Nine Crystal Structures Determine the Substrate Envelope of the MDR HIV-1 Protease

Under drug selection pressure, emerging mutations render HIV-1 protease drug resistant, leading to the therapy failure in anti-HIV treatment. It is known that nine substrate cleavage site peptides bind to wild type (WT) HIV-1 protease in a conserved pattern. However, how the multidrug-resistant (MDR) HIV-1 protease binds to the substrate cleavage site peptides is yet to be determined. MDR769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, and 90) was selected for present study to understand the binding to its natural substrates. MDR769 HIV-1 protease was co-crystallized with nine substrate cleavage site hepta-peptides. Crystallographic studies show that MDR769 HIV-1 protease has an expanded substrate envelope with wide open flaps. Furthermore, ligand binding energy calculations indicate weaker binding in MDR769 HIV-1 protease-substrate complexes. These results help in designing the next generation of HIV-1 protease inhibitors by targeting the MDR HIV-1 protease.     

Substitution of proline with pipecolic acid at the scissile bond converts a peptide substrate of HIV proteinase into a selective inhibitor

The nonapeptide H-Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-NH2 containing the retroviral Tyr-Pro cleavage site is a good substrate for the proteinase of human immunodeficiency viruses but it is not readily hydrolyzed by other nonviral proteinases including the structurally related pepsin-like aspartic proteinases. Replacing the Pro by L-pipecolic acid (2-piperidinecarboxylic acid) converted the substrate into an effective inhibitor of HIV-1 and HIV-2 proteinases with IC50 of approximately 1 microM. This compound showed a high degree of selectivity in that it did not inhibit cathepsin D and renin.     

Predicting human immunodeficiency virus protease cleavage sites in nonlinear projection space

HIV-1 protease has a broad and complex substrate specificity. The discovery of an accurate, robust, and rapid method for predicting the cleavage sites in proteins by HIV protease would greatly expedite the search for inhibitors of HIV protease. During the last two decades, various methods have been developed to explore the specificity of HIV protease cleavage activity. However, because little advancement has been made in the understanding of HIV-1 protease cleavage site specificity, not much progress has been reported in either extracting effective methods or maintaining high prediction accuracy. In this article, a theoretical framework is developed, based on the kernel method for dimensionality reduction and prediction for HIV-1 protease cleavage site specificity. A nonlinear dimensionality reduction kernel method, based on manifold learning, is proposed to reduce the high dimensions of protease specificity. A support vector machine is applied to predict the protease cleavage. Superior performance in comparison to that previously published in literature is obtained using numerical simulations showing that the basic specificities of the HIV-1 protease are maintained in reduction feature space, and by combining the nonlinear dimensionality reduction algorithm with a support vector machine classifier.     

Mechanism of inhibition of the retroviral protease by a Rous sarcoma virus peptide substrate representing the cleavage site between the gag p2 and p10 proteins.

The activity of the avian myeloblastosis virus (AMV) or the human immunodeficiency virus type 1 (HIV-1) protease on peptide substrates which represent cleavage sites found in the gag and gag-pol polyproteins of Rous sarcoma virus (RSV) and HIV-1 has been analyzed. Each protease efficiently processed cleavage site substrates found in their cognate polyprotein precursors. Additionally, in some instances heterologous activity was detected. The catalytic efficiency of the RSV protease on cognate substrates varied by as much as 30-fold. The least efficiently processed substrate, p2-p10, represents the cleavage site between the RSV p2 and p10 proteins. This peptide was inhibitory to the AMV as well as the HIV-1 and HIV-2 protease cleavage of other substrate peptides with Ki values in the 5-20 microM range. Molecular modeling of the RSV protease with the p2-p10 peptide docked in the substrate binding pocket and analysis of a series of single-amino acid-substituted p2-p10 peptide analogues suggested that this peptide is inhibitory because of the potential of a serine residue in the P1' position to interact with one of the catalytic aspartic acid residues. To open the binding pocket and allow rotational freedom for the serine in P1', there is a further requirement for either a glycine or a polar residue in P2' and/or a large amino acid residue in P3'. The amino acid residues in P1-P4 provide interactions for tight binding of the peptide in the substrate binding pocket.     

