Non-active Site Mutations Research Articles

Contrasting the effect of hinge region insertions and non-active site mutations on HIV protease-inhibitor interactions: Insights from altered flap dynamics

HIV-1 protease (PR) enzyme is a viable antiretroviral drug target due to its crucial role in HIV maturation. Over many decades, the HIV-1 PR enzyme has exhibited mutations brought on by drug pressure and error-prone nature of HIV-1 reverse transcriptase. Non-active site mutations have played a pivotal role in drug resistance; however, their mechanism of action has not been fully elucidated. We investigated how non-active site mutations affect the conformational stability and drug binding ability of HIV-1 PR. In light of this, we studied a novel HIV-1 subtype C protease variant containing an insertion of valine (↑V) in the hinge region. We analysed the mutations in the presence and absence of ten background mutations. Molecular dynamics simulations revealed that both with and without the background mutations, the PR exhibited increased flexibility of hinge, flaps and fulcrum regions. This allowed the PR to adopt a wider flap conformation when in complex with several inhibitors. Additionally, the simulations revealed that the protease inhibitors (PIs) could not bring the mutated variant proteases into a stable, closed conformation, resulting in increased solvent exposure of the inhibitors. Together, these results suggest that the mutations decrease the favourability of binding by altering the dynamics of the flap regions. Notably, the insertion mutation increased PR hinge flexibility and the introduction of background mutations compensated for this by stabilising the cantilever and hinge regions. Together, these findings provide insight into how non-active site mutations affect PR conformational dynamics in critical areas of the PR thus impacting on drug binding capacity and potentially contributing to drug resistance.

Non-active site mutations in the HIV protease: Diminished drug binding affinity is achieved through modulating the hydrophobic sliding mechanism

The global HIV/AIDS epidemic still currently affects approximately 38 million individuals globally. The protease enzyme of the human immunodeficiency virus is a major drug target in antiviral therapy, however, under the influence of reverse transcriptase and in the context of drug pressure, the rapid PR mutation rate contributes significantly to clinical failure. The set of cooperative non-active site mutations, I13V/I62V/V77I, have been associated with reduced inhibitor susceptibility and are the focus of the current study. When compared to the wild-type protease the mutant protease exhibited decreased binding affinities towards ATV and DRV by 64- and 12-fold, respectively, and decreased the overall favourable Gibbs free energy for ATV, DRV, RTV and SQV. Moreover, these mutations decreased the thermal stability of the protease when in complex with ATV and DRV by approximately 6.4 and 4.2 °C, respectively. The crystal structure of the mutant protease revealed that the location of these mutations and their effect on the hydrophobic sliding mechanism may be crucial in their role in resistance.

Drug Resistance Mechanism of M46I-Mutation-Induced Saquinavir Resistance in HIV-1 Protease Using Molecular Dynamics Simulation and Binding Energy Calculation.

Drug-resistance-associated mutation in essential proteins of the viral life cycle is a major concern in anti-retroviral therapy. M46I, a non-active site mutation in HIV-1 protease has been clinically associated with saquinavir resistance in HIV patients. A 100 ns molecular dynamics (MD) simulation and MM-PBSA calculations were performed to study the molecular mechanism of M46I-mutation-based saquinavir resistance. In order to acquire deeper insight into the drug-resistance mechanism, the flap curling, closed/semi-open/open conformations, and active site compactness were studied. The M46I mutation significantly affects the energetics and conformational stability of HIV-1 protease in terms of RMSD, RMSF, Rg, SASA, and hydrogen formation potential. This mutation significantly decreased van der Waals interaction and binding free energy (∆G) in the M46I–saquinavir complex and induced inward flap curling and a wider opening of the flaps for most of the MD simulation period. The predominant open conformation was reduced, but inward flap curling/active site compactness was increased in the presence of saquinavir in M46I HIV-1 protease. In conclusion, the M46I mutation induced structural dynamics changes that weaken the protease grip on saquinavir without distorting the active site of the protein. The produced information may be utilized for the discovery of inhibitor(s) against drug-resistant HIV-1 protease.

