Chronology of sodium channel mutations associated with pyrethroid resistance in Aedes aegypti

Aedes aegypti is the primary mosquito vector of dengue, yellow fever, Zika and chikungunya. Current strategies to control Ae. aegypti rely heavily on insecticide interventions. Pyrethroids are a major class of insecticides used for mosquito control because of their fast acting, highly insecticidal activities and low mammalian toxicity. However, Ae. aegypti populations around the world have begun to develop resistance to pyrethroids. So far, more than a dozen mutations in the sodium channel gene have been reported to be associated with pyrethroid resistance in Ae. aegypti. Co-occurrence of resistance-associated mutations is common in pyrethroid-resistant Ae. aegypti populations. As global use of pyrethroids in mosquito control continues, new pyrethroid-resistant mutations keep emerging. In this microreview, we compile pyrethroid resistance-associated mutations in Ae. aegypti in a chronological order, as they were reported, and summarize findings from functional evaluation of these mutations in an in vitro sodium channel expression system. We hope that the information will be useful for tracing possible evolution of pyrethroid resistance in this important human disease vector, in addition to the development of methods for global monitoring and management of pyrethroid resistance in Ae. aegypti.     

Allostery in Orai1 binding to calmodulin revealed from conformational thermodynamics

Here, we study microscopic mechanism of complex formation between Ca²⁺-bound calmodulin (holoCaM) and Orai1 that regulates Ca²⁺-dependent inactivation process in eukaryotic cells. We compute conformational thermodynamic changes in holoCaM with respect to complex of Orai1 bound to C-terminal domain of holoCaM using histograms of dihedral angles of the proteins over trajectories from molecular dynamics simulations. Our analysis shows that the N-terminal domain residues L4, T5, Q41, N42, T44 and E67 of holoCaM get destabilized and disordered due to Orai1 binding to C-terminal domain of calmodulin affect the N-terminal domain residues. Among these residues, polar T44, having maximum destabilization and disorder via backbone fluctuations, shows the largest change in solvent exposure. This suggests that N-terminal domain is allosterically regulated via T44 by the binding of Orai1 to the C-terminal domain.     

Mutations in exons 10 and 11 of human glucokinase result in conformational variations in the active site of the structure contributing to poor substrate binding – explains hyperglycemia in type 2 diabetic patients

Mutations in the glucokinase (GK) gene play a critical role in the establishment of type 2 diabetes. In our earlier study, R308K mutation in GK in a clinically proven type 2 diabetic patient showed, structural and functional variations that contributed immensely to the hyperglycemic condition. In the extension of this work, a cohort of 30 patients with established type 2 diabetic condition were chosen and the exons 10 and 11 of GK were PCR-amplified and sequenced. The sequence alignment showed A379S, D400Y, E300A, E395A, E395G, H380N, I348N, L301M, M298I, M381G, M402R, R308K, R394P, R397S, and S398R mutations in 12 different patients. The structural analysis of these mutated GKs, showed a variable number of β-α-β units, hairpins, β-bulges, strands, helices, helix–helix interactions, β-turns, and γ-turns along with the RMSD variations when compared to wild-type GK. Molecular modeling studies revealed that the substrate showed variable binding orientations and could not fit into the active site of these mutated structures; moreover, it was expelled out of the conformations. Therefore, these structural variations in GK due to mutations could be one of the strongest reasons for the hyperglycemic levels in these type 2 diabetic patients.     

昆虫钠离子通道基因突变及其与杀虫剂抗性关系的研究进展

昆虫电压门控钠离子通道(voltage-gated sodium channel)存在于所有可兴奋细胞的细胞膜上,在动作电位的产生和传导上起重要作用,是有机氯和拟除虫菊酯杀虫剂的靶标位点。在农业和医学害虫控制过程中,由于有机氯和拟除虫菊酯杀虫剂的广泛使用,抗药性问题日益突出。其中,由于钠离子通道基因突变,降低了钠离子通道对有机氯和拟除虫菊酯类杀虫剂的亲和性,从而产生击倒抗性(knock-down resistance, kdr),已成为抗性产生的重要机制之一。本文综述了昆虫钠离子通道的跨膜拓扑结构、功能、进化及其基因的克隆;更重要的是总结了已报道的40多种昆虫40个钠离子通道基因非同义突变,以及钠离子通道基因选择性mRNA剪接和编辑,以及它们与杀虫剂抗性的关系;也评述了钠离子通道基因突变引起蛋白质结构的改变,从而对杀虫剂抗性的影响机制。这些研究对于进一步鉴定与杀虫剂抗性相关的突变及抗性机制,开发有机氯和拟除虫菊酯类杀虫剂抗性分子监测方法具有重要意义。     