Comprehensive bioinformatic analysis of the specificity of human immunodeficiency virus type 1 protease

Rapidly developing viral resistance to licensed human immunodeficiency virus type 1 (HIV-1) protease inhibitors is an increasing problem in the treatment of HIV-infected individuals and AIDS patients. A rational design of more effective protease inhibitors and discovery of potential biological substrates for the HIV-1 protease require accurate models for protease cleavage specificity. In this study, several popular bioinformatic machine learning methods, including support vector machines and artificial neural networks, were used to analyze the specificity of the HIV-1 protease. A new, extensive data set (746 peptides that have been experimentally tested for cleavage by the HIV-1 protease) was compiled, and the data were used to construct different classifiers that predicted whether the protease would cleave a given peptide substrate or not. The best predictor was a nonlinear predictor using two physicochemical parameters (hydrophobicity, or alternatively polarity, and size) for the amino acids, indicating that these properties are the key features recognized by the HIV-1 protease. The present in silico study provides new and important insights into the workings of the HIV-1 protease at the molecular level, supporting the recent hypothesis that the protease primarily recognizes a conformation rather than a specific amino acid sequence. Furthermore, we demonstrate that the presence of 1 to 2 lysine residues near the cleavage site of octameric peptide substrates seems to prevent cleavage efficiently, suggesting that this positively charged amino acid plays an important role in hindering the activity of the HIV-1 protease.     

Synthetic non-peptide inhibitors of HIV protease

We have studied the inhibition of HIV protease by the antifungal antibiotic cerulenin, as well as by several related synthetic, structurally simpler analogs. The effect of these compounds on HIV protease was conveniently studied by monitoring the cleavage of an authentic single peptide bond in a synthetic nonapeptide corresponding to a natural cleavage site in HIV-1 gag precursor polyprotein. The relative inhibitory effects of these compounds have afforded an insight into the structural characteristics which impart antiprotease activity.     

Ribonuclease A as a substrate of the protease from human immunodeficiency virus-1

Bovine pancreatic ribonuclease A (RNase) contains two bonds, Met29-Met30 and Tyr92-Pro93 which are representative of sites in the human immunodeficiency virus-1 (HIV-1) gag polyprotein precursors that are cleaved by the HIV-1 protease during viral maturation. Nevertheless, neither native nor performic acid-oxidized RNase is a substrate for the protease. However, RNase derivatives obtained by reduction and S-alkylation with iodoacetate or iodoacetamide undergo cleavage by the HIV-1 protease at a single site, Ala109-alkyl-Cys110, that is distinct from either of the two predicted bonds mentioned above. The neutral carboxyamido-methylcysteinyl derivative is cleaved 8 times faster than that containing the negatively charged carboxy-methyl substituent at P1'. Succinylation of these S-alkylated RNase derivatives creates a second site of cleavage by the protease between succinyl-Lys7 and Phe8. Thus, the pattern of cleavage of denatured RNase by the HIV-1 protease can be manipulated by chemical derivatization of the substrate, and the new sites of hydrolysis revealed by these studies add to our understanding of the specificity of this important enzyme.     

Interdomain hydrolysis of a truncated Pseudomonas exotoxin by the human immunodeficiency virus-1 protease

