Proposed Binding Site Research Articles

The Conserved Candida albicans CA3427 Gene Product Defines a New Family of Proteins Exhibiting the Generic Periplasmic Binding Protein Structural Fold

Nosocomial diseases due to Candida albicans infections are in constant rise in hospitals, where they cause serious complications to already fragile intensive care patients. Antifungal drug resistance is fast becoming a serious issue due to the emergence of strains resistant to currently available antifungal agents. Thus the urgency to identify new potential protein targets, the function and structure of which may guide the development of new antifungal drugs. In this context, we initiated a comparative genomics study in search of promising protein coding genes among the most conserved ones in reference fungal genomes. The CA3427 gene was selected on the basis of its presence among pathogenic fungi contrasting with its absence in the non pathogenic Saccharomyces cerevisiae. We report the crystal 3D-structure of the Candida albicans CA3427 protein at 2.1 Å resolution. The combined analysis of its sequence and structure reveals a structural fold originally associated with periplasmic binding proteins. The CA3427 structure highlights a binding site located between the two protein domains, corresponding to a sequence segment conserved among fungi. Two crystal forms of CA3427 were found, suggesting that the presence or absence of a ligand at the proposed binding site might trigger a “Venus flytrap” motion, coupled to the previously described activity of bacterial periplasmic binding proteins. The conserved binding site defines a new subfamily of periplasmic binding proteins also found in many bacteria of the bacteroidetes division, in a choanoflagellate (a free-living unicellular and colonial flagellate eukaryote) and in a placozoan (the closest multicellular relative of animals). A phylogenetic analysis suggests that this gene family originated in bacteria before its horizontal transfer to an ancestral eukaryote prior to the radiation of fungi. It was then lost by the Saccharomycetales which include Saccharomyces cerevisiae.

Three hydrophobic amino acids in Escherichia coli HscB make the greatest contribution to the stability of the HscB-IscU complex

BackgroundGeneral iron-sulfur cluster biosynthesis proceeds through assembly of a transient cluster on IscU followed by its transfer to a recipient apo-protein. The efficiency of the second step is increased by the presence of HscA and HscB, but the reason behind this is poorly understood. To shed light on the function of HscB, we began a study on the nature of its interaction with IscU. Our work suggested that the binding site of IscU is in the C-terminal domain of HscB, and two different triple alanine substitutions ([L92A, M93A, F153A] and [E97A, E100A, E104A]) involving predicted binding site residues had detrimental effects on this interaction. However, the individual contribution of each substitution to the observed effect remains to be determined as well as the possible involvement of other residues in the proposed binding site.ResultsIn the work reported here, we used isothermal titration calorimetry to characterize the affinity of single alanine HscB mutants for IscU, and subsequently confirmed our results with nuclear magnetic resonance spectroscopy. Alanine substitutions of L92, L96, and F153 severely impaired the ability of HscB to form a complex with IscU; substitutions of R87, R99, and E100 had more modest effects; and substitutions of T89, M93, E97, D103, E104, R152, K156, and S160 had only minor or no detectable effects.ConclusionsOur results show that the residues of HscB most important for strong interaction with IscU include three hydrophobic residues (L92, L96, and F153); in addition, we identified a number of other residues whose side chains contribute to a lesser extent to the interaction. Our results suggest that the triple alanine substitution at HscB positions 92, 96, and 153 will destabilize the HscB-IscU complex by ΔΔGb≅ 5.7 kcal/mol, equivalent to a ≅ 15000-fold reduction in the affinity of HscB for IscU. We propose that this triple mutant could provide a more definitive test of the functional importance of the HscB-IscU interaction in vivo than those used previously that yielded inconclusive results.

Coexistence of two adamantane binding sites in the influenza A M2 ion channel

The influenza A virus contains a proton-selective ion channel (M2) that is the target of the adamantane family of drug inhibitors. Two recently published studies relating to adamantane binding of the M2 ion channel using X-ray crystallography and solution NMR have reignited interest in the potential use of adamantanes in combating the spread of influenza A. However, these two studies propose different binding sites for the adamantane drugs with the X-ray M2/amantadine structure favoring an ion channel pore-binding model and the solution NMR M2/rimantadine structure suggesting the existence of a lipid-facing binding pocket. We conducted a series of surface plasmon resonance (SPR) experiments designed to accurately measure the affinity of amantadine and rimantadine to M2 ion channels embedded in 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC) liposomes. We find that this class of drug is capable of binding M2 with two different affinities in the order of 10(-4) and 10(-7) M, suggesting that both proposed binding sites are feasible. Furthermore, by examining drug binding to M2 mutant constructs (V27A, S31N, and D44A), it was possible to probe the location of the two binding sites. We show that a high-affinity binding site corresponds to the M2 ion channel pore whereas the secondary, low-affinity binding site can be attributed to the lipid face of the pore. These SPR results are in excellent agreement with the most recent solid-state NMR study of amantadine-bound M2 in lipid bilayers and provide independent support that the ion channel pore-binding site is responsible for the pharmacological activity elicited by the adamantane drugs.

A Solid-State NMR Study Reveals Structure and Dynamics in Copper(ii)-Binding to Alzheimer's Beta-Amyloid Fibrils

β-amyloid (Aβ) peptide associated with Alzheimer's diseases exhibit neural toxicity upon aggregation. One of the most widespread hypotheses on the origin of the toxicity of Aβ aggregates is the binding of Cu2+ ions to Aβ fibrils and subsequent generation of reactive oxygen species (ROS) such as H2O2 by Cu2+-bound Aβ. Although a variety of studies have been performed on Cu2+-binding to Aβ, the proposed binding sites or models have been controversial partly because non-crystalline and insoluble nature of Aβ fibrils have limited access to site-specific structure and dynamic properties on Cu2+-bound Aβ fibrils. Here, we examine the effect of Cu2+ binding to amyloid fibrils of 40-residue Aβ(1-40) by UV-VIS spectroscopy and solid-state NMR (SSNMR). Specifically, we will answer the following questions (i) Is the Cu2+ binding is site specific? (ii) If so, which sites are involved in binding? (iii) Are there any major structural changes introduced by Cu2+ binding or oxidization due to Cu2+?UV-VIS spectroscopy showed that Cu2+ binds to Aβ(1-40) fibrils almost completely when the ratio of Cu2+ to Aβ(1-40) is less than 1. Based on the result, we performed high-resolution SSNMR experiments on Cu2+-bound Aβ(1-40) fibrils. First, the 13C T1 paramagnetic relaxation enhancement (PRE) due to Cu2+ binding on Aβ was measured for different residues; the PRE data highlight possible binding sites, where the relaxation enhancements are notable. The analysis indicates that the binding is specific, and Cu2+ most likely binds to His-13/14 and His-6. Second, the comparison of 2D 13C/13C correlation spectra of Aβ fibrils with and without Cu2+ revealed that the secondary structure of Aβ(1-40) fibrils is largely unaltered by Cu2+ binding. Third, we tested a previously proposed hypothesis that Met-35/Gly-33 can be oxidized[4] by Cu2+ to produce ROS by SSNMR.

Isolation and Characterization of AaWRKY1, an Artemisia annua Transcription Factor that Regulates the Amorpha-4,11-diene Synthase Gene, a Key Gene of Artemisinin Biosynthesis

Amorpha-4,11-diene synthase (ADS) of Artemisia annua catalyzes the conversion of farnesyl diphosphate into amorpha-4,11-diene, the first committed step in the biosynthesis of the antimalarial drug artemisinin. The promoters of ADS contain two reverse-oriented TTGACC W-box cis-acting elements, which are the proposed binding sites of WRKY transcription factors. A full-length cDNA (AaWRKY1) was isolated from a cDNA library of the glandular secretory trichomes (GSTs) in which artemisinin is synthesized and sequestered. AaWRKY1 encodes a 311 amino acid protein containing a single WRKY domain. AaWRKY1 and ADS genes were highly expressed in GSTs and both were strongly induced by methyl jasmonate and chitosan. Transient expression analysis of the AaWRKY1-GFP (green fluorescent protein) reporter revealed that AaWRKY1 was targeted to nuclei. Biochemical analysis demonstrated that the AaWRKY1 protein was capable of binding to the W-box cis-acting elements of the ADS promoters, and it demonstrated transactivation activity in yeast. Co-expression of the effector construct 35S::AaWRKY1 with a reporter construct ADSpro1::GUS greatly activated expression of the GUS (beta-glucuronidase) gene in stably transformed tobacco. Furthermore, transient expression experiments in agroinfiltrated Nicotiana benthamiana and A. annua leaves showed that AaWRKY1 protein transactivated the ADSpro2 promoter activity by binding to the W-box of the promoter; disruption of the W-box abolished the activation. Transient expression of AaWRKY1 cDNA in A. annua leaves clearly activated the expression of the majority of artemisinin biosynthetic genes. These results strongly suggest the involvement of the AaWRKY1 transcription factor in the regulation of artemisinin biosynthesis, and indicate that ADS is a target gene of AaWRKY1 in A. annua.

Structural modeling identified the tRNA-binding domain of Utp8p, an essential nucleolar component of the nuclear tRNA export machinery of Saccharomyces cerevisiae

Utp8p is an essential 80 kDa intranuclear tRNA chaperone that transports tRNAs from the nucleolus to the nuclear tRNA export receptors in Saccharomyces cerevisiae. To help understand the mechanism of Utp8p function, predictive tools were used to derive a partial model of the tertiary structure of Utp8p. Secondary structure prediction, supported by circular dichroism measurements, indicated that Utp8p is divided into 2 domains: the N-terminal beta sheet and the C-terminal alpha helical domain. Tertiary structure prediction was more challenging, because the amino acid sequence of Utp8p is not directly homologous to any known protein structure. The tertiary structures predicted by threading and fold recognition had generally modest scores, but for the C-terminal domain, threading and fold recognition consistently pointed to an alpha-alpha superhelix. Because of the sequence diversity of this fold type, no single structural template was an ideal fit to the Utp8p sequence. Instead, a composite template was constructed from 3 different alpha-alpha superhelix structures that gave the best matches to different portions of the C-terminal domain sequence. In the resulting model, the most conserved sequences grouped in a tight cluster of positive charges on a protein that is otherwise predominantly negative, suggesting that the positive-charge cleft may be the tRNA-binding site. Mutations of conserved positive residues in the proposed binding site resulted in a reduction in the affinity of Utp8p for tRNA both in vivo and in vitro. Models were also derived for the 10 fungal homologues of Utp8p, and the localization of the positive charges on the conserved surface was found in all cases. Taken together, these data suggest that the positive-charge cleft of the C-terminal domain of Utp8p is involved in tRNA-binding.

Homology modeling of MCH1 receptor and validation by docking/scoring and protein-aligned CoMFA

Homology modeling is becoming a valid method for obtaining three-dimensional coordinates for proteins. However, it is hard to judge the qualities of the resulting models warranting robