A new topological method to measure protein structure similarity

A method for the quantitative evaluation of structural similarity between protein pairs is developed that makes use of a Delaunay-based topological mapping. The result of the mapping is a three-dimensional array which is representative of the global structural topology and whose elements can be used to construe an integral scoring scheme. This scoring scheme was tested for its dependence on the protein length difference in a pairwise comparison, its ability to provide a reasonable means for structural similarity comparison within a family of structural neighbors of similar length, and its sensitivity to the differences in protein conformation. It is shown that such a topological evaluation of similarity is capable of providing insight into these points of interest. Protein structure comparison using the method is computationally efficient and the topological scores, although providing different information about protein similarity, correlate well with the distance root-mean-square deviation values calculated by rigid-body structural alignment.     

Knowledge-based computational mutagenesis for predicting the disease potential of human non-synonymous single nucleotide polymorphisms

Certain genetic variations in the human population are associated with heritable diseases, and single nucleotide polymorphisms (SNPs) represent the most common form of such differences in DNA sequence. In particular, substantial interest exists in determining whether a non-synonymous SNP (nsSNP), leading to a single residue replacement in the translated protein product, is neutral or disease-related. The nature of protein structure-function relationships suggests that nsSNP effects, either benign or leading to aberrant protein function possibly associated with disease, are dependent on relative structural changes introduced upon mutation. In this study, we characterize a representative sampling of 1790 documented neutral and disease-related human nsSNPs mapped to 243 diverse human protein structures, by quantifying environmental perturbations in the associated proteins with the use of a computational mutagenesis methodology that relies on a four-body, knowledge-based, statistical contact potential. These structural change data are used as attributes to generate a vector representation for each nsSNP, in combination with additional features reflecting sequence and structure of the corresponding protein. A trained model based on the random forest supervised classification algorithm achieves 76% cross-validation accuracy. Our classifier performs at least as well as other methods that use significantly larger datasets of nsSNPs for model training, and the novelty of our attributes differentiates the model as an orthogonal approach that can be utilized in conjunction with other techniques. A dedicated server for obtaining predictions, as well as supporting datasets and documentation, is available at http://proteins.gmu.edu/automute.     

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.     

Flap opening dynamics in HIV-1 protease explored with a coarse-grained model

We present a one-bead coarse-grained model that enables dynamical simulations of proteins on the time scale of tens of microseconds. The parameterization of the force field includes accurate conformational terms that allow for fast and reliable exploration of the configurational space. The model is applied to the dynamics of flap opening in HIV-1 protease. The experimental structure of the recently crystallized semi-open conformation of HIV-1 protease is well reproduced in the simulation, which supports the accuracy of our model. Thanks to very long simulations and extensive sampling of opening and closing events, we also investigate the thermodynamics and kinetics of the opening process. We have shown that the effect of the solvent slows down the dynamics to the experimentally observed time scales. The model is found to be reliable for application to substrate docking simulations, which are currently in progress.     

Molecular dynamics studies on HIV-1 protease: a comparison of the flap motions between wild type protease and the M46I/G51D double mutant

The emergence of drug-resistant mutants of HIV-1 is a tragic effect associated with conventional long-treatment therapies against acquired immunodeficiency syndrome. These mutations frequently involve the aspartic protease encoded by the virus; knowledge of the molecular mechanisms underlying the conformational changes of HIV-1 protease mutants may be useful in developing more effective and longer lasting treatment regimes. The flap regions of the protease are the target of a particular type of mutations occurring far from the active site. These mutations modify the affinity for both substrate and ligands, thus conferring resistance. In this work, molecular dynamics simulations were performed on a native wild type HIV-1 protease and on the drug-resistant M46I/G51D double mutant. The simulation was carried out for a time of 3.5 ns using the GROMOS96 force field, with implementation of the SPC216 explicit solvation model. The results show that the flaps may exist in an ensemble of conformations between a “closed” and an “open” conformation. The behaviour of the flap tips during simulations is different between the native enzyme and the mutant. The mutation pattern leads to stabilization of the flaps in a semi-open configuration.     

