Crystallographic Analysis and Molecular Modeling Studies of HIV-1 Protease and Drug Resistant Mutants

HIV-1 protease (PR) is an effective target protein for drugs in anti-retroviral therapy (ART). Using PR inhibitors (PIs) in clinical therapy successfully reduces mortality of HIV infected patients. However, drug resistant variants are selected in AIDS patients because of the fast evolution of the viral genome. Structural, kinetic and MD simulations of PR variants with or without substrate or PIs were used to better understand the molecular basis of drug resistance. Information obtained from these extensive studies will benefit the design of more effective inhibitor in ART. Amprenavir (APV) inhibition of PRWT, and single mutants of PRV32I, PRI50V, PRI54M, PRI54V, PRI84V and PRL90M were studied and X-ray crystal structures of PR variants complexes with APV were solved at resolutions of 1.02-1.85 Å to identify structural alterations. Crystal structures of PRWT, PRV32I and PRI47V were solved at resolutions of 1.20-1.40 Å. Reaction intermediates were captured in the substrate binding cavity, which represent three consecutive steps in the catalytic reaction of HIV PR. HIV-1 PR20 variant is a multi-drug resistant variant from a clinical isolate and it is of utility to investigate the mechanisms of resistance. The crystal structures of PR20 with inactivating mutation D25N have been determined at 1.45-1.75 Å resolution, and three distinct flap conformations, open, twisted and tucked, were observed. These studies help understand molecular basis of drug resistance and provide clues for design of inhibitors to combat multi-drug resistant PR. The evaluation of electrostatic force in MD simulations is the computationally intensive work, which is of order theta(N2) with integration of all atom pairs. AMMP invokes Amortized FMM in summation of electrostatic force, which reduced work load to theta(N). A hybrid, CPU and GPU, parallel implementation of Amortized FMM was developed and improves the elapsed time of MD simulation 20 fold faster than CPU based parallelization

Drug resistance mechanisms and drug design strategies for human immunodeficiency virus and hepatitis c virus proteases

The antiviral drug development has improved steadily to treat the infections of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) which represent heavy public health burdens. The viral protease plays an indispensable role in viral maturation and therefore becomes one of the most important targets for drug design. Nine HIV-1 protease inhibitors and two HCV protease inhibitors have been developed and approved by the U.S. Food and Drug Administration. However, mutations in the protease decrease reduce the efficacy the drugs. In this study, the enzyme assays indicate that darunavir and tipranavir exhibit the most potent inhibition against the multi-drug resistant (MDR) HIV-1 protease, which is supported by the co-crystal structures of the MDR protease-darunavir complex and the MDR protease-tipranavir complex. The MDR HIV-1 protease not only decreases the susceptibility to drugs but also impedes the formation of protease-substrate complex. Molecular dynamics simulation results show that the MDR HIV-1 protease needs to conquer a higher desolvation energy barrier to bind the substrate. Besides the study of drug resistance mechanisms, two drug discovery methods have been carried out in the study. One method is the modification of a current drug, lopinavir. The potency of the lopinavir analog against the MDR HIV-1 protease increases. The other method is the identification of a novel HIV-1 protease inhibitor scaffold to increase the structural diversity of inhibitors. In the HCV section, the study focuses on an HCV NS3/4A protease inhibitor, telaprevir, which is approved for the treatment of patients infected with the HCV genotype 1. The enzyme assays and molecular dynamic studies suggest that telaprevir may retain sufficient potency to treat the non-genotype 1 HCV strains

Structure and Dynamics of Viral Substrate Recognition and Drug Resistance: A Dissertation

Drug resistance is a major problem in quickly evolving diseases, including the human immunodeficiency (HIV) and hepatitis C viral (HCV) infections. The viral proteases (HIV protease and HCV NS3/4A protease) are primary drug targets. At the molecular level, drug resistance reflects a subtle change in the balance of molecular recognition; the drug resistant protease variants are no longer effectively inhibited by the competitive drug molecules but can process the natural substrates with enough efficiency for viral survival. Therefore, the inhibitors that better mimic the natural substrate binding features should result in more robust inhibitors with flat drug resistance profiles. The native substrates adopt a consensus volume when bound to the enzyme, the substrate envelope. The most severe resistance mutations occur at protease residues that are contacted by the inhibitors outside the substrate envelope. To guide the design of robust inhibitors, we investigate the shared and varied properties of substrates with the protein dynamics taken into account to define the dynamic substrate envelope of both viral proteases. The NS3/4A dynamic substrate envelope is compared with inhibitors to detect the structural and dynamic basis of resistance mutation patterns. Comparative analyses of substrates and inhibitors result in a solid list of structural and dynamic features of substrates that are not shared by inhibitors. This study can help guiding the development of novel inhibitors by paying attention to the subtle differences between the binding properties of substrates versus inhibitors

HIV-1 protease-substrate coevolution in nelfinavir resistance

Resistance to various human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. The virus accumulates mutations within the protease (PR) that render the PIs less potent. Occasionally, Gag sequences also coevolve with mutations at PR cleavage sites contributing to drug resistance. In this study, we investigated the structural basis of coevolution of the p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations by determining crystal structures of wild-type and NFV-resistant HIV-1 protease in complex with p1-p6 substrate peptide variants with L449F and/or S451N. Alterations of residue 30\u27s interaction with the substrate are compensated by the coevolving L449F and S451N cleavage site mutations. This interdependency in the PR-p1-p6 interactions enhances intermolecular contacts and reinforces the overall fit of the substrate within the substrate envelope, likely enabling coevolution to sustain substrate recognition and cleavage in the presence of PR resistance mutations. IMPORTANCE: Resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. Mutations in HIV-1 protease selected under the pressure of protease inhibitors render the inhibitors less potent. Occasionally, Gag sequences also mutate and coevolve with protease, contributing to maintenance of viral fitness and to drug resistance. In this study, we investigated the structural basis of coevolution at the Gag p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations. Our structural analysis reveals the interdependency of protease-substrate interactions and how coevolution may restore substrate recognition and cleavage in the presence of protease drug resistance mutations

On the entropy of protein families

Proteins are essential components of living systems, capable of performing a huge variety of tasks at the molecular level, such as recognition, signalling, copy, transport, ... The protein sequences realizing a given function may largely vary across organisms, giving rise to a protein family. Here, we estimate the entropy of those families based on different approaches, including Hidden Markov Models used for protein databases and inferred statistical models reproducing the low-order (1-and 2-point) statistics of multi-sequence alignments. We also compute the entropic cost, that is, the loss in entropy resulting from a constraint acting on the protein, such as the fixation of one particular amino-acid on a specific site, and relate this notion to the escape probability of the HIV virus. The case of lattice proteins, for which the entropy can be computed exactly, allows us to provide another illustration of the concept of cost, due to the competition of different folds. The relevance of the entropy in relation to directed evolution experiments is stressed.Comment: to appear in Journal of Statistical Physic

Molecular dynamics simulations of HIV-1 protease complexed with saquinavir

Inhibition of the Human Immunode�ficiency virus type-1 (HIV-1) protease enzyme blocks HIV-1 replication. Protease inhibitor drugs have successfully been used as a therapy for HIV-infected individuals to reduce their viral loads and slow the progression to Acquired Immune Defi�ciency Syndrome (AIDS). However, mutations readily and rapidly accrue in the protease gene resulting in a reduced sensitivity of the protein to the inhibitor. In this thesis, molecular dynamics simulations (MDS) were run on HIV proteases complexed with the protease inhibitor saquinavir, and the strength of affinity calculated through MMPBSA and normal mode analysis. We show in this thesis that at least 13 residues can be computationally mutated in the proteases sequence without adversely aff�ecting its structure or dynamics, and can still replicate the change in binding affinity to saquinavir caused by said mutations. Using 6 protease genotypes with an ordered decrease in saquinavir sensitivity we use MDS to calculate drug binding affinity. Our results show that single 10ns simulations of the systems resulted in good concurrence for the wild-type (WT) system, but an overall strong anti-correlation to biochemically derived results. Extension of the WT and multi-drug resistant (MDR) systems to 50ns yielded no improvement in the correlation to experimental. However, expansion of these systems to a 10-repetition ensemble MDS considerably improved the MDR binding affinity compared to the biochemical result. Principle components analysis on the simulations revealed that a much greater confi�gurational sampling was achieved through ensemble MD than simulation extension. These data suggest a possible mechanism for saquinavir resistance in the MDR system, where a transitioning to a lower binding-affinity configuration than WT occurs. Furthermore, we show that ensembles of 1ns in length sample a significant proportion of the con�figurations adopted over 10ns, and generate sufficiently similar binding affinities

Investigating the relationship between core stability and early life cycle events in HIV-1

HIV-1 capsid (CA) plays a vital role in the early stages of HIV-1 infection. The CA lattice surrounding the viral core is predominantly assembled from CA hexamers and stabilised by intra-hexamer and inter-hexamer interactions. Optimal stability of the lattice is known to be critical for efficient infection; however, a comprehensive screen of the effects of stabilising all lattice interfaces has not been performed. Disulphide cross-linking of residues across lattice interfaces has been used in vitro to stabilise CA assemblies. In this study, putatively stabilising cysteine CA mutations were designed at each interface of the CA lattice and their effects on early life cycle events, including reverse transcription and nuclear entry, assessed. The introduction of cysteine mutations at intra-hexamer (both NTD-NTD and NTD-CTD) and inter-hexamer (dimeric CTD-CTD only) lattice interfaces resulted in cross-linking and hyperstable viral cores in infected cells. These cores were minimally infectious and encountered sequential blocks to infectivity at reverse transcription, nuclear entry and post-nuclear entry. The infectivity defect of hyper-stable core mutant, A14C/E45C, was partially compensated – without an observable decrease in stability – by addition of mutations reported to perturb interactions with CPSF6. In contrast, Nup153 and CypA mutations were unable to compensate the infectivity defect suggesting that this was a CPSF6-specific effect. Proximal ligation assays were performed to visualise and quantify interactions between CA and host factors, indicating that hyper-stable cores encountered a block to nuclear entry in G1/S arrested cells. Overall, the results of this study suggest that mutations at different lattice interfaces can result in global changes to the intrinsic stability of the viral core and results in fitness defects at multiple stages of the HIV-1 early life cycle

Role of Protein Flexibility in Function, Resistance Pathways and Substrate Recognition Specificity in HIV-1 Protease: A Dissertation

In the 30 years since the Center for Disease Control\u27s Morbidity and Mortality Weekly Report published the first mention of what later was determined to be AIDS (Acquired immunodeficiency syndrome) and HIV (Human immunodeficiency virus) recognized as the causative pathogen, much has been done to understand this disease’s pathogenesis, development of drugs and emergence of drug resistance under selective drug therapy. Highly Active Antiretroviral Therapy (HAART), a combination of drugs that includes HIV-1 reverse transcriptase, protease, and more recently, integrase and entry inhibitors, have helped stabilize the HIV prevalence at extraordinarily high levels. Despite the recent stabilization of this global epidemic, its dimensions remain staggering with estimated (33-36 million) people living with HIV-AIDS in 2007 alone. This is because the available drugs against AIDS provide treatment for infected individuals, but HIV evolves rapidly under drug pressure and develops resistant strains, rendering the therapy ineffective. Therefore, a better understanding underlying the molecular mechanisms of viral infection and evolution is required to tackle drug resistance and develop improved drugs and treatment regimens. HIV-1 protease is an important target for developing anti-HIV drugs. However, resistant mutations rapidly emerge within the active site of the protease and greatly reduce its affinity for the protease inhibitors. Frequently, these active site drug resistant mutations co-occur with secondary/ non-active site/ associated or compensatory mutations distal to the active site. The role of these accessory mutations is often suggested to be in maintaining viral fitness and stability of protease. Many of the non-active site drug resistant mutations are clustered in the hydrophobic core in each monomer of the protease. Molecular dynamic simulation studies suggest that the hydrophobic core residues facilitate the conformational changes that occur in protease upon ligand binding. There is a complex interdependence and interplay between the inherent adaptability, drug resistant mutations and substrate recognition by the protease. Protease is inherently dynamic and has wide substrate specificity. The PI (protease inhibitor) resistant mutations, perhaps, modulate this dynamics and bring about changes in molecular recognition, such that, in resistant proteases, the substrates are recognized specifically over the PIs for the same binding site. In this thesis research, I have investigated these three complementary phenomena in concert. Chapter II examines the importance of hydrophobic core dynamics in modulating protease function. The hydrophobic core in the WT protease is intrinsically flexible and undergoes conformational changes required for protease to bind its substrates. This study investigated if dynamics is important for protease function by engineering restricted vs. flexible hydrophobic core region in each monomer of the protease, using disulfide chemistry. Under oxidizing conditions, disulfide bond established cross-link at the interface of putative moving domains in each monomer, thereby, restricting motion in this region. Upon reduction of the disulfide bond, the constraining influence was reversed and flexibility returned to near WT. The disulfide cross-linked protease showed significant loss of function when tested in functional cleavage assay. Two protease variants (G16C/L38C) and (R14C/E65C) were engineered and examined for changes in structure and enzymatic activity under oxidizing and reducing conditions. (R14C/E65C) was engineered as an internal control variant, such that cysteines were engineered between putative non-moving domains. Structurally, both the variants were very similar with no structural perturbations under oxidizing or reducing conditions. While significant loss in function was observed for (G16C/L38C) only under oxidizing conditions, (R14C/E65C) did not show any loss of function under oxidizing or reduced conditions, as expected. Successful regain of function for cross-linked (G16C/L38C) was obtained upon reversible reduction of the disulfide bond. Taken together, these data demonstrate that the hydrophobic core dynamics modulates protease function and support the hypothesis that the distal drug resistant mutations, possibly causing drug resistance by modulating hydrophobic core dynamics via long range structural perturbations. Since protease recognizes and cleaves more than 10 substrates at different rates, our further interest is to investigate if there is a differential loss of activity for some specific substrates over the others, and whether the order of polypeptide cleavage is somehow affected by restricted core mobility. In order to better answer these questions it is essential to understand: what determines the substrate binding specificity in protease? A two-pronged approach was applied to address this question as described in chapter III and IV respectively. In chapter III, I investigated the determinants of substrate specificity in HIV-1 protease by using computational positive design and engineered specificity-designed asymmetric protease (Pr3, A28S/D30F/G48R) that would preferentially bind to one of its natural substrates, RT-RH over two other substrates, p2-NC and CA-p2, respectively. The designed protease was expressed, purified and analyzed for changes in structure and function relative to WT. Kinetic studies on Pr3 showed that the specificity of Pr3 for RT-RH was increased significantly compared to the wild-type (WT), as predicted by the positive design. ITC (Isothermal Titration Calorimetry) studies confirmed the kinetic data on RT-RH. Crystal structural of substrate complexes of WT protease and Pr3 variant with RT-RH, CA-p2 and p2-NC were further obtained and analyzed. The structural analysis, however, only partially confirmed to the positive design due to the inherent structural pliability of the protease. Overall, this study supports the positive computational design approach as an invaluable tool in facilitating our understanding of complex proteins such as HIV 1 protease and also proposes the integration of internal protein flexibility in the design algorithms to make the in-silico designs more robust and dependable. Chapter IV probed the substrate specificity determining factors in HIV-1protease system by focusing on the substrate sequences. Previous studies have demonstrated that three N-terminal residues immediate to the scissile bond (P1-P3) are important in determining recognition specificity. This work investigated the structural basis of substrate binding to the protease. Catalytically active WT protease was crystallized with decameric polypeptides corresponding to five of the natural cleavage sites of protease. The structural analyses of these complexes revealed distinct P side product bound in all the structures, demonstrating the higher binding affinity of N terminal substrate for protease. This thesis research successfully establishes that intrinsic hydrophobic core flexibility modulates function in HIV-1 protease and proposes a potential mechanism to explain the role of non-active site mutations in conferring drug resistance in protease. Additionally, the work on specificity designed and N terminal product bound protease complexes advances our understanding of substrate recognition in HIV protease

Structural Studies of the Anti-HIV Human Protein APOBEC3G Catalytic Domain: A Dissertation

HIV/AIDS is a disease of grave global importance with over 33 million people infected world-wide and nearly 2 million deaths each year. The rapid emergence of drug resistance, due to viral mutation, renders anti-retroviral drug candidates ineffective with alarming speed and regularity. Instead of targeting mutation prone viral proteins, an alternative approach is to target host proteins that interact with viral proteins and are critical for the HIV life-cycle. APOBEC3G is a host anti-HIV restriction factor that can exert tremendous negative pressure by hypermutating the viral genome and has the potential to be a promising candidate for anti-retroviral therapeutic research. The work presented in this thesis is focused on investigating the A3G catalytic domain structure and implications of various observed structural features for biological function. High-resolution crystal structures of the A3G catalytic domain were solved using data from macromolecular X-ray crystallographic experiments, revealing a novel intermolecular zinc coordinating motif unique to A3G. Major intermolecular interfaces observed in the crystal structure were investigated for relevance to biochemical activity and biological function. Co-crystallization with a small-molecule A3G inhibitor, discovered using high-throughput screening assays, revealed a cysteine residue near the active site that is critical for inhibition of catalytic activity by catechol moieties. The serendipitous discovery of covalent interactions between this inhibitor and a surface cysteine residue led to further biochemical experiments that revealed the other cysteine, near the active site, to be critical for inhibition. Computational modeling was used to propose a steric-hinderance based mechanism of action that was supported by mutational experiments. Structures of other human APOBEC3 homologs were modeled using in-silico methods examined for similarities and differences with A3G catalytic domain crystal structures. Comparisons based on these homology models suggest putative structural features that may endow substrate specificity and other characteristics to the APOBEC3 family members

Investigating the Structural Basis for Human Disease: APOBEC3A and Profilin

Analyzing protein tertiary structure is an effective method to understanding protein function. In my thesis study, I aimed to understand how surface features of protein can affect the stability and specificity of enzymes. I focus on 2 proteins that are involved in human disease, Profilin (PFN1) and APOBEC3A (A3A). When these proteins are functioning correctly, PFN1 modulates actin dynamics and A3A inhibits retroviral replication. However, mutations in PFN1 are associated with amyotrophic lateral sclerosis (ALS) while the over expression of A3A are associated with the development of cancer. Currently, the pathological mechanism of PFN1 in this fatal disease is unknown and although it is known that the sequence context for mutating DNA vary among A3s, the mechanism for substrate sequence specificity is not well understood. To understand how the mutations in Profilin could lead to ALS, I solved the structure of WT and 2 ALS-related mutants of PFN1. Our collaborators demonstrated that ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in cells. This mutation-induced destabilization can account for the high propensity of ALS-linked variants to aggregate and also provides rationale for their reported loss-of-function phenotypes in cell-based assays. The source of this destabilization was illuminated by my X-ray crystal structures of several PFN1 proteins. I found an expanded cavity near the protein core of the destabilized M114T variant. In contrast, the E117G mutation only modestly perturbs the structure and stability of PFN1, an observation that reconciles the occurrence of this mutation in the control population. These findings suggest that a destabilized form of PFN1 underlies PFN1-mediated ALS pathogenesis. To characterize A3A’s substrate specificity, we solved the structure of apo and bound A3A. I then used a systematic approach to quantify affinity for substrate as a function of sequence context, pH and substrate secondary structure. I found that A3A preferred ssDNA binding motif is T/CTCA/G, and that A3A can bind RNA in a sequence specific manner. The affinity for substrate increased with a decrease in pH. Furthermore, A3A binds tighter to its substrate binding motif when in the loop region of folded nucleic acid compared to a linear sequence. This result suggests that the structure of DNA, and not just its chemical identity, modulates A3 affinity and specificity for substrate