Differential resistance of insect sodium channels with kdr mutations to deltamethrin, permethrin and DDT

Knockdown resistance (kdr) in insects, caused by inherited nucleotide polymorphisms in the voltage-gated sodium channel (VGSC) gene, is a major threat to the efficacy of pyrethroid insecticides. Classic kdr, resulting from an L1014F substitution in the VGSC is now present in numerous pest species. Two other substitutions at the L1014 locus have also been reported, L1014S and L1014H. Here we have used expression of L1014 modified Drosophila para VGSCs in Xenopus oocytes with two-electrode voltage clamp to characterise all three mutations. The mutations L1014F and L1014H caused significant depolarizing shifts in the half activation voltage (V_50,act) from −17.3 mV (wild-type) to −13.1 and −13.5 mV respectively, whereas L1014S caused no shift in V_50,act but its currents decayed significantly faster than wild-type channels. Treatment of the wild-type channel with deltamethrin (≥1 nM), permethrin (≥30 nM) or DDT (≥1 ??M) resulted in hyperpolarizing shifts in V_50,act. Deltamethrin, permethrin and DDT also produced “tail currents” with EC₅₀s of 0.043, 0.40 and 65 ??M and maximum modifications of 837, 325 and 7% respectively. L1014F provided a high level of resistance against all insecticides for both measured parameters. L1014H most effectively combated deltamethrin induced tail currents while L1014S strongly resisted the large DDT induced shifts in V_50,act. We conclude that L1014H and L1014S may have arisen through heavy exposure to specific pyrethroids and DDT respectively.     

The relationship between the flexibility of proteins and their conformational states on forming protein-protein complexes with an application to protein-protein docking

We investigate the extent to which the conformational fluctuations of proteins in solution reflect the conformational changes that they undergo when they form binary protein-protein complexes. To do this, we study a set of 41 proteins that form such complexes and whose three-dimensional structures are known, both bound in the complex and unbound. We carry out molecular dynamics simulations of each protein, starting from the unbound structure, and analyze the resulting conformational fluctuations in trajectories of 5 ns in length, comparing with the structure in the complex. It is found that fluctuations take some parts of the molecules into regions of conformational space close to the bound state (or give information about it), but at no point in the simulation does each protein as whole sample the complete bound state. Subsequent use of conformations from a clustered MD ensemble in rigid-body docking is nevertheless partially successful when compared to docking the unbound conformations, as long as the unbound conformations are themselves included with the MD conformations and the whole globally rescored. For one key example where sub-domain motion is present, a ribonuclease inhibitor, principal components analysis of the MD was applied and was also able to produce conformations for docking that gave enhanced results compared to the unbound. The most significant finding is that core interface residues show a tendency to be less mobile (by size of fluctuation or entropy) than the rest of the surface even when the other binding partner is absent, and conversely the peripheral interface residues are more mobile. This surprising result, consistent across up to 40 of the 41 proteins, suggests different roles for these regions in protein recognition and binding, and suggests ways that docking algorithms could be improved by treating these regions differently in the docking process.     

Molecular modeling study for conformational changes of Sirtuin 2 due to substrate and inhibitor binding

Sirtuin is a member of NAD⁺-dependent deacetylase family. The structural details of Sirtuin 2 (SIRT2) complex will be very useful to discover the drug which might have beneficial effects on various diseases like cancer, diabetes, etc. Unfortunately, SIRT2 complex structure is not available yet, hence molecular docking was carried out to dock the substrate (NAD⁺ and acetylated lysine) and inhibitor (sirtinol) in the NAD⁺ binding site. The suitable binding orientation of substrate and inhibitor in the SIRT2 active site was selected and subjected to 5?ns molecular dynamics simulations to adjust the binding orientation of inhibitor and substrate as well as to identify the conformational changes in the active site. The result provides an insight about 3D SIRT2 structural details as well as the importance of F96 in deacetylation function. In addition, our simulations revealed the displacement of F96 upon substrate and inhibitor binding, inducing an extended conformation of loop3 and changing its interactions with the rest of SIRT2. We believe that our study could be helpful to gain a structural insight of SIRT2 and to design the receptor-based inhibitors.     

昆虫击倒抗性基因突变对钠通道功能的影响

该文综述了昆虫钠通道基因的表达与功能特性、击倒抗性突变的功能和这些突变对钠通道门控的影响,以及钠通道基因突变与抗性表现型之间的因果关系;还讨论了这些突变增强击倒抗性的分子机理。     

Cooperative symmetric to asymmetric conformational transition of the apo-form of scavenger decapping enzyme revealed by simulations

Decapping is a central step in eukaryotic mRNA turnover and in gene expression regulation. The human scavenger decapping enzyme, DcpS, catalyses cap hydrolysis following mRNA degradation. DcpS is a dimeric enzyme, with two active sites. Crystal structures suggest that DcpS must undergo significant conformational changes upon ligand binding, but the mechanism of this transition is unknown. Here, we report two long timescale (20 ns) molecular dynamics simulations of the apo-form of DcpS. The dimer is observed to undergo a strikingly cooperative motion, with one active site closing while the other opens. The amplitude of the conformational change is 6-21 A and the apparent timescale is 4-13 ns. These findings indicate that the crystallographically observed symmetric conformation of apo-form of DcpS is only a minor conformation in solution. The simulations also show that active sites are structurally connected via the domain-swapped dimer structure of the N-terminal domain, even in the absence of a bound ligand. These findings suggest a functional reason for the enzyme existing as a dimer, and may be widely relevant, also for other dimeric proteins.     

Equivalent intensity but differential dominance of sodium channel blocker insecticide resistance conferred by F1845Y and V1848I mutations of the voltage-gated sodium channel in Plutella xylostella

Two point mutations (F1845Y and V1848I) in the voltage-gated sodium channel gene of Plutella xylostella are involved in the target-site resistance to sodium channel blocker insecticides (SCBIs). The contribution of the individual mutations to the SCBI resistance and the associated inheritance modes is as yet unclear. Through 2 rounds of single-pair crossing and marker-assisted selection, 2 P. xylostella strains (1845Y and 1848I) bearing homozygous F1845Y or V1848I mutant alleles were successfully established from a field-collected population, and the contribution of each mutation to SCBI resistance, as well as associated inheritance patterns, was determined. When compared with the susceptible SZPS strain, each of the mutations individually conferred equally high-level resistance to indoxacarb (378 and 313 fold) and metaflumizone (734 and 674 fold), respectively. However, dominance levels of resistance to SCBIs were significantly different between the 2 resistant strains. Resistance of the 1845Y strain to indoxacarb and metaflumizone was inherited as an autosomal and incompletely dominant trait (D values ranged from 0.43 to 0.76). In contrast, that of the 1848I strain followed an autosomal but incompletely recessive to semidominant mode (D values: −0.24 to 0.09). Our findings enriched the current understanding of inheritance and mechanisms of SCBI resistance in P. xylostella, and will help develop resistance management programs for P. xylostella and other economic pests.     

细胞色素P450介导的昆虫抗药性的分子机制

细胞色素P450(简称P450) 对杀虫剂的代谢作用直接影响到昆虫对杀虫剂的耐受性和杀虫剂对昆虫的选择性,由P450介导的杀虫剂代谢解毒作用的增强是昆虫产生抗药性的常见而重要的机制。P450介导的杀虫剂代谢抗性具有普遍性、交互抗性与进化可塑性的特点,涉及P450基因重复与基因扩增、基因转录上调以及结构基因的变异等多样化的分子机制,并且多重机制的共同作用可以导致高水平抗药性。这些研究发现说明,无论是昆虫抗药性机制的研究,还是抗药性监测与治理都要有动态的、因地制宜的理念。     

Conserved tertiary couplings stabilize elements in the PDZ fold,leading to characteristic patterns of domain conformational flexibility

Single‐domain allostery has been postulated to occur through intramolecular pathways of signaling within a protein structure. We had previously investigated these pathways by introducing a local thermal perturbation and analyzed the anisotropic propagation of structural changes throughout the protein. Here, we develop an improved approach, the Rotamerically Induced Perturbation (RIP), that identifies strong couplings between residues by analyzing the pathways of heat‐flow resulting from thermal excitation of rotameric rotations at individual residues. To explore the nature of these couplings, we calculate the complete coupling maps of 5 different PDZ domains. Although the PDZ domain is a well conserved structural fold that serves as a scaffold in many protein–protein complexes, different PDZ domains display unique patterns of conformational flexibility in response to ligand binding: some show a significant shift in a set of α‐helices, while others do not. Analysis of the coupling maps suggests a simple relationship between the computed couplings and observed conformational flexibility. In domains where the α‐helices are rigid, we find couplings of the α‐helices to the body of the protein, whereas in domains having ligand‐responsive α‐helices, no couplings are found. This leads to a model where the α‐helices are intrinsically dynamic but can be damped if sidechains interact at key tertiary contacts. These tertiary contacts correlate to high covariation contacts as identified by the statistical coupling analysis method. As these dynamic modules are exploited by various allosteric mechanisms, these tertiary contacts have been conserved by evolution.     