Cantilever-centric mechanism of cooperative non-active site mutations in HIV protease: Implications for flap dynamics

The HIV-1 protease is an important drug target in antiretroviral therapy due to the crucial role it plays in viral maturation. A greater understanding of the dynamics of the protease as a result of drug-induced mutations has been successfully elucidated using computational models in the past. We performed induced-fit docking studies and molecular dynamics simulations on the wild-type South African HIV-1 subtype C protease and two non-active site mutation-containing protease variants; HP3 PR and HP4 PR. The HP3 PR contained the I13V, I62V, and V77I mutations while HP4 PR contained the same mutations with the addition of the L33F mutation. The simulations were initiated in a cubic cell universe containing explicit solvent, with the protease variants beginning in the fully closed conformation. The trajectory for each simulation totalled 50 ns. The results indicate that the mutations increase the dynamics of the flap, hinge, fulcrum and cantilever regions when compared to the wild-type protease while in complex with protease inhibitors. Specifically, these mutations result in the protease favouring the semi-open conformation when in complex with inhibitors. Moreover, the HP4 PR adopted curled flap tip conformers which coordinated several water molecules into the active site in a manner that may reduce inhibitor binding affinity. The mutations affected the thermodynamic landscape of inhibitor binding as there were fewer observable chemical contacts between the mutated variants and saquinavir, atazanavir and darunavir. These data help to elucidate the biophysical basis for the selection of cooperative non-active site mutations by the HI virus.

Structural insight into conformational dynamics of non-active site mutations in KasA: A Mycobacterium tuberculosis target protein

The enzyme β-Ketoacyl ACP synthase I (KasA) is a potent drug target in mycolic acid pathway of Mycobacterium tuberculosis (Mtb). In the present study, we investigated the structural dynamics of wild-type (WT) and mutants KasA (D66N, G269S, G312S, and F413L) in both monomer and dimer form to provide insight into protein structural stability. To gain better understanding of structural flexibility of KasA, combined molecular dynamics and essential dynamics were employed to analyze the conformational changes induced by non-active site mutations. The results confirm that non-active site mutations lower the structural stability in dimer KasA as compared to WT. The protein network topology and close residue interactions of WT and mutant residues of KasA have been predicted through residue interaction network analysis (RIN). Non-active site mutations distort RIN architecture and subsequently affect the drug binding landscape. T-pad associated with mode vector analysis comprehensively pronounces the structural impact caused by non-active site mutations. It also identified the critical fluctuating residues present in the gate segment (GS) region (115–147). The non-active site mutations altered the structural stability of the mutant protein structures, and these mutations may be a cause for the resistance mechanism of KasA against anti-tuberculosis drugs. Further, it is observed that dimer mutant KasA proteins display much more structural flexibility than WT at the ligand binding site which is evident from the binding site analysis and hydrogen bond interaction patterns. This study provides a better understanding of the structural dynamic behaviour of KasA mutants, thereby facilitating the need to find a novel and potent inhibitor against Mtb.

Exploring the drug resistance mechanism of active site, non-active site mutations and their cooperative effects in CRF01_AE HIV-1 protease: molecular dynamics simulations and free energy calculations

Human immunodeficiency virus type 1 protease is essential for virus replication and maturation and has been considered as one of the important drug target for the antiretroviral treatment of HIV infection. The majority of HIV infections are caused due to non-B subtypes in developing countries. Subtype AE is spreading rapidly and infecting huge population worldwide. Understanding the interdependence of active and non-active site mutations in conferring drug resistance is crucial for the development effective inhibitors in subtype AE protease. In this work, we have investigated the mechanism of resistance against indinavir (IDV) due to therapy selected active site mutation V82F, non-active site mutations PF82V and their cooperative effects PV82F in subtype AE-protease using molecular dynamics simulations and binding free energy calculations. The simulations suggested all the three complexes lead to decrease in binding affinity of IDV, whereas the PF82V complex resulted in an enhanced binding affinity compared to V82F and PV82F complexes. Large positional deviation of IDV was observed in V82F complex. The preservation of hydrogen bonds of IDV with active site Asp25/Asp25′ and flap residue Ile50/50′ via a water molecule is crucial for effective binding. Owing to the close contact of 80s loop with Ile50′ and Asp25, the alteration between residues Thr80 and Val82, further induces conformational change thereby resulting in loss of interactions between IDV and the residues in the active site cavity, leading to drug resistance. Our present study shed light on the effect of active, non-active site mutations and their cooperative effects in AE protease.Communicated by Ramaswamy H. Sarma

Non-active site mutation (Q123A) in New Delhi metallo-β-lactamase (NDM-1) enhanced its enzyme activity

New Delhi metallo β-lactamase-1 is one of the carbapenemases, causing hydrolysis of almost all β-lactamase antibiotics. Seventeen different NDM variants have been reported so far, they varied in their sequences either by single or multiple amino acid substitutions. Hence, it is important to understand its structural and functional relation. In the earlier studies role of active site residues has been studied but non-active site residues has not studied in detail. Therefore, we have initiated to further comprehend its structure and function relation by mutating some of its non-active site residues. A laboratory mutant of NDM-1 was generated by PCR-based site-directed mutagenesis, replacing Q to A at 123 position. The MICs of imipenem and meropenem for NDM-1Q123A were found increased by 2 fold as compare to wild type and so the hydrolytic activity was enhanced (Kcat/Km) as compared to NDM-1 wild type. GOLD fitness scores were also found in favour of kinetics data. Secondary structure for α-helical content was determined by Far-UV circular dichroism (CD), which showed significant conformational changes. We conclude a noteworthy role of non-active-site amino acid residues in the catalytic activity of NDM-1. This study also provides an insight of emergence of new variants through natural evolution.

Activity loss by H46A mutation in Mycobacterium tuberculosis isocitrate lyase is due to decrease in structural plasticity and collective motions of the active site

Mycobacterium tuberculosis isocitrate lyase (MtbICL) is a crucial enzyme of the glyoxylate cycle and is a validated anti-tuberculosis drug target. Structurally distant, non-active site mutation (H46A) in MtbICL has been found to cause loss of enzyme activity. The aim of the present work was to explore the structural alterations induced by H46A mutation that caused the loss of enzyme activity. The structural and dynamic consequences of H46A mutation were studied using multiple computational methods such as docking, molecular dynamics simulation and residue interaction network analysis (RIN). Principal component analysis and cross correlation analysis revealed the difference in conformational flexibility and collective modes of motions between the wild-type and mutant enzyme, particularly in the active site region. RIN analysis revealed that the active site geometry was disturbed in the mutant enzyme. Thus, the dynamic perturbation of the active site led to enzyme transition from its active form to inactive form upon mutation. The computational analyses elucidated the mutant-specific conformational alterations, differential dominant motions, and anomalous residue level interactions that contributed to the abrogated function of mutant MtbICL. An understanding of interactions of mutant enzymes may help in modifying the existing drugs and designing improved drugs for successful control of tuberculosis.

F1174V mutation alters the ALK active conformation in response to Crizotinib in NSCLC: Insight from molecular simulations

Crizotinib is an efficient antineoplastic drug for treatment of non-small cell lung carcinoma (NSCLC), which is identified as an anaplastic lymphoma kinase (ALK) inhibitor. F1174V is a recently identified acquired point mutation relating to the Crizotinib resistance in NSCLC patients. The mechanism of Crizotinib resistance relating to F1174V mutation as a non-active site mutation remains unclear. In this study, the molecular dynamic simulation was used to investigate the possible mechanisms by which F1174V mutation may affect the structure and activity of ALK kinase domain. The results suggested that F1174V mutation could cause two important secondary structure alterations, which led to the local conformational change in ALK kinase domain. This causes more positive free energy in the mutant complex in comparison with the wild-type one. In addition, our structural analyses illustrated that F1174V mutation could result in some important interactions, which represent the key characteristics of the ALK active conformation. This study provided a molecular mechanism for ALK Crizotinib resistance caused by F1174V mutation,which could facilitate designing more efficient drugs.

Drug Resistance Mechanism of L10F, L10F/N88S and L90M mutations in CRF01_AE HIV-1 protease: Molecular dynamics simulations and binding free energy calculations

HIV-1 protease plays a crucial role in viral replication and maturation, which makes it one of the most attractive targets for anti-retroviral therapy. The majority of HIV infections in developing countries are due to non-B subtype. Subtype AE is spreading rapidly and infecting huge population worldwide. The mutations in the active site of subtype AE directly impair the interactions with the inhibitor. The non-active site mutations influence the binding of the inhibitor indirectly and their resistance mechanism is not well understood. It is important to design new effective inhibitors that combat drug resistance in subtype AE protease. In this work, we examined the effect of non active site mutations L10F, L10F/N88S and L90M with nelfinavir using molecular dynamics simulation and binding free energy calculations. The simulations suggested that the L10F and L10F/N88S mutants decrease the binding affinity of nelfinavir, whereas the L90M mutant increases the binding affinity. The formation of hydrogen bonds between nelfinavir and Asp30 is crucial for effective binding. The benzamide moiety of nelfinavir shows large positional deviation in L10F and L10F/N88S complexes and the L10F/N88S mutation changes the hydrogen bond between the side chain atoms of 30th residue and the 88th residue. Consequently the hydrogen bond interaction between Asp30 and nelfinavir are destroyed leading to drug resistance. Our present study shed light on the resistance mechanism of the strongly linked mutation L10F/N88S observed experimentally in AE subtype.

Non-active site mutations disturb the loop dynamics, dimerization, viral budding and egress of VP40 of the Ebola virus.

The first account of the dynamic features of the loop region of VP40 of the Ebola virus (EboV) using accelerated molecular dynamics (aMD) simulations is reported herein. Due to its major role in the Ebola life cycle, VP40 is considered a promising therapeutic target. The available experimental data on the N-terminal domain (NTD) loop indicates that mutations K127A, T129A and N130A demonstrate an unrecognized role for NTD-plasma membrane (PM) interaction for efficient VP40-PM localization, oligomerization, matrix assembly and egress. Despite experimental results, the molecular description of VP40 and the information it can provide still remain vague. Therefore, to gain further molecular insight into the effect of mutations on the loop region of VP40 and its effects on the overall protein conformation and VP40 dimerization, aMD simulations and post-dynamic analyses were employed for wildtype (WT) and mutant systems. The results showed significant variations in the presence of mutations as per RMSF, RMSD, Rg, PCA and distance calculations in comparison to the WT. These results could provide researchers with insight with regards to the conformational aspects concerning VP40 and its close relation to the experimental data. We believe that the results presented in this study will ultimately provide a useful understanding of the structural landscape of the loop region of VP40, which would contribute towards the discovery of novel EboV inhibitors.

Coumarin Derivatives as Substrate Probes of Mammalian Cytochromes P450 2B4 and 2B6: Assessing the Importance of 7-Alkoxy Chain Length, Halogen Substitution, and Non-Active Site Mutations.

Using a combined structural and biochemical approach, the functional importance of a recently described peripheral pocket bounded by the E-, F-, G-, and I-helices in CYP2B4 and 2B6 was probed. Three series of 4-substituted-7-alkoxycoumarin derivatives with -H, -CH3, or -CF3 at the 4 position of the coumarin core were used initially to monitor functional differences between CYP2B4 and 2B6. 7-Ethoxy-4-(trifluoromethyl)coumarin (7-EFC) displayed the highest catalytic efficiency among these substrates. Mutants were made to alter side-chain polarity (V/E194Q) or bulk (F/Y244W) to alter access to the peripheral pocket. Modest increases in catalytic efficiency of 7-EFC O-deethylation by the mutants were magnified considerably by chlorination or bromination of the substrate ethoxy chain. A structure of CYP2B6 Y244W in complex with (+)-α-pinene was solved at 2.2 Å and showed no CYMAL-5 in the peripheral pocket. A ligand free structure of CYP2B4 F244W was solved at 3.0 Å with CYMAL-5 in the peripheral pocket. In both instances, comparison of the respective wild-type and mutant CYP2B enzymes revealed that CYMAL-5 occupancy of the peripheral pocket had little effect on the topology of active site residue side-chains, despite the fact that the peripheral pocket and active site are located on opposite sides of the I-helix. Analysis of available CYP2B structures suggest that the effect of the amino acid substitutions within the peripheral pocket derive from altered interactions between the F and G helices.

Structural studies on molecular mechanisms of Nelfinavir resistance caused by non-active site mutation V77I in HIV-1 protease.

BackgroundThe human immunodeficiency virus (HIV-1) is a retrovirus causing acquired immunodeficiency syndrome (AIDS), which has become a serious problem across the world and has no cure reported to date. Human immunodeficiency virus (HIV-1) protease is an attractive target for antiviral treatment and a number of therapeutically useful inhibitors have been designed against it. The emergence of drug resistant mutants of HIV-1 poses a serious problem for conventional therapies that have been used so far. Until now, thirteen protease inhibitors (PIs), major mutation sites and many secondary mutations have been listed in the HIV Drug Resistance Database. In this study, we have studied the effect of the V77I mutation in HIV-PR along with the co-occurring mutations L33F and K20T through multi-nanosecond molecular dynamics simulations. V77I is known to cause Nelfinavir (NFV) resistance in the subtype B population of HIV-1 protease. We have for the first time reported the effect of this clinically relevant mutation on the binding of Nelfinavir and the conformational flexibility of the protease.ResultsTwo HIV-PR mutants have been considered in this study - the Double Mutant Protease (DBM) V77I-L33F and Triple Mutant Protease (TPM) V77I-K20T-L33F. The molecular dynamics simulation studies were carried out and the RMSD trajectories of the unliganded wild type and mutated protease were found to be stable. The binding affinity of NFV with wild type HIV-PR was very high with a Glide XP docking score of -9.3 Kcal/mol. NFV showed decreased affinity towards DBM with a docking score of -8.0 Kcal/mol, whereas its affinity increased towards TPM (Glide XP score: -10.3). Prime/MM-GBSA binding free energy of the wild type, DBM and TPM HIV-PR docked structures were calculated as -38.9, -11.1 and -42.6 Kcal/mol respectively. The binding site cavity volumes of wild type, DBM and TPM protease were 1186.1, 1375.5 and 1042.5 Å3 respectively.ConclusionIn this study, we have studied the structural roles of the two HIV-PR mutations by conducting molecular dynamics simulation studies of the wild type and mutant HIV-1 PRs. The present study proposes that DBM protease showed greater flexibility and the flap separation was greater with respect to the wild type protease. The cavity size of the MD-stabilized DBM was also found to be increased, which may be responsible for the decreased interaction of Nelfinavir with the cavity residues, thus explaining the decreased binding affinity. On the other hand, the binding affinity of NFV for TPM was found to be enhanced, accounted for by the decrease in cavity size of the mutant which facilitated strong interactions with the flap residues. The flap separation of TPM was less than the wild type protease and the decreased cavity size may be responsible for its lower resistance, and hence, may be the reason for its lower clinical relevance.

Dynamical Network in HIV-1 Protease and its Mutants: Implications on Drug Resistance

Drugs targeting HIV protease have long been used in AIDS therapy. However, emergence of multi-drug resistance, due to active and non-active site mutations, intensifies the need to understand the drug resistance mechanism of the enzyme. Here, we employ molecular dynamics (MD) simulations and network analyses to unravel the drug resistance mechanisms. Results from MD simulations suggest that the mutants modulate either the ligand binding envelop in the active site or the dynamics of flaps in HIV-1 protease. While the mutations at the active site or flap region account for drug resistance directly, the mechanism of allosteric mutations could not be explained. Network analyses show that the allosteric mutations affect the functional sites by modulating the stress centrality. Stress interference data ascribe the residues 71, 90 and 93 as the active site modulators and 15, 20 and 36 as the flap modulators. The integrity of the global network, however, was not affected by deletion of the mutant-prone nodes, indicating that the function of HIV-1 protease is preserved.

