Study of the impact of the T877A mutation on ligand‐induced helix‐12 positioning of the androgen receptor resulted in design and synthesis of novel antiandrogens

Antiandrogen flutamide, an antagonist of the wild‐type androgen receptor (AR), is used in the clinics for treating metastatic prostate cancer. However, the T877A mutated AR is paradoxically activated by hydroxyflutamide, an active form of flutamide. Despite of crystallographic studies, how the T877A mutation results in antagonist‐agonist conversion of hydroxyflutamide remains a puzzle. Here, started from a structural model of the apo form of AR ligand‐binding domain (AR‐LBD), we have investigated the impact of the T877A mutation on ligand‐induced helix‐12 positioning by replica‐exchange molecular dynamics (REMD) simulations with an unique protocol, which is capable of simulating the H12 dynamics and keeping the main body of AR‐LBD unchanged. Specifically, (i) we have computationally demonstrated that on the binding of hydroxyflutamide, the apo form of H12 rearranges into the agonistic form in the T877A mutant, but into the antagonistic forms in the wild‐type receptor, shedding light on hydroxyflutamide agonism/antagonism; (ii) By REMD simulations, we have predicted antiandrogen SC184 is a non‐agonist of the T877A mutant. This was confirmed by luciferase assays; and (iii) on the basis of the binding modes of hydroxyflutamide and SC184 from the simulations, we designed a novel flutamide derivative called SC333, which was subsequently predicted to be a pure antagonist of the T877A mutant. We then synthesized and experimentally confirmed SC333 is a pan‐antiandrogen effective against the wild‐type and the T877A and W741C mutated ARs, showing low micromolar cytotoxicity in LNCaP cells. Importantly, we demonstrated that distribution of the H12 conformations from REMD simulations is correlated with ligand agonist/antagonist activity. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Transient sampling of aggregation‐prone conformations causes pathogenic instability of a parkinsonian mutant of DJ‐1 at physiological temperature

Various missense mutations in the cytoprotective protein DJ‐1 cause rare forms of inherited parkinsonism. One mutation, M26I, diminishes DJ‐1 protein levels in the cell but does not result in large changes in the three‐dimensional structure or thermal stability of the protein. Therefore, the molecular defect that results in loss of M26I DJ‐1 protective function is unclear. Using NMR spectroscopy near physiological temperature, we found that the picosecond–nanosecond dynamics of wild‐type and M26I DJ‐1 are similar. In contrast, elevated amide hydrogen/deuterium exchange rates indicate that M26I DJ‐1 is more flexible than the wild‐type protein on longer timescales and that hydrophobic regions of M26I DJ‐1 are transiently exposed to solvent. Tryptophan fluorescence spectroscopy and thiol crosslinking analyzed by mass spectrometry also demonstrate that M26I DJ‐1 samples conformations that differ from the wild‐type protein at 37°C. These transiently sampled conformations are unstable and cause M26I DJ‐1 to aggregate in vitro at physiological temperature but not at lower temperatures. M26I DJ‐1 aggregation is correlated with pathogenicity, as the structurally similar but non‐pathogenic M26L mutation does not aggregate at 37°C. The onset of dynamically driven M26I DJ‐1 instability at physiological temperature resolves conflicting literature reports about the behavior of this disease‐associated mutant and illustrates the pitfalls of characterizing proteins exclusively at room temperature or below, as key aspects of their behavior may not be apparent.     

Molecular dynamics simulation of PNPLA3 I148M polymorphism reveals reduced substrate access to the catalytic cavity

A missense mutation I148M in PNPLA3 (patatin‐like phospholipase domain‐containing 3 protein) is significantly correlated with nonalcoholic fatty liver disease (NAFLD). To glean insights into mutation's effect on enzymatic activity, we performed molecular dynamics simulation and flexible docking studies. Our data show that the size of the substrate‐access entry site is significantly reduced in mutants, which limits the access of palmitic acid to the catalytic dyad. Besides, the binding free energy calculations suggest low affinity for substrate to mutant enzyme. The substrate‐bound system simulations reveal that the spatial arrangement of palmitic acid is distinct in wild‐type from that in mutant. The substrate recognition specificity is lost due to the loop where the I148M mutation was located. Our results provide strong evidence for the mechanism by which I148M affects the enzyme activity and suggest that mediating the dynamics may offer a potential avenue for NAFLD. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