Molecular modeling,docking and ADMET studies towards development of novel Disopyramide analogs for potential inhibition of human voltage gated sodium channel proteins

The sodium “channelopathies” are the first among the ion channel diseases identified and have attracted widespread clinical and scientific interests. Human voltage gated sodium channels are sites of action of several antiarrhythmic drugs, local anesthetics and related antiepileptic drugs. The present study aims to optimize the activity of Disopyramide, by modification in its structures which may improve the drug action by reducing its side effects. Herein, we have selected Human voltage-gated sodium channel protein type 5 as a potent molecular target. Nearly eighty analogs of Disopyramide are designed and optimized. Thirty are selected for energy minimization using Discovery studio and the LigPrep 2.5. Prior to docking, the active sites of all the proteins are identified. The processing, optimization and minimization of all the proteins is done in Protein preparation wizard. The docking study is performed using the GLIDE. Finally top five ranked lead molecules with better dock scores are identified as having strong binding affinity to 2KAV protein than Disopyramide based on XP G scores. These five leads are further docked with other similar voltage gated sodium channel proteins (PDB IDs: 2KBI, 4DCK, 2L53 and 4DJC) and the best scoring analog with each protein is identified. Drug likeliness and comparative bioactivity analysis for all the analogs is done using QikProp 3.4. Results have shown that the top five lead molecules would have the potential to act as better drugs as compared to Disopyramide and would be of interest as promising starting point for designing compounds against various Sodium channelopathies.     

Shift in nucleotide conformational equilibrium contributes to increased rate of catalysis of GpAp versus GpA in barnase

The microbial ribonuclease barnase exhibits low catalytic activity toward GpN dinucleotides, where G is guanosine, p is phosphate and N represents any nucleoside. When a phosphate is added to the 3'-end, as in GpNp, substrate affinity is enhanced by one order of magnitude, and the catalytic rate by two. In order to gain insight into this phenomenon, we analyzed the nucleotide conformations and protein-nucleotide interactions of 4 ns molecular dynamics (MD) trajectories of complexes of barnase with guanylyl(3'-5') adenosine (GpA) and guanylyl(3'-5') adenosine 3'-monophosphate (GpAp), respectively, in the presence of solvent and counter ions. We found that, in a majority of the bound GpA conformations, the guanine base was firmly bound to the recognition site. The phosphate and adenosine moieties pointed into the solvent, and interactions with key catalytic residues were absent. In contrast, the bound GpAp adopted conformations in which all of the nucleotide portions remained tightly bound to the enzyme and interactions with key catalytic residues were maintained. These observations indicate that, for GpA, a significant proportion of the bound nucleotide adopts non-productive conformations and that adding the terminal phosphate as in GpAp shifts the equilibrium of the bound conformations towards structures capable of undergoing catalysis. Incorporating this property into the kinetic equations yields an increase in both the apparent rate constant (kcat) and the apparent dissociation constant (K(M)) for GpAp versus GpA. The increase in K(M), caused by the presence of additional non-productive binding modes for GpA, should however be counterbalanced by the propensity of free GpA to adopt folded conformations in solution, which are unable to bind the enzyme and by the tighter binding of GpAp (Giraldo J, Wodak SJ, Van Belle D. Conformational analysis of GpA and GpAp in aqueous solution by molecular dynamics and statistical methods. J Mol Biol 1998; 283:863-882). Addition of the terminal phosphate is shown to significantly influence the collective motion of the enzyme in a manner that fosters interactions with key catalytic residues, representing a further likely contribution to the catalytic rate enhancement.     

Molecular dynamics study of conformational changes in human serum albumin by binding of fatty acids

Human serum albumin (HSA) binds with fatty acids under normal physiologic conditions. To date, there is little published information on the tertiary structure of HSA-fatty acid complex in aqueous solution. In the present study, we used molecular dynamics (MD) simulations to elucidate possible structural changes of HSA brought about by the binding of fatty acids. Both unliganded HSA and HSA-fatty acid complex models for MD calculations were constructed based on the X-ray crystal structures. Five myristates (MYRs) were bound in the HSA-fatty acid complex model. In the present MD study, the motion of domains I and III caused by the binding of MYR molecules increased the radius of gyration of HSA. Root-mean-square fluctuations from the MD simulations revealed that the atomic fluctuations of the specific amino acids at drug-binding site I that can regulate the drug-binding affinity were increased by the binding of MYR molecules. Primary internal motions, characterized by the first three principal components, were observed mainly at domains I and III in the principal component analysis for trajectory data. The directional motion projected on the first principal component of unliganded HSA was conserved in HSA-MYR complex as the third principal directional motion with higher frequency. However, the third principal directional motion in unliganded HSA turned into the first principal directional motion with lower frequency in the HSA-MYR complex. Thus, the present MD study provides insights into the possible conformational changes of HSA caused by the binding of fatty acids.