结合分子相似性、药效团和分子对接筛选新的HIV-1蛋白酶抑制剂

结合分子相似性、药效团和分子对接建立兼顾计算效率和预测准确度的HIV-1蛋白酶抑制剂筛选方法。首先通过对现有HIV-1蛋白酶抑制剂分子进行相似性分析,选取代表性的HIV-1蛋白酶抑制剂作为模板分子,构建和优化药效团模型,并从1万个化合物中优先筛选出500个化合物。而后采用分子对接方法进一步考察化合物与HIV-1蛋白酶结合情况,得到4个新的活性候选化合物,并进行其结合自由能计算和抗突变性分析。结果表明新候选化合物ST025723和HIV-1蛋白酶表现出较好的相互作用和抗突变性,具有深入研究的价值,同时也证明分子相似性、药效团和分子对接相结合能够快速有效地发现新颖活性候选化合物。     

Analysis of the structure of HIV-1 protease complexed with a hexapeptide inhibitor. Part II: Molecular dynamic studies of the active site region

Six models of the catalytic site of HIV-1 protease complexed with a reduced peptide inhibitor, MVT-101, were investigated. These studies focused on the details of protonation of the active site, its total net charge and hydrogen bonding pattern, which was consistent with both the observed coplanar configuration of the acidic groups of the catalytic aspartates (Asp-25 and Asp-125) and the observed binding mode of the inhibitor. Molecular dynamic simulations using AMBER 4.0 indicated that the active site should be neutral. The planarity of the aspartate dyad may be due to the formation of two hydrogen bonds: one between the inner O_δ1oxygen atoms of the two catalytic aspartates and another between the O_δ2atom of Asp-125 and the nitrogen atom of the reduced peptide bond of the bound inhibitor. This would require two additional protonations, either of both aspartates, or of one Asp and the amido nitrogen atom of Nle-204. Our results favor the Asp-inhibitor protonation but the other one is not excluded. Implications of these findings for the mechanism of enzymatic catalysis are discussed. Dynamic properties of the hydrogen bond network in the active site and an analysis of the interaction energy between the inhibitor and the protease are presented. © 1997 Wiley-Liss, Inc.     

Design of a folding inhibitor of the HIV-1 protease

A novel way to inhibit HIV-1 protease by destabilizing its native state is discussed. A simplified protein model is used together with Monte Carlo simulations, to assess the destabilizing effect of peptides displaying the same sequence as specific fragments of the protein which are essential for its stability. Model calculations also show that it is unlikely that the protein can escape the inhibitory peptide by point mutations.     

Structural engineering of the HIV-1 protease molecule with a beta-turn mimic of fixed geometry.

An important goal in the de novo design of enzymes is the control of molecular geometry. To this end, an analog of the protease from human immunodeficiency virus 1 (HIV-1 protease) was prepared by total chemical synthesis, containing a constrained, nonpeptidic type II' beta-turn mimic of predetermined three-dimensional structure. The mimic beta-turn replaced residues Gly16,17 in each subunit of the homodimeric molecule. These residues constitute the central amino acids of two symmetry-related type I' beta-turns in the native, unliganded enzyme. The beta-turn mimic-containing enzyme analog was fully active, possessed the same substrate specificity as the Gly16,17-containing enzyme, and showed enhanced resistance to thermal inactivation. These results indicate that the precise geometry of the beta-turn at residues 15-18 in each subunit is not critical for activity, and that replacement of the native sequence with a rigid beta-turn mimic can lead to enhanced protein stability. Finally, the successful incorporation of a fixed element of secondary structure illustrates the potential of a "molecular kit set" approach to protein design and synthesis.     

HIV-1 protease inhibition potential of functionalized polyoxometalates

Polyoxometalates (POMs) are interesting biomedical agents due to their versatile anticancer and antiviral properties, such as remarkable anti-HIV activity. Although POMs are tunable and easily accessible inorganic drug prototypes in principle, their full potential can only be tapped by enhancing their biocompatibility, for example, through organic functionalization. We have therefore investigated the HIV-1 protease inhibition potential of functionalized Keggin- and Dawson-type POMs with organic side chains. Their inhibitory performance was furthermore compared to other POM types, and the buffer dependence of the results is discussed. In addition, chemical shift mapping NMR experiments were performed to exclude POM-substrate interactions. Whereas the introduction of organic side chains into POMs is a promising approach in principle, the influence of secondary effects on the reaction system also merits detailed investigation.     

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.     

Post-translational modification of glutamine and lysine residues of HIV-1 aspartyl protease by transglutaminase increases its catalytic activity

The human immunodeficiency virus type 1 aspartyl protease (HIV-1 PR) is a homodimeric aspartyl endopeptidase that is required for virus replication. HIV-1 PR was shown to act invitro as acyl-donor and -acceptor for both guinea pig liver transglutaminase (TG, EC 2.3.2.13) and human Factor XIIIa. These preliminary evidences suggested that the HIV-1 PR contains at least three TG-reactive glutaminyl and one lysyl residues. We report here that the incubation of HIV-1 PR with TG increases its catalytic activity. This increase is dependent upon the time of incubation, the concentration of TG and the presence of Ca²⁺. Identification of ε-(γ-glutamyl)lysine in the proteolytic digest of the TG-modified HIV-1 PR suggested intramolecular covalent cross-linking of this protease which may promote a non-covalent dimerization and subsequent activation of this enzyme via a conformational change. This hypothesis is supported by the observation that the TG-catalyzed activation of HIV-1 PR was completely abolished by spermidine (SPD) which acts as a competitive inhibitor of ε-(γ-glutamyl)lysine formation. Indeed, in the presence of 1 mM SPD the formation of the isopeptide was decreased of about 80%. The main products of the TG-catalyzed modification of HIV-1 PR in the presence of SPD were N₁-mono(γ-glutamyl)SPD and N₈-mono(γ-glutamyl)SPD. Negligible amount of N₁,N₈-bis(γ-glutamyl)SPD were found. The significance of these results is discussed with respect to the activation of the protease by post-translational modification and design of potential inhibitors.     

Overcoming drug resistance in HIV-1 chemotherapy: the binding thermodynamics of Amprenavir and TMC-126 to wild-type and drug-resistant mutants of the HIV-1 protease

Amprenavir is one of six protease inhibitors presently approved for clinical use in the therapeutic treatment of AIDS. Biochemical and clinical studies have shown that, unlike other inhibitors, Amprenavir is severely affected by the protease mutation I50V, located in the flap region of the enzyme. TMC-126 is a second-generation inhibitor, chemically related to Amprenavir, with a reported extremely low susceptibility to existing resistant mutations including I50V. In this paper, we have studied the thermodynamic and molecular origin of the response of these two inhibitors to the I50V mutation and the double active-site mutation V82F/I84V that affects all existing clinical inhibitors. Amprenavir binds to the wild-type HIV-1 protease with high affinity (5.0 x 10(9) M(-1) or 200 pM) in a process equally favored by enthalpic and entropic contributions. The mutations I50V and V82F/I84V lower the binding affinity of Amprenavir by a factor of 147 and 104, respectively. TMC-126, on the other hand, binds to the wild-type protease with extremely high binding affinity (2.6 x 10(11) M(-1) or 3.9 pM) in a process in which enthalpic contributions overpower entropic contributions by almost a factor of 4. The mutations I50V and V82F/I84V lower the binding affinity of TMC-126 by only a factor of 16 and 11, respectively, indicating that the binding affinity of TMC-126 to the drug-resistant mutants is still higher than the affinity of Amprenavir to the wild-type protease. Analysis of the data for TMC-126 and KNI-764, another second-generation inhibitor, indicates that their low susceptibility to mutations is caused by their ability to compensate for the loss of interactions with the mutated target by a more favorable entropy of binding.     

Interchain hydrophobic clustering promotes rigidity in HIV-1 protease flap dynamics: new insights from Molecular Dynamics

The dynamics of HIV-1 protease (HIV-pr), a drug target for HIV infection, has been studied extensively by both computational and experimental methods. The flap dynamics of HIV-pr is considered to be more important for better ligand binding and enzymatic actions. Moreover, it has been demonstrated that the drug-induced mutations can change the flap dynamics of HIV-pr affecting the binding affinity of the ligands. Therefore, detailed understanding of flap dynamics is essential for designing better inhibitors. Previous computational investigations observed significant variation in the flap opening in nanosecond time scale indicating that the dynamics is highly sensitive to the simulation protocols. To understand the sensitivity of the flap dynamics on the force field and simulation protocol, molecular dynamics simulations of HIV-pr have been performed with two different AMBER force fields, ff99 and ff02. Two different trajectories (20?ns each) were obtained using the ff99 and ff02 force field. The results showed polarizable force field (ff02) make the flap tighter than the nonpolarizable force field (ff99). Some polar interactions and hydrogen bonds involving flap residues were found to be stronger with ff02 force field. The formation of interchain hydrophobic cluster (between flap tip of one chain and active site wall of another chain) was found to be dominant in the semi-open structures obtained from the simulations irrespective of the force field. It is proposed that an inhibitor, which will promote this interchain hydrophobic clustering, may make the flaps more rigid, and presumably the effect of mutation would be small on ligand binding.     

An immunoenzymatic solid-phase assay for quantitative determination of HIV-1 protease activity

A novel immunoenzymatic procedure for the quantitative determination of HIV protease activity is provided. An N-terminal biotinylated peptide (DU1) that comprises an HIV-1 protease (HIV-PR) cleavage sequence was bound to streptavidin-coated microtiter plates. The bound peptide can be quantified by an immunoenzymatic procedure (enzyme-linked immunosorbent assay, ELISA) that includes a monoclonal antibody (Mab 332) against the peptide (DU1) C-terminal. The incubation of the bound peptide with HIV-PR in solution resulted in a signal decrement, as the peptide was hydrolyzed and the released C-terminal segment washed away. An equation that relates the amount of added enzyme to the kinetics of the reaction was written in order to describe this heterogeneous enzyme-quasi-saturable system. This equation allows quantitative determination of protease activity, a feature widely underrated in previous similar assays. The assay also allows evaluation of the inhibitory activity of HIV-PR inhibitors. Due to the intrinsic advantages of the ELISA format, this method could be used in high-throughput screening of HIV protease inhibitors. The assay can be extended to other proteolytic enzymes.     

High-performance liquid chromatographic assay of a new HIV-1 protease inhibitor, LB71350, in the plasma of dogs

A reliable reversed-phase high-performance liquid chromatographic method has been developed for the determination of LB71350 in the plasma of dogs. The analyte was deproteinized with 1.5 volumes of methanol and 0.5 volumes of 10% zinc sulfate, and the supernatant was injected into a 5-μm Capcell Pak C₁₈ column (150×4.6 mm I.D.). The mobile phase was a stepwise gradient mixture of acetonitrile and 0.2% triethylamine–HCl with a flow-rate of 1 ml/min and detection at UV 245 nm. The proportion of acetonitrile was kept at 52% for the first 6 min, increased to 100% for the next 0.5 min, kept at 100% for the next 2 min, decreased to 52% for the next 0.5 min, and finally kept at 52% for the next 7 min. The retention time of LB71350 was 6.9 min. The calibration was linear over the concentration range of 0.1–100 mg/l for dog plasma (r>0.997) and the limit of quantitation was 0.1 mg/l using 0.1 ml plasma. The quality control samples were reproducible with acceptable accuracy and precision at 0.1, 1, 10 and 100 mg/l concentrations. The within-day recovery (n=5) was 90.2–93.9%, the between-day recovery (n=5) was 89.5–93.5%, and the absolute between-day recovery (n=5) was 77–81%. The within-day precision (n=5) and between-day precision (n=5) were 2.59–5.82% and 3.17–4.55%, respectively. No interferences from endogenous substances were observed. Taken together, the above HPLC assay method by deproteinization and UV detection was suitable for the determination of LB71350 in the preclinical pharmacokinetics.     

Exploration of the effects of sequence variations between HIV-1 and HIV-2 proteases on their three-dimensional structures

Abstract

HIV protease inhibitors (PIs) approved by the FDA (US Food and Drug Administration) are a major class of antiretroviral. HIV-2 protease (PR2) is naturally resistant to most of them as PIs were designed for HIV-1 protease (PR1). In this study, we explored the impact of amino-acid substitutions between PR1 and PR2 on the structure of protease (PR) by comparing the structural variability of 13 regions using 24 PR1 and PR2 structures complexed with diverse ligands. Our analyses confirmed structural rigidity of the catalytic region and highlighted the important role of three regions in the conservation of the catalytic region conformation. Surprisingly, we showed that the flap region, corresponding to a flexible region, exhibits similar conformations in PR1 and PR2. Furthermore, we identified regions exhibiting different conformations in PR1 and PR2, which could be explained by the intrinsic flexibility of these regions, by crystal packing, or by PR1 and PR2 substitutions. Some substitutions induce structural changes in the R2 and R4 regions that could have an impact on the properties of PI-binding site and could thus modify PI binding mode. Substitutions involved in structural changes in the elbow region could alter the flexibility of the PR2 flap regions relative to PR1, and thus play a role in the transition from the semi-open form to the closed form, and have an impact on ligand binding. These results improve the understanding of the impact of sequence variations between PR1 and PR2 on the natural resistance of HIV-2 to commercially available PIs.

Communicated by Ramaswamy H. Sarma     

Structural insights into the mechanisms of drug resistance in HIV-1 protease NL4-3

The development of resistance to anti-retroviral drugs targeted against HIV is an increasing clinical problem in the treatment of HIV-1-infected individuals. Many patients develop drug-resistant strains of the virus after treatment with inhibitor cocktails (HAART therapy), which include multiple protease inhibitors. Therefore, it is imperative that we understand the mechanisms by which the viral proteins, in particular HIV-1 protease, develop resistance. We have determined the three-dimensional structure of HIV-1 protease NL4-3 in complex with the potent protease inhibitor TL-3 at 2.0 A resolution. We have also obtained the crystal structures of three mutant forms of NL4-3 protease containing one (V82A), three (V82A, M46I, F53L) and six (V82A, M46I, F53L, V77I, L24I, L63P) point mutations in complex with TL-3. The three protease mutants arose sequentially under ex vivo selective pressure in the presence of TL-3, and exhibit fourfold, 11-fold, and 30-fold resistance to TL-3, respectively. This series of protease crystal structures offers insights into the biochemical and structural mechanisms by which the enzyme can overcome inhibition by TL-3 while recovering some of its native catalytic activity.