The function and the expression of forebrain GABAA receptors change with hormonal state in the adult mouse

Neurotransmission mediated by gamma‐aminobutyric acid type A (GABA_A) receptors in the mammalian medial preoptic area (mPOA) plays a pivotal role in the expression of hormone‐sensitive behaviors. Hand in hand with GABAergic control of reproduction, hormone treatments that activate gonadal steroid signaling pathways in gonadectomized rats are known to regulate the expression of specific GABA_A receptor subunit mRNAs. While the effects of exogenous hormone treatments have been well documented, little information is available as to how GABA_A receptor‐mediated transmission in the mPOA is altered by endogenous changes in hormonal state in gonadally‐intact adult animals or if those changes can be ascribed to hormone‐dependent changes in receptor subunit composition. In the present study, we found that both the peak amplitudes of GABA_A receptor‐mediated synaptic currents in the mPOA, as well as the ability of the endogenous neurosteroids to modulate those currents, varied as a function of the estrous cycle. Moreover, we found that the degree of neurosteroid modulation was also significantly different between wild‐type and the androgen‐insensitive testicular feminization (Tfm) mutant male mice. Semiquantitative RT‐PCR analysis performed to assess levels of GABA_A receptor subunit mRNAs indicated that levels of specific subunits varied over the course of the estrous cycle and between wild‐type and Tfm male mice. The variations in GABA_A receptor expression and function in the mPOA that are associated with differences in gonadal steroid signaling may contribute to the dynamic nature of GABAergic control of neuroendocrine pathways. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 137–149, 2002; DOI 10.1002/neu.10021     

Chorein deficiency leads to upregulation of gephyrin and GABA(A) receptor

Chorea-acanthocytosis (ChAc) is a hereditary neurodegenerative disorder caused by loss of function mutations in the VPS13A gene encoding chorein. Recently, using a gene-targeting technique to delete exons 60-61, we produced a ChAc-model mouse that corresponds to a human disease mutation. In this study, a comparative microarray analysis of gene expression in the striatum revealed an increased level of gephyrin gene expression in the ChAc-model mice compared with wild type mice. Since gephyrin is known as a GABA(A) receptor-anchoring protein, we compared the protein-level expression and localization of gephyrin and the GABA(A) receptor alpha1 (GABRA1) and gamma2 (GABRG2) subunits. Gephyrin and GABRG2 immunoreactivities in the striatum and hippocampus of the ChAc-model mice were significantly higher than those in the wild types. Our results suggest that chorein functional loss may lead to a compensatory upregulation of gephyrin and GABRG2 in the pathologic condition in ChAc.     

Insights into allosteric control of microtubule dynamics from a buried β‐tubulin mutation that causes faster growth and slower shrinkage

αβ‐tubulin subunits cycle through a series of different conformations in the polymer lattice during microtubule growing and shrinking. How these allosteric responses to different tubulin:tubulin contacts contribute to microtubule dynamics, and whether the contributions are evolutionarily conserved, remains poorly understood. Here, we sought to determine whether the microtubule‐stabilizing effects (slower shrinking) of the β:T238A mutation we previously observed using yeast αβ‐tubulin would generalize to mammalian microtubules. Using recombinant human microtubules as a model, we found that the mutation caused slow microtubule shrinking, indicating that this effect of the mutation is indeed conserved. However, unlike in yeast, β:T238A human microtubules grew faster than wild‐type and the mutation did not appear to attenuate the conformational change associated with guanosine 5′‐triphosphate (GTP) hydrolysis in the lattice. We conclude that the assembly‐dependent conformational change in αβ‐tubulin can contribute to determine the rates of microtubule growing as well as shrinking. Our results also suggest that an allosteric perturbation like the β:T238A mutation can alter the behavior of terminal subunits without accompanying changes in the conformation of fully surrounded subunits in the body of the microtubule.     