The specificity of HIV-1 (human immunodeficiency virus-1) protease has been evaluated relative to its ability to cleave the three-domain Pseudomonas exotoxin (PE66) and related proteins in which the first domain has been deleted or replaced by a segment of CD4. Native PE66 is not hydrolyzed by the HIV-1 protease. However, removal of its first domain produces a molecule which is an excellent substrate for the enzyme. The major site of cleavage in this truncated exotoxin, called LysPE40, occurs in a segment that connects its two major domains, the translocation domain (II), and the ADP-ribosyltransferase (III). This interdomain region contains the sequence ...Asn-Tyr-Pro-Thr... which is similar to that surrounding the scissile Tyr-Pro bond in the gag precursor polyprotein, a natural substrate of the HIV-1 protease. Nevertheless, it is not this sequence that is recognized and cleaved by the enzyme, but one 6 residues away, ...Ala-Leu-Leu-Glu... in which the Leu-Leu peptide bond is hydrolyzed. A second, slower cleavage takes place at the Leu-Ala bond 3 residues in from the NH2 terminus of LysPE40. When domain I of PE66 is replaced by a segment comprising the first two domains of CD4, the resulting chimeric protein is hydrolyzed at the same Leu-Leu bond by HIV-1 protease. Enzyme activities toward synthetic peptides modeled after the sequences defined above in LysPE40 are in complete accord, relative to specificity, kinetics, and pH optimum, with results obtained in the hydrolysis of the parent protein. These findings demonstrate that ideas concerning the specificity of the HIV-1 protease that are based solely upon its processing of natural viral polyproteins can be expanded by evaluation of other multidomain proteins as substrates. Moreover, it would appear that it is not a particular conformation, but sequence and accessibility that play the dominant role in defining sites in a protein substrate that are susceptible to hydrolysis by the enzyme.     

Enzymatic activity of a synthetic 99 residue protein corresponding to the putative HIV-1 protease

A protein corresponding to the putative protease of the human immunodeficiency virus 1 (HIV-1) has been prepared by total chemical synthesis. This 99 residue synthetic enzyme showed specific proteolytic activity on fragments of the natural gag precursor and on synthetic peptide substrates, two of which released fragments corresponding to the N terminus and C terminus of the protease molecule itself. The observed substrate specificity was not restricted to cleavage at Phe/Tyr-Pro bonds. Inhibition studies provided direct evidence that the HIV-1 protease belongs to the family of aspartic proteases. The availability of the HIV-1 protease as a defined molecular species has important implications for the design of specific inhibitors that do not interfere with the host cell metabolism as a possible route to antiviral agents against acquired immunodeficiency syndrome (AIDS).     

Substrate shape determines specificity of recognition for HIV-1 protease: analysis of crystal structures of six substrate complexes

The homodimeric HIV-1 protease is the target of some of the most effective antiviral AIDS therapy, as it facilitates viral maturation by cleaving ten asymmetric and nonhomologous sequences in the Gag and Pol polyproteins. Since the specificity of this enzyme is not easily determined from the sequences of these cleavage sites alone, we solved the crystal structures of complexes of an inactive variant (D25N) of HIV-1 protease with six peptides that correspond to the natural substrate cleavage sites. When the protease binds to its substrate and buries nearly 1000 A2 of surface area, the symmetry of the protease is broken, yet most internal hydrogen bonds and waters are conserved. However, no substrate side chain hydrogen bond is conserved. Specificity of HIV-1 protease appears to be determined by an asymmetric shape rather than a particular amino acid sequence.     

Computational proteomics analysis of HIV-1 protease interactome

HIV-1 protease is a small homodimeric enzyme that ensures maturation of HIV virions by cleaving the viral precursor Gag and Gag-Pol polyproteins into structural and functional elements. The cleavage sites in the viral polyproteins share neither sequence homology nor binding motif and the specificity of the HIV-1 protease is therefore only partially understood. Using an extensive data set collected from 16 years of HIV proteome research we have here created a general and predictive rule-based model for HIV-1 protease specificity based on rough sets. We demonstrate that HIV-1 protease specificity is much more complex than previously anticipated, which cannot be defined based solely on the amino acids at the substrate's scissile bond or by any other single substrate amino acid position only. Our results show that the combination of at least three particular amino acids is needed in the substrate for a cleavage event to occur. Only by combining and analyzing massive amounts of HIV proteome data it was possible to discover these novel and general patterns of physico-chemical substrate cleavage determinants. Our study is an example how computational biology methods can advance the understanding of the viral interactomes.     

