this agent prevented the cleavage of Gag protein at the site between p17 and p24 in HIV-1 chronically infected MOLT-4 cells as well as in the released virus. Passage of HIV-1 in the presence of gradually increasing concentrations of this protease inhibitor resulted in emergence of a variant that could evade the drug effects. In the resistant variant the maturation of Gag proteins appeared normal, but its infectivity was reduced compared with that of the parent virus. The nucleotides coding the amino acids at and around the cleavage site between Gag proteins p17 and p24 were not changed. One point mutation (A-->G) at site 2082 of the pol gene that resulted in one amino acid change at site 84 of the protease from isoleucine to valine (I-84-->V) could be detected in the resistant variant. An HIV-1 infectious DNA clone with the I-84-->V mutation also showed reduced sensitivity to this protease inhibitor. The findings that the resistant variant had lower infectivity and was still affected by higher doses of the drug support the speculation that resistance to protease inhibitors may not be as problematic as other drug resistance.     

Purification of HIV-1 wild-type protease and characterization of proteolytically inactive HIV-1 protease mutants by pepstatin A affinity chromatography

Recombinant wild-type protease of human immunodeficiency virus, type [(HIV-1) expressed in E. coli was purified by pepstatin A affinity chromatography. An 88-fold purification was achieved giving a protease preparation with a specific enzymatic activity of approximately 3700 pmol/min/μg. Two proteolytically inactive HIV-1 mutant proteases (Arg-87 → Lys; Asn-88 → Glu) were found to bind to pepstatin A agarose, and they were purified as the wild-type protease. A third mutant protease (Arg-87 → Glu) was apparently unable to bind to pepstatin A under similar conditions. Binding to pepstatin A indicates the binding ability of the substrate binding site and the ability to form dimers. These features may be used to purify and to characterize other mutated HIV-1 proteases.     

Purification of recombinant HIV-1 protease.

A method is described to purify recombinant HIV-1 protease from soluble extracts of Escherichia coli. The isolation involves QAE-Sepharose anion exchange chromatography, hexyl agarose hydrophobic interaction chromatography, MonoS cation exchange chromatography, and Superose 6 size exclusion chromatography. Approximately 100 micrograms of protease was obtained from 18 g E. coli paste. The protein was judged to be homogeneous due to the presence of a single band on a silver-stained SDS polyacrylamide gel.     

Molecular dynamics of HIV-1 protease.

Molecular dynamics simulations have been carried out based on the GROMOS force field on the aspartyl protease (PR) of the human immunodeficiency virus HIV-1. The principal simulation treats the HIV-1 PR dimer and 6990 water molecules in a hexagonal prism cell under periodic boundary conditions and was carried out for a trajectory of 100 psec. Corresponding in vacuo simulations, i.e., treating the isolated protein without solvent, were carried out to study the influence of solvent on the simulation. The results indicate that including waters explicitly in the simulation results in a model considerably closer to the crystal structure than when solvent is neglected. Detailed conformational and helicoidal analysis was performed on the solvated form to determine the exact nature of the dynamical model and the exact points of agreement and disagreement with the crystal structure. The calculated dynamical model was further elucidated by means of studies of the time evolution of the cross-correlation coefficients for atomic displacements of the atoms comprising the protein backbone. The cross-correlation analysis revealed significant aspects of structure originating uniquely in the dynamical motions of the molecule. In particular, an unanticipated through-space, domain-domain correlation was found between the mobile flap region covering the active site and a remote regions of the structure, which collectively act somewhat like a molecular cantilever. The significance of these results is discussed with respect to the inactivation of the protease by site-specific mutagenesis, and in the design of inhibitors.     

Characterization of the NiFeCO complex of carbon monoxide dehydrogenase as a catalytically competent intermediate in the pathway of acetyl-coenzyme A synthesis.

Many anaerobic bacteria fix CO2 via the acetyl-CoA pathway. Carbon monoxide dehydrogenase (CODH), a key enzyme in the pathway, condenses a methyl group, a carbonyl group from CO, CO2, or the carboxyl group of pyruvate, and CoA to form acetyl-CoA. When treated with CO, CODH exhibits an EPR signal which results from an organometallic complex containing nickel, at least 3 iron, and CO and has been referred to as the NiFeC signal. Although this EPR signal has been presumed to be the spectroscopic signature of the enzyme-bound C-1 precursor of the carbonyl group of acetyl-CoA, its catalytic relevance had not been rigorously studied. We have demonstrated the catalytic competence of this NiFeC species by showing that the rate of formation of the NiFeC EPR signal is faster than the rate of an isotope exchange reaction between CO and acetyl-CoA, a partial reaction in the overall synthesis. Generation of the NiFeC signal in the absence of CO by acetyl-CoA has been demonstrated and requires a one-electron reduction at a midpoint potential of -541 mV versus the standard hydrogen electrode. In addition, we have observed and characterized an isotope exchange reaction between the carbonyl group of acetyl-CoA and the carbonyl group of the NiFeC complex, indicating that the C in the NiFeC complex is in the form of CO. These combined results demonstrate that the NiFeCO complex exhibits the characteristics expected of the precursor of the carbonyl group of acetyl-CoA.     