A positive‐charged patch and stabilized hydrophobic core are essential for avirulence function of AvrPib in the rice blast fungus

Fungal avirulence effectors, a key weapon utilized by pathogens to promote their infection, are recognized by immune receptors to boost host R gene‐mediated resistance. Many avirulence effectors share sparse sequence homology to proteins with known functions, and their molecular and biochemical functions together with the evolutionary relationship among different members remain largely unknown. Here, the crystal structure of AvrPib, an avirulence effector from Magnaporthe oryzae, was determined and showed a high degree of similarity to the 相似文献   

Molecular modeling of Gly80 and Ser80 variants of human group IID phospholipase A2 and their receptor complexes: potential basis for weight loss in chronic obstructive pulmonary disease

Weight loss is a well known systemic manifestation of chronic obstructive pulmonary disease (COPD). A Gly80Ser mutation on human group IID secretory phospholipase A2 (sPLA₂) enhances expression of the cytokines that are responsible for weight loss. In this study, we seek to establish a structural correlation of wild type sPLA₂ and the Gly80Ser mutation with function. sPLA₂ with glycine and serine at the 80th positions and the M-type receptor were modelled. The enzymes were docked to the receptor and molecular dynamics was carried out to 70 ns. Structural analysis revealed the enzymes to comprise three helices (H1–H3), two short helices (SH1 and SH2), and five loops including a calcium binding loop (L1–L5), and to be stabilized by seven disulfide bonds. The overall backbone folds of the two models are very similar, with main chain RMSD of less than 1 Å. The active site within the substrate binding channel shows a catalytic triad of water–His67–Asp112, showing a hydrogen bonded network. Major structural differences between wild type and mutant enzymes were observed locally at the site of the mutation and in their global conformations. These differences include: (1) loop-L3 between H2 and H3, which bears residue Gly80 in the wild type, is in a closed conformation with respect to the channel opening, while in the mutant enzyme it adopts a relatively open conformation; (2) the mutant enzyme is less compact and has higher solvent accessible surface area; and (3) interfacial binding contact surface area is greater, and the quality of interactions with the receptor is better in the mutant enzyme as compared to the wild type. Therefore, the structural differences delineated in this study are potential biophysical factors that could determine the increased potency of the mutant enzyme with macrophage receptor for cytokine secreting function, resulting in exacerbation of cachexia in COPD.     

Role of the helix capping in the stability of the mouse prion (180-213) segment: investigation through molecular dynamics simulations.

Molecular dynamics simulation of the 180-213 segment, forming the B and C helices in the mouse prion protein, and of three mutants, where the capping box residues or the hydrophobic staple motif residues were selectively mutated, have been carried out. The results indicate that the wild type segment is stable over all the trajectory, whilst the mutants display different degrees of destabilization. In detail mutation of Asp202 brings to a rapid unfolding of helix C likely because of the concomitant loss of a hydrogen bond and of a negative charge able to stabilize the dipole in the first turn of the helix. A lower destabilizing effect is observed upon mutation Thr199. On the other hand mutation of Phe198 and Val203, the hydrophobic staple residues, brings to an incorrect orientation of the first helix relative to the second one due to a weakening of the hydrophobic interaction. The results confirm the importance of the presence of both motifs for the structural integrity of the isolated fragment and suggest that these residues may have a main role in the structural transition observed in the inherited human prion diseases.     

Endoplasmic reticulum retention and associated degradation of a GABAA receptor epilepsy mutation that inserts an aspartate in the M3 transmembrane segment of the alpha1 subunit

A GABA(A) receptor alpha1 subunit epilepsy mutation (alpha1(A322D)) introduces a negatively charged aspartate residue into the hydrophobic M3 transmembrane domain of the alpha1 subunit. We reported previously that heterologous expression of alpha1(A322D)beta2gamma2 receptors in mammalian cells resulted in reduced total and surface alpha1 subunit protein. Here we demonstrate the mechanism of this reduction. Total alpha1(A322D) subunit protein was reduced relative to wild type protein by a similar amount when expressed alone (86 +/- 6%) or when coexpressed with beta2 and gamma2S subunits (78 +/- 6%), indicating an expression reduction prior to subunit oligomerization. In alpha1beta2gamma2S receptors, endoglycosidase H deglycosylated only 26 +/- 5% of alpha1 subunits, consistent with substantial protein maturation, but in alpha1(A322D)beta2gamma2S receptors, endoglycosidase H deglycosylated 91 +/- 4% of alpha1(A322D) subunits, consistent with failure of protein maturation. To determine the cellular localization of wild type and mutant subunits, the alpha1 subunit was tagged with yellow (alpha1-YFP) or cyan (alpha1-CFP) fluorescent protein. Confocal microscopic imaging demonstrated that 36 +/- 4% of alpha1-YFPbeta2gamma2 but only 5 +/- 1% alpha1(A322D)-YFPbeta2gamma2 colocalized with the plasma membrane, whereas the majority of the remaining receptors colocalized with the endoplasmic reticulum (55 +/- 4% alpha1-YFPbeta2gamma2S, 86 +/- 3% alpha1(A322D)-YFP). Heterozygous expression of alpha1-CFPbeta2gamma2S and alpha1(A322D)-YFPbeta2gamma2S or alpha1-YFPbeta2gamma2S and alpha1(A322D)-CFPbeta2gamma2S receptors showed that membrane GABA(A) receptors contained primarily wild type alpha1 subunits. These data demonstrate that the A322D mutation reduces alpha1 subunit expression after translation, but before assembly, resulting in endoplasmic reticulum-associated degradation and membrane alpha1 subunits that are almost exclusively wild type subunits.     

Packing interface energetics in different crystal forms of the λ Cro dimer

Variation among crystal structures of the λ Cro dimer highlights conformational flexibility. The structures range from a wild type closed to a mutant fully open conformation, but it is unclear if each represents a stable solution state or if one may be the result of crystal packing. Here we use molecular dynamics (MD) simulation to investigate the energetics of crystal packing interfaces and the influence of site‐directed mutagenesis on them in order to examine the effect of crystal packing on wild type and mutant Cro dimer conformation. Replica exchange MD of mutant Cro in solution shows that the observed conformational differences between the wild type and mutant protein are not the direct consequence of mutation. Instead, simulation of Cro in different crystal environments reveals that mutation affects the stability of crystal forms. Molecular Mechanics Poisson‐Boltzmann Surface Area binding energy calculations reveal the detailed energetics of packing interfaces. Packing interfaces can have diverse properties in strength, energetic components, and some are stronger than the biological dimer interface. Further analysis shows that mutation can strengthen packing interfaces by as much as ～5 kcal/mol in either crystal environment. Thus, in the case of Cro, mutation provides an additional energetic contribution during crystal formation that may stabilize a fully open higher energy state. Moreover, the effect of mutation in the lattice can extend to packing interfaces not involving mutation sites. Our results provide insight into possible models for the effect of crystallization on Cro conformational dynamics and emphasize careful consideration of protein crystal structures. Proteins 2014; 82:1128–1141. © 2013 Wiley Periodicals, Inc.     

Staurosporine scaffold–based rational discovery of the wild‐type sparing reversible inhibitors of EGFR T790M gatekeeper mutant in lung cancer with analog‐sensitive kinase technology