Bioluminescence detection of proteolytic bond cleavage by using recombinant aequorin

Detection of proteolytic bond cleavage was achieved by taking advantage of the bioluminescence emission generated by the photoprotein aequorin. A genetically engineered HIV-1 protease substrate was coupled with a cysteine-free mutant of aequorin by employing the polymerase chain reaction to produce a fusion protein that incorporates an optimum natural protease cleavage site. The fusion protein was immobilized on a solid phase and employed as the substrate for the HIV-1 protease. Proteolytic bond cleavage was detected by a decrease in the bioluminescence generated by the aequorin fusion protein on the solid phase. A dose-response curve for HIV-1 protease was constructed by relating the decrease in bioluminescence signal with varying amounts of the protease. The system was also used to evaluate two competitive and one noncompetitive inhibitor of the HIV-1 protease. Among the advantages of this assay is that by using recombinant methods a complete bioluminescently labeled protease recognition site can be designed and produced. The assay yields very sensitive detection limits, which are inherent to bioluminescence-based methods. An application of this system may be in the high-throughput screening of biopharmaceutical drugs that are potential inhibitors of a target protease.     

A cleavage enzyme-cytometric bead array provides biochemical profiling of resistance mutations in HIV-1 Gag and protease

Most protease-substrate assays rely on short, synthetic peptide substrates consisting of native or modified cleavage sequences. These assays are inadequate for interrogating the contribution of native substrate structure distal to a cleavage site that influences enzymatic cleavage or for inhibitor screening of native substrates. Recent evidence from HIV-1 isolates obtained from individuals resistant to protease inhibitors has demonstrated that mutations distal to or surrounding the protease cleavage sites in the Gag substrate contribute to inhibitor resistance. We have developed a protease-substrate cleavage assay, termed the cleavage enzyme- cytometric bead array (CE-CBA), which relies on native domains of the Gag substrate containing embedded cleavage sites. The Gag substrate is expressed as a fluorescent reporter fusion protein, and substrate cleavage can be followed through the loss of fluorescence utilizing flow cytometry. The CE-CBA allows precise determination of alterations in protease catalytic efficiency (k(cat)/K(M)) imparted by protease inhibitor resistance mutations in protease and/or gag in cleavage or noncleavage site locations in the Gag substrate. We show that the CE-CBA platform can identify HIV-1 protease present in cellular extractions and facilitates the identification of small molecule inhibitors of protease or its substrate Gag. Moreover, the CE-CBA can be readily adapted to any enzyme-substrate pair and can be utilized to rapidly provide assessment of catalytic efficiency as well as systematically screen for inhibitors of enzymatic processing of substrate.     

A radiometric assay for HIV-1 protease

A rapid, high-throughput radiometric assay for HIV-1 protease has been developed using ion-exchange chromatography performed in 96-well filtration plates. The assay monitors the activity of the HIV-1 protease on the radiolabeled form of a heptapeptide substrate, [tyrosyl-3,5-3H]Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, which is based on the p17-p24 cleavage site found in the viral polyprotein substrate Pr55gag. Specific cleavage of this uncharged heptapeptide substrate by HIV-1 protease releases the anionic product [tyrosyl-3,5-3H]Ac-Ser-Gln-Asn-Tyr, which is retained upon minicolumns of the anion-exchange resin AG1-X8. Protease activity is determined from the recovery of this radiolabeled product following elution with formic acid. This facile and highly sensitive assay may be utilized for steady-state kinetic analysis of the protease, for measurements of enzyme activity during its purification, and as a routine assay for the evaluation of protease inhibitors from natural product or synthetic sources.     