Uncovering and Redesigning a Key Amino Acid of Glucose Oxidase for Improved Biotechnological Applications

AbstractHere we uncover a key amino acid at the entrance of the active site of glucose oxidase from Penicilium amagasakiense and we successfully redesign it by nonactive site mutations, leading to enzymatic anodes with 2.4 fold higher current densities. The increase in current density could be correlated with a better interaction between the negatively charged amino acid located in position 424 and the positively charged redox mediator. We also demonstrate that this increase in current was not specific to Osmium and that further introduction of negative charges around the active site of glucose oxidase lead to a decrease in current density, illustrating the uniqueness of this amino acid in position 424.

A molecular dynamics investigation on the crizotinib resistance mechanism of C1156Y mutation in ALK

Crizotinib is an anaplastic lymphoma kinase (ALK) inhibitor that has recently been approved in the US for the treatment of non-small cell lung carcinoma (NSCLC). Despite its outstanding safety and efficacy, several resistant mutations against crizotinib have been detected in the treatment of NSCLC. However, in contrast to the widely accepted mechanism of steric hindrance by mutations at the active site, the mechanism by which the C1156Y non-active site mutation confers resistance against crizotinib remains unclear. In the present study, the resistance mechanism of C1156Y in ALK was investigated using molecular dynamics simulations. The results suggest that despite the non-active site mutation, C1156Y causes the dislocation of crizotinib as well as the indirect conformational changes in the binding cavity, which results in a marked decrease in the van der Waals and electrostatic interactions between crizotinib and ALK. The obtained results provide a detailed explanation of the resistance caused by C1156Y and may give a vital clue for the design of drugs to combat crizotinib resistance.

A Comparative Study of Drug Resistance Mechanism Associated with Active Site and Non-Active Site Mutations: I388N and D425G Mutants of Acetyl-Coenzyme-A Carboxylase

A major concern in the development of acetyl-CoA carboxylase-inhibiting (ACCase; EC 6.4.1.2) herbicides is the emergence of resistance as a result of the selection of distinct mutations within the CT domain. Mutations associated with resistance have been demonstrated to include both active sites and non-active sites, including Ile-1781-Leu, Trp- 2027-Cys, Ile-2041-Asn, Asp-2078-Gly, and Gly-2096-Ala (numbered according to the Alopecurus myosuroides plastid ACCase). In the present study, extensive computational simulations, including molecular dynamics (MD) simulations and molecular mechanics-Poisson-Boltzmann surface area (MM/PBSA) calculations, were carried out to compare the molecular mechanisms of active site mutation (I388N) and non-active site mutation (D425G) in Alopecurus myosuroides resistance to some commercial herbicides targeting ACCase, including haloxyfop (HF), diclofop (DF) and fenoxaprop (FR). All of the computational model and energetic results indicated that both I388N and D425G mutations have effects on the conformational change of the binding pocket. The π-π interaction between ligand and Phe377 and Tyr161' residues, which make an important contribution to the binding affinity, was decreased after mutation. As a result, the mutant-type ACCase has a lower affinity for the inhibitor than the wild-type enzyme, which accounts for the molecular basis of herbicidal resistance. The structural and mechanistic insights obtained from the present study will deepen our understanding of the interactions between ACCase and herbicides, which provides a molecular basis for the future design of a promising inhibitor with low resistance risk.

Evolved streptavidin mutants reveal key role of loop residue in high‐affinity binding

We have performed a detailed analysis of streptavidin variants with altered specificity towards desthiobiotin. In addition to changes in key residues which widen the ligand binding pocket and accommodate the more structurally flexible desthiobiotin, the data revealed the role of a key, non-active site mutation at the base of the flexible loop (S52G) which slows dissociation of this ligand by approximately sevenfold. Our data suggest that this mutation results in the loss of a stabilizing contact which keeps this loop open and accessible in the absence of ligand. When this mutation was introduced into the wild-type protein, destabilization of the opened loop conferred a ∼10-fold decrease in both the on-rate and off-rate for the ligand biotin-4-fluoroscein. A similar effect was observed when this mutation was added to a monomeric form of this protein. Our results provide key insight into the role of the streptavidin flexible loop in ligand binding and maintaining high affinity interactions.

Double Electron-Electron Resonance and 1H-15N HSQC Spectral Analysis as Means to Understand the Effects of Single and Multiple Site Mutations in the HIV-1 protease

In this study, site-directed spin labeling coupled with electron paramagnetic resonance (SDSL-EPR) was used to probe changes in the conformational sampling of subtype B human immunodeficiency virus type 1 protease (HIV-1PR) attributed to the active site mutation D30N and the non-active site mutations M36I and A71V. Double electron-electron resonance (DEER) was employed to derive distance distribution profiles for each MTSL-labeled construct. Those with double mutations (D30N M36I and D30N A71V) exhibited higher percentage of the closed conformation while that with triple mutation (D30N M36I A71V) has a higher percentage of wide-open conformation relative to wild-type. The percentage semi-open conformation markedly decreased in the former constructs while it is comparable to wild-type for the latter.Meanwhile, 1H-15N heteronuclear single quantum coherence (HSQC) spectra were obtained for HIV-1PR constructs with single (M36I), double (D30N M36I) and triple mutations (D30N M36I A71V), and were compared to that of the wild-type. HIV-1PR residues that have marked chemical shift perturbations as a result of an active or non-active site mutation were identified. Non-active site mutations occurring at the flap elbow (M36I) or beta-sheet core (A71V) of the protease were found to affect chemical shift more than the mutation at the active site pocket (D30N). Information gained from single-point mutation-induced changes in the conformational sampling and dynamics of the HIV-1PR may provide key insights on the mechanism of drug resistance.

Resistant mechanism against nelfinavir of subtype C human immunodeficiency virus type 1 proteases

Human immunodeficiency virus type 1 protease (HIV-1 PR) is one of the major targets of anti-AIDS drug discovery, and the subtype C HIV-1 is infecting more and more humans. In this work, we executed computational simulations of subtype B (labeled B(WT)) HIV-1 PR, D30N mutant B (labeled B(D30N)) HIV-1 PR, subtype C (labeled C(Ref)) HIV-1 PR, D30N mutant C (labeled C(D30N)) HIV-1 PR, and D30N/N83T mutant C (labeled C(D30N/N83T)) HIV-1 PR with drug nelfinavir (NFV), aiming at clarifying (1) the resistant mechanism against NFV due to D30N mutation in subtype C HIV-1 PR; (2) the reason that the emergence rate of N83T mutation in C(D30N) HIV-1 PR is higher than that in B(D30N) HIV-1 PR; (3) the affinity of NFV with C(D30N/N83T) HIV-1 PR is higher than B(D30N) and C(D30N) HIV-1 PRs. The results indicate: (1) D30N mutation in subtype C HIV-1 PR reduces the hydrogen bond between the 30th residue and NFV, and the binding free energy contributions of some residues decrease; (2) the hydrogen bonds between the 83th/83′th residue and the 34th/34′th residue exist in both B(D30N) and C(D30N/N83T) HIV-1 PRs, while they disappear in C(D30N) HIV-1 PR. Meanwhile, the binding free energy contribution of N30 in C(D30N) is lower than that in B(D30N) and C(D30N/N83T); (3) N83T mutation makes some residues dislocate, and the contributions of these residues to binding free energy in C(D30N/N83T) increase comparing to those in B(D30N) and C(D30N). Our findings suggest that despite the nonactive site mutations, the polymorphisms regulate the emergence rates of these drug-resistant mutants.