The human epidermal growth factor receptor (EGFR) has been established as an attractive target for lung cancer therapy. However, an acquired EGFR T790M gatekeeper mutation is frequently observed in patients treated with first‐line anticancer agents such as gefitinib and erlotinib to cause drug resistance, largely limiting the application of small‐molecule kinase inhibitors in EGFR‐targeted chemotherapy. Previously, the reversible pan‐kinase inhibitor staurosporine and its several analogs such as Gö6976 and K252a have been reported to selectively inhibit the EGFR T790M mutant (EGFR^T790M) over wild‐type kinase (EGFR^WT), suggesting that the staurosporine scaffold is potentially to develop the wild‐type sparing reversible inhibitors of EGFR^T790M. Here, we systematically evaluated the inhibitor response of 28 staurosporine scaffold–based compounds to EGFR T790M mutation at structural, energetic, and molecular levels by using an integrated in silico–in vitro analog‐sensitive (AS) kinase technology. With the strategy, we were able to identify 4 novel wild‐type sparing inhibitors UCN‐01, UCN‐02, AFN941, and SB‐218078 with high or moderate selectivity of 30‐, 45‐, 5‐, and 8‐fold for EGFR^T790M over EGFR^WT, respectively, which are comparable with or even better than that of the parent compound staurosporine (24‐fold). Molecular modeling and structural analysis revealed that van der Waals contacts and hydrophobic forces can form between the side chain of mutated residue Met790 and the pyrrolidinone moiety of inhibitor ligand UCN‐02, which may simultaneously improve the favorable interaction energy between the kinase and inhibitor, and reduce the unfavorable desolvation penalty upon the kinase‐inhibitor binding. A hydroxyl group of UCN‐02 additional to staurosporine locates at the pyrrolidinone moiety, which can largely alter the electronic distribution of pyrrolidinone moiety and thus promote the intermolecular interaction with Met790 residue. This can well explain the measured higher selectivity of UCN‐02 than staurosporine for mutant over wild‐type kinase.     

Three amino acids in the D2 dopamine receptor regulate selective ligand function and affinity

The D₂ dopamine receptor is an important therapeutic target for the treatment of psychotic, agitated, and abnormal behavioral states. To better understand the specific interactions of subtype‐selective ligands with dopamine receptor subtypes, seven ligands with high selectivity (>120‐fold) for the D₄ subtype of dopamine receptor were tested on wild‐type and mutant D₂ receptors. Five of the selective ligands were observed to have 21‐fold to 293‐fold increases in D₂ receptor affinity when three non‐conserved amino acids in TM2 and TM3 were mutated to the corresponding D₄ amino acids. The two ligands with the greatest improvement in affinity for the D₂ mutant receptor [i.e., 3‐{[4‐(4‐iodophenyl) piperazin‐1‐yl]methyl}‐1H‐pyrrolo[2,3‐b]pyridine (L‐750,667) and 1‐[4‐iodobenzyl]‐4‐[N‐(3‐isopropoxy‐2‐pyridinyl)‐N‐methyl]‐aminopiperidine (RBI‐257)] were investigated in functional assays. Consistent with their higher affinity for the mutant than for the wild‐type receptor, concentrations of L‐750,667 or RBI‐257 that produced large reductions in the potency of quinpirole’s functional response in the mutant did not significantly reduce quinpirole’s functional response in the wild‐type D₂ receptor. In contrast to RBI‐257 which is an antagonist at all receptors, L‐750,667 is a partial agonist at the wild‐type D₂ but an antagonist at both the mutant D₂ and wild‐type D₄ receptors. Our study demonstrates for the first time that the TM2/3 microdomain of the D₂ dopamine receptor not only regulates the selective affinity of ligands, but in selected cases can also regulate their function. Utilizing a new docking technique that incorporates receptor backbone flexibility, the three non‐conserved amino acids that encompass the TM2/3 microdomain were found to account in large part for the differences in intermolecular steric contacts between the ligands and receptors. Consistent with the experimental data, this model illustrates the interactions between a variety of subtype‐selective ligands and the wild‐type D₂, mutant D₂, or wild‐type D₄ receptors.     

Examining the critical roles of human CB2 receptor residues Valine 3.32 (113) and Leucine 5.41 (192) in ligand recognition and downstream signaling activities

We performed molecular modeling and docking to predict a putative binding pocket and associated ligand–receptor interactions for human cannabinoid receptor 2 (CB2). Our data showed that two hydrophobic residues came in close contact with three structurally distinct CB2 ligands: CP-55,940, SR144528 and XIE95-26. Site-directed mutagenesis experiments and subsequent functional assays implicated the roles of Valine residue at position 3.32 (V113) and Leucine residue at position 5.41 (L192) in the ligand binding function and downstream signaling activities of the CB2 receptor. Four different point mutations were introduced to the wild type CB2 receptor: V113E, V113L, L192S and L192A. Our results showed that mutation of Val113 with a Glutamic acid and Leu192 with a Serine led to the complete loss of CB2 ligand binding as well as downstream signaling activities. Substitution of these residues with those that have similar hydrophobic side chains such as Leucine (V113L) and Alanine (L192A), however, allowed CB2 to retain both its ligand binding and signaling functions. Our modeling results validated by competition binding and site-directed mutagenesis experiments suggest that residues V113 and L192 play important roles in ligand binding and downstream signaling transduction of the CB2 receptor.     

Role of ELA region in auto-activation of mutant KIT receptor: a molecular dynamics simulation insight

KIT receptor is the prime target in gastrointestinal stromal tumor (GISTs) therapy. Second generation inhibitor, Sunitinib, binds to an inactivated conformation of KIT receptor and stabilizes it in order to prevent tumor formation. Here, we investigated the dynamic behavior of wild type and mutant D816H KIT receptor, and emphasized the extended A-loop (EAL) region (805–850) by conducting molecular dynamics simulation (～100?ns). We analyzed different properties such as root mean square cutoff or deviation, root mean square fluctuation, radius of gyration, solvent-accessible surface area, hydrogen bonding network analysis, and essential dynamics. Apart from this, clustering and cross-correlation matrix approach was used to explore the conformational space of the wild type and mutant EAL region of KIT receptor. Molecular dynamics analysis indicated that mutation (D816H) was able to alter intramolecular hydrogen bonding pattern and affected the structural flexibility of EAL region. Moreover, flexible secondary elements, specially, coil and turns were dominated in EAL region of mutant KIT receptor during simulation. This phenomenon increased the movement of EAL region which in turn helped in shifting the equilibrium towards the active kinase conformation. Our atomic investigation of mutant KIT receptor which emphasized on EAL region provided a better insight into the understanding of Sunitinib resistance mechanism of KIT receptor and would help to discover new therapeutics for KIT-based resistant tumor cells in GIST therapy.     

Side‐chain hydrophobicity and the stability of Aβ16–22 aggregates

Recent mutagenesis studies using the hydrophobic segment of Aβ suggest that aromatic π‐stacking interactions may not be critical for fibril formation. We have tested this conjecture by probing the effect of Leu, Ile, and Ala mutation of the aromatic Phe residues at positions 19 and 20, on the double‐layer hexametric chains of Aβ fragment Aβ_16–22 using explicit solvent all‐atom molecular dynamics. As these simulations rely on the accuracy of the utilized force fields, we first evaluated the dynamic and stability dependence on various force fields of small amyloid aggregates. These initial investigations led us to choose AMBER99SB‐ILDN as force field in multiple long molecular dynamics simulations of 100 ns that probe the stability of the wild‐type and mutants oligomers. Single‐point and double‐point mutants confirm that size and hydrophobicity are key for the aggregation and stability of the hydrophobic core region (Aβ_16–22). This suggests as a venue for designing Aβ aggregation inhibitors the substitution of residues (especially, Phe 19 and 20) in the hydrophobic region (Aβ_16–22) with natural and non‐natural amino acids of similar size and hydrophobicity.     

Molecular simulation of the Kv7.4[ΔS269] mutant channel reveals that ion conduction in the cavity is perturbed due to hydrophobic gating

Mutations in the voltage-gated potassium channel Kv7.4 (encoded as KCNQ4) lead to the early onset of non-syndromic hearing loss, which is significant during language acquisition. The deletion of the S269 pore residue (genetic Δ mutation) in Kv7.4 has been reported to be associated with hearing loss. So far, there is no mechanistic understanding of how this mutation modulates channel function. To understand the role of S269 in ion conduction, we performed molecular dynamics simulations for both wild type and ΔS269 mutant channels. Simulations indicate that the ΔS269 mutation suppresses the fluctuations in the neighboring Y269 residue and thereby consolidates the ring formed by I307 and F310 residues in the adjacent S6 helixes in the cavity region. We show that the long side chains of I307 near the entrance to the cavity form a hydrophobic gate. Comparison of the free energy profiles of a cavity ion in Kv7.4 and Kv7.4[ΔS269] channels reveals a sizable energy barrier in the latter case, which suppresses ion conduction. Thus the simulation studies reveal that the hydrophobic gate resulting from the ΔS269 mutation appears to be responsible for sensorineural hearing loss.     

The beta subunit determines the ion selectivity of the GABAA receptor

The gamma-aminobutyric acid, type A (GABA(A)) receptor is a chloride-conducting receptor composed of alpha, beta, and gamma subunits assembled in a pentameric structure forming a central pore. Each subunit has a large extracellular agonist binding domain and four transmembrane domains (M1-M4), with the second transmembrane (M2) domain lining the pore. Mutation of five amino acids in the M1-M2 loop of the beta(3) subunit to the corresponding amino acids of the alpha(7) nicotinic acetylcholine subunit rendered the GABA(A) receptor cation-selective upon co-expression with wild type alpha(2) and gamma(2) subunits. Similar mutations in the alpha(2) or gamma(2) subunits did not lead to such a change in ion selectivity. This suggests a unique role for the beta(3) subunit in determining the ion selectivity of the GABA(A) receptor. The pharmacology of the mutated GABA(A) receptor is similar to that of the wild type receptor, with respect to muscimol binding, Zn(2+) and bicuculline sensitivity, flumazenil binding, and potentiation of GABA-evoked currents by diazepam. There was, however, an increase in GABA sensitivity (EC(50) = 1.3 microm) compared with the wild type receptor (EC(50) = 6.4 microm) and a loss of desensitization to GABA of the mutant receptor.     

Structural,molecular motions,and free-energy landscape of Leishmania sterol-14α-demethylase wild type and drug resistant mutant: a comparative molecular dynamics study

Sterol-14α-demethylase (CYP51) is an ergosterol pathway enzyme crucial for the survival of infectious Leishmania parasite. Recent high-throughput metabolomics and whole genome sequencing study revealed amphotericin B resistance in Leishmania is indeed due to mutation in CYP51. The residue of mutation (asparagine 176) is conserved across the kinetoplastidae and not in yeast or humans, portraying its functional significance. In order to understand the possible cause for the resistance, knowledge of structural changes due to mutation is of high importance. To shed light on the structural changes of wild and mutant CYP51, we conducted comparative molecular dynamics simulation study. The active site, substrate biding cavity, substrate channel entrance (SCE), and cavity involving the mutated site were studied based on basic parameters and large concerted molecular motions derived from essential dynamics analyses of 100 ns simulation. Results indicated that mutant CYP51 is stable and less compact than the wild type. Correspondingly, the solvent accessible surface area (SASA) of the mutant was found to be increased, especially in active site and cavities not involving the mutation site. Free-energy landscape analysis disclosed mutant to have a rich conformational diversity than wild type, with various free-energy conformations of mutant having SASA greater than wild type with SCE open. More residues were found to interact with the mutant CYP51 upon docking of substrate to both the wild and mutant CYP51. These results indicate that, relative to wild type, the N176I mutation of CYP51 in Leishmania mexicana could possibly favor increased substrate binding efficiency.     

Structure‐function relationships of brazzein variants with altered interactions with the human sweet taste receptor

Brazzein (Brz) is a small (54 amino acid residue) sweet tasting protein with physical and taste properties superior to other non‐carbohydrate sweeteners. In an investigation of sequence‐dependent functional properties of the protein, we used NMR spectroscopy to determine the three‐dimensional structures and dynamic properties of two Brz variants: one with a single‐site substitution (D40K), which is three‐fold sweeter than wild‐type Brz, and one with a two‐residue insertion between residues 18 and 19 (ins₁₈RI₁₉), which is devoid of sweetness. Although the three‐dimensional folds of the two variants were very similar to wild‐type Brz, they exhibited local conformational and dynamic differences. The D40K substitution abolished the strong inter‐stand H‐bond between the side chains of residues Gln46 and Asp40 present in wild‐type Brz and increased the flexibility of the protein especially at the mutation site. This increased flexibility presumably allows this site to interact more strongly with the G‐protein coupled human sweet receptor. On the other hand, the Arg‐Ile insertion within Loop9–19 leads to distortion of this loop and stiffening of the adjacent site whose flexibility appears to be required for productive interaction with the sweet receptor.