A catalogue of putative HIV-1 protease host cell substrates

Abstract Processing of human immunodeficiency virus (HIV) proteins by the HIV-1 protease is essential for HIV infectivity. In addition, several studies have revealed cleavage of human proteins by this viral protease during infection; however, no large-scale HIV-1 protease degradomics study has yet been performed. To identify putative host substrates in an unbiased manner and on a proteome-wide scale, we used positional proteomics to identify peptides reporting protein processing by the HIV-1 protease, and a catalogue of over 120 cellular HIV-1 protease substrates processed in vitro was generated. This catalogue includes previously reported substrates as well as recently described interaction partners of HIV-1 proteins. Cleavage site alignments revealed a specificity profile in good correlation with previous studies, even though the ELLE consensus motif was not cleaved efficiently when incorporated into peptide substrates due to subsite cooperativity. Our results are further discussed in the context of HIV-1 infection and the complex substrate recognition by the viral protease.     

Antiviral agent based on the non-structural protein targeting the maturation process of HIV-1: expression and susceptibility of chimeric Vpr as a substrate for cleavage by HIV-1 protease

The processing of precursor proteins (Gag and Gag-pol) by the viral protease is absolutely required in order to generate infectious particles. This prompted us to consider novel strategies that target viral maturation. Towards this end, we have engineered an HIV-1 virion associated protein, Vpr, to contain protease cleavage signal sequences from Gag and Gag-pol precursor proteins. We previously reported that virus particles derived from HIV-1 proviral DNA, encoding chimeric Vpr, showed a lack of infectivity, depending on the fusion partner. As an extension of that work, the potential of chimeric Vpr as a substrate for HIV-1 protease was tested utilizing an epitope-based assay. Chimeric Vpr molecules were modified such that the Flag epitope is removed following cleavage, thus allowing us to determine the efficiency of protease cleavage. Following incubation with the protease, the resultant products were analyzed by radioimmunoprecipitation using antibodies directed against the Flag epitope. Densitometric analysis of the autoradiograms showed processing to be both rapid and specific. Further, the analysis of virus particles containing chimeric Vpr by immunoblot showed reactivities to antibodies against the Flag epitope similar to the data observed in vitro. These results suggest that the pseudosubstrate approach may provide another avenue for developing antiviral agents.     

Structural basis for coevolution of a human immunodeficiency virus type 1 nucleocapsid-p1 cleavage site with a V82A drug-resistant mutation in viral protease

Maturation of human immunodeficiency virus (HIV) depends on the processing of Gag and Pol polyproteins by the viral protease, making this enzyme a prime target for anti-HIV therapy. Among the protease substrates, the nucleocapsid-p1 (NC-p1) sequence is the least homologous, and its cleavage is the rate-determining step in viral maturation. In the other substrates of HIV-1 protease, P1 is usually either a hydrophobic or an aromatic residue, and P2 is usually a branched residue. NC-p1, however, contains Asn at P1 and Ala at P2. In response to the V82A drug-resistant protease mutation, the P2 alanine of NC-p1 mutates to valine (AP2V). To provide a structural rationale for HIV-1 protease binding to the NC-p1 cleavage site, we solved the crystal structures of inactive (D25N) WT and V82A HIV-1 proteases in complex with their respective WT and AP2V mutant NC-p1 substrates. Overall, the WT NC-p1 peptide binds HIV-1 protease less optimally than the AP2V mutant, as indicated by the presence of fewer hydrogen bonds and fewer van der Waals contacts. AlaP2 does not fill the P2 pocket completely; PheP1' makes van der Waals interactions with Val82 that are lost with the V82A protease mutation. This loss is compensated by the AP2V mutation, which reorients the peptide to a conformation more similar to that observed in other substrate-protease complexes. Thus, the mutant substrate not only binds the mutant protease more optimally but also reveals the interdependency between the P1' and P2 substrate sites. This structural interdependency results from coevolution of the substrate with the viral protease.     

Chemical synthesis and expression of the HIV-1 protease gene in E. coli

The 297bp HIV-1 protease gene was constructed from five discrete synthetic fragments and expressed in E. coli. A soluble protein product of 11.5 Kd was detected by immunoblotting using protease specific antisera. A quantitative assay system, utilizing a synthetic nonapeptide spanning the cleavage site between p17-p24 in the gag polyprotein, was used to measure the specific protease activity in crude extracts. The protease hydrolyzed tyrosyl-proline bonds with an approximate specific activity of 43 pmoles/min/micrograms of total protein. The chemical synthesis of the protease gene and it's expression provides a feasible method for rapid mutant analysis, important for structure-function studies and rational design of potential inhibitors.     

Performance of random forest when SNPs are in linkage disequilibrium

Background

Proteases of human pathogens are becoming increasingly important drug targets, hence it is necessary to understand their substrate specificity and to interpret this knowledge in practically useful ways. New methods are being developed that produce large amounts of cleavage information for individual proteases and some have been applied to extract cleavage rules from data. However, the hitherto proposed methods for extracting rules have been neither easy to understand nor very accurate. To be practically useful, cleavage rules should be accurate, compact, and expressed in an easily understandable way.

Results

A new method is presented for producing cleavage rules for viral proteases with seemingly complex cleavage profiles. The method is based on orthogonal search-based rule extraction (OSRE) combined with spectral clustering. It is demonstrated on substrate data sets for human immunodeficiency virus type 1 (HIV-1) protease and hepatitis C (HCV) NS3/4A protease, showing excellent prediction performance for both HIV-1 cleavage and HCV NS3/4A cleavage, agreeing with observed HCV genotype differences. New cleavage rules (consensus sequences) are suggested for HIV-1 and HCV NS3/4A cleavages. The practical usability of the method is also demonstrated by using it to predict the location of an internal cleavage site in the HCV NS3 protease and to correct the location of a previously reported internal cleavage site in the HCV NS3 protease. The method is fast to converge and yields accurate rules, on par with previous results for HIV-1 protease and better than previous state-of-the-art for HCV NS3/4A protease. Moreover, the rules are fewer and simpler than previously obtained with rule extraction methods.

Conclusion

A rule extraction methodology by searching for multivariate low-order predicates yields results that significantly outperform existing rule bases on out-of-sample data, but are more transparent to expert users. The approach yields rules that are easy to use and useful for interpreting experimental data.     

Crystal structures of highly constrained substrate and hydrolysis products bound to HIV-1 protease. Implications for the catalytic mechanism

HIV-1 protease is a key target in treating HIV infection and AIDS, with 10 inhibitors used clinically. Here we used an unusual hexapeptide substrate, containing two macrocyclic tripeptides constrained to mimic a beta strand conformation, linked by a scissile peptide bond, to probe the structural mechanism of proteolysis. The substrate has been cocrystallized with catalytically active synthetic HIV-1 protease and an inactive isosteric (D25N) mutant, and three-dimensional structures were determined (1.60 A). The structure of the inactive HIVPR(D25N)/substrate complex shows an intact substrate molecule in a single orientation that perfectly mimics the binding of conventional peptide ligands of HIVPR. The structure of the active HIVPR/product complex shows two monocyclic hydrolysis products trapped in the active site, revealing two molecules of the N-terminal monocyclic product bound adjacent to one another, one molecule occupying the nonprime site, as expected, and the other monocycle binding in the prime site in the reverse orientation. The results suggest that both hydrolysis products are released from the active site upon cleavage and then rebind to the enzyme. These structures reveal that N-terminal binding of ligands is preferred, that the C-terminal site is more flexible, and that HIVPR can recognize substrate shape rather than just sequence alone. The product complex reveals three carboxylic acids in an almost planar orientation, indicating an unusual hexagonal homodromic complex between three carboxylic acids. The data presented herein regarding orientation of catalytic aspartates support the cleavage mechanism proposed by Northrop. The results imply strategies for design of inhibitors targeting the N-terminal side of the cleavage site or taking advantage of the flexibility in the protease domain that accommodates substrate/inhibitor segments C-terminal to the cleavage site.