Adaptability and flexibility of HIV-1 protease.

Even though more than 200 three-dimensional structures of HIV-1 protease complexed to a variety of inhibitors are available in the Protein Data Bank; very few structures of unliganded protein have been determined. We have recently solved structures of unliganded HIV-1 protease tethered dimer mutants to resolutions of 1.9 A and 2.1 A, and have found that the flaps assume closed-flap conformation even in the absence of any bound ligand. We report comparison of the unliganded closed-flap structure with structures of HIV-1 protease inhibitor complexes with a view to accurately identifying structural changes that the ligand can induce on binding to HIV-1 protease in the crystal. These studies reveal that the least flexible region present in the active site of HIV-1 protease need not also be the least adaptable to external stress, thus highlighting the conceptual difference between flexibility and adaptability of proteins in general.     

Characterization of a folding intermediate from HIV-1 ribonuclease H.

The RNase H domain from HIV-1 (HIV RNase H) encodes an essential retroviral activity. Refolding of the isolated HIV RNase H domain shows a kinetic intermediate detectable by stopped-flow far UV circular dichroism and pulse-labeling H/D exchange. In this intermediate, strands 1, 4, and 5 as well as helices A and D appear to be structured. Compared to its homolog from Escherichia coli, the rate limiting step in refolding of HIV RNase H appears closer to the native state. We have modeled this kinetic intermediate using a C-terminal deletion fragment lacking helix E. Like the kinetic intermediate, this variant folds rapidly and shows a decrease in stability. We propose that inhibition of the docking of helix E to this folding intermediate may present a novel strategy for anti HIV-1 therapy.     

Overexpression and characterization of carboxyl-terminal processing protease for precursor D1 protein: regulation of enzyme-substrate interaction by molecular environments

CtpA, which is classified as a novel type of serine protease with a Ser/Lys catalytic dyad, is responsible for the C-terminal processing of precursor D1 protein (pD1) of the photosystem II reaction center, a process that is indispensable for the integration of water-splitting machinery in photosynthesis. In this study, overexpression in Escherichia coli and one-step purification of spinach CtpA were carried out to analyze the characteristics of this new type of protease and to elucidate the molecular interactions in the C-terminal processing of pD1 on the thylakoid membrane. The successful accumulation of functional CtpA in E. coli may argue against the possibility, based on homology to E. coli Tsp, that the enzyme is involved in the degradation of incomplete proteins in chloroplasts, e.g. by utilizing the ssrA-tagging system. Analysis using a synthetic pD1 oligopeptide demonstrated that the enzymatic properties (including substrate recognition) of overexpressed CtpA with an extra sequence of GSHMLE at the N terminus were indistinguishable from those of the native enzyme. CtpA was insensitive to penem, which has been shown to inhibit some Ser/Lys-type proteases, suggesting that the catalytic center of CtpA is quite unique. By using the substrate in different molecular environments (i.e. synthetic pD1 oligopeptide in solution and pD1 in photosystem II-enriched thylakoid membrane), we observed a dramatic difference in the pH profile and affinity for the substrate, suggesting the presence of a specific interaction of CtpA with a factor(s) that modulates the pH dependence of proteolytic action in response to physiological conditions.     

Bacterial luciferase: demonstration of a catalytically competent altered conformational state following a single turnover

Ziegler-Nicoli et al. [Ziegler-Nicoli, M., Meighen, E. A., & Hastings, J. W. (1974) J. Biol. Chem. 249, 2385-2392] reported that a highly reactive cysteinyl residue on the alpha subunit of bacterial luciferase resides in or near the flavin binding site such that the enzyme-flavin complex is protected from inactivation by alkylating reagents. These authors also observed that injection of reduced flavin mononucleotide (FMNH2) into an air-equilibrated solution of enzyme protected the enzyme from alkylation for much longer than the lifetime of the 4a-peroxydihydroflavin intermediate resulting from reaction of enzyme-bound FMNH2 with O2. Two related explanations were offered: either the product flavin mononucleotide dissociated from the enzyme much more slowly following a catalytic cycle than would be predicted from the Kd measured by equilibrium binding or the enzyme itself, without bound flavin, was in an altered conformational state in which the thiol was less reactive following a catalytic cycle. Either explanation involves a slow return of the enzyme to its initial state following a catalytic cycle. We have investigated this phenomenon in more detail and found that rapid removal of the flavin from the enzyme by chromatography following catalytic turnover did not return the enzyme to its original state of susceptibility to either alkylating reagents or proteolytic enzymes. The flavin-free enzyme returned to the susceptible conformation with a half-time of ca. 25 min at 0 degree C. Inactivation of the enzyme at intermediate times of relaxation by either a proteolytic enzyme or an alkylating reagent showed biphasic kinetics, indicative of a mixture of the protected and susceptible forms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comprehensive mutagenesis of HIV-1 protease: a computational geometry approach

A computational geometry technique based on Delaunay tessellation of protein structure, represented by C(alpha) atoms, is used to study effects of single residue mutations on sequence-structure compatibility in HIV-1 protease. Profiles of residue scores derived from the four-body statistical potential are constructed for all 1881 mutants of the HIV-1 protease monomer and compared with the profile of the wild-type protein. The profiles for an isolated monomer of HIV-1 protease and the identical monomer in a dimeric state with an inhibitor are analyzed to elucidate changes to structural stability. Protease residues shown to undergo the greatest impact are those forming the dimer interface and flap region, as well as those known to be involved in inhibitor binding.     

Reversible S-nitrosation and inhibition of HIV-1 protease.

Nitric oxide (*NO) is a short-lived free radical with many functions including vasoregulation, synaptic plasticity, and immune modulation and has recently been associated with AIDS pathology. Various pathophysiological conditions, such as viral infection, trigger inducible nitric oxide synthase (iNOS) to synthesize NO in the cell. NO-derived species can react with thiols of proteins and form nitrosothiol adducts. HIV-1 protease (HIV-PR) contains two cysteine residues, Cys67 and Cys95, which are believed to serve a regulatory function. We have found that HIV-PR is inactivated by nitric oxide produced in vitro by NO donors and by iNOS. Sodium nitroprusside inhibited HIV-PR by 70%, and S-nitroso-N-acetylpenicillamine completely inhibited the enzyme. Furthermore, iNOS generated sufficient NO to inhibit HIV-PR activity by almost 90%. This inactivation was reversed by the addition of reducing agents. Treatment of HIV-PR with NO donors and ritonavir (a competitive peptide inhibitor) indicates that NO exerts its effect through a site independent of the active site of HIV-PR. Using electrospray ionization mass spectrometry, we found that NO forms S-nitrosothiols on Cys67 and Cys95 of HIV-PR which directly correlate with a loss of activity. These data indicate that NO may suppress HIV-1 replication by directly inhibiting HIV-PR.     

Lipopeptides as dimerization inhibitors of HIV-1 protease.

In AIDS therapy, attempts have been made to inhibit the virus-encoded enzymes, e.g. HIV-1 protease, using active site-directed inhibitors. This approach is questionable, however, due to virus mutations and the high toxicity of the drugs. An alternative method to inhibit the dimeric HIV protease is the targeting of the interface region of the protease subunits in order to prevent subunit dimerization and enzyme activity. This approach should be less prone to inactivation by mutation. A list of improved 'dimerization inhibitors' of HIV-1 protease is presented. The main structural features are a short 'interface' peptide segment, including non-natural amino acids, and an aliphatic N-terminal blocking group. The high inhibitory power of some of the lipopeptides [e.g. palmitoyl-Tyr-Glu-Leu-OH, palmitoyl-Tyr-Glu-(L-thyronine)-OH, palmitoyl-Tyr-Glu-(L-biphenyl-alanine)-OH] with low nanomolar Ki values in the enzyme test suggests that mimetics with good bio-availability can be derived for AIDS therapy.     

Synthetic HIV-2 protease cleaves the GAG precursor of HIV-1 with the same specificity as HIV-1 protease

A 99-amino acid protein having the deduced sequence of the protease from human immunodeficiency virus type 2 (HIV-2) was synthesized by the solid phase method and tested for specificity. The folded peptide catalyzes specific processing of a recombinant 43-kDa GAG precursor protein (F-16) of HIV-1. Although the protease of HIV-2 shares only 48% amino acid identity with that of HIV-1, the HIV-2 enzyme exhibits the same specificity toward the HIV-1 GAG precursor. Fragments of 34, 32, 24, 10, and 9 kDa were generated from F-16 GAG incubated with the protease. N-terminal amino acid sequence analysis of proteolytic fragments indicate that cleavage sites recognized by HIV-2 protease are identical to those of HIV-1 protease. The verified cleavage sites in F-16 GAG appear to be processed independently, as indicated by the formation of the intermediate fragments P32 and P34 in nearly equal ratios. The site nearest the amino terminus is quite conserved between the two viral GAG proteins (...VSQNY-PIVQN...in HIV-1,...KGGNY-PVQHV...in HIV-2). In contrast, the putative second site (...IPFAA-AQQKG...) of HIV-2 GAG shares minimal sequence identity with site 2 of HIV-1 GAG (...SATIM-MQRGN...). These sequence variations in the substrates suggest higher order structural features that may influence recognition by the proteases. Pepstatin A inhibits HIV-2 protease, whereas 1,10-phenanthroline and phenylmethylsulfonylfluoride do not; these results are in agreement with the finding that proteases of HIV and other retroviruses are aspartyl proteases.     

Dimerization inhibitors of HIV-1 protease

By targeting the highly conserved antiparallel beta-sheet formed by the interdigitation of the N- and C-terminal strands of each monomer, dimerization inhibitors of HIV-1 protease may be useful to overcome the drug resistance observed with current active-site directed antiproteases. Sequestration of the monomer by the inhibitor (or disruption of the dimer interface) prevents the correct assembly of the inactive monomers to active enzyme. Strategies for the design of drugs targeting the dimer interface are described. Various dimerization inhibitors are reported including N- and C-terminal mimetics, lipopeptides and cross-linked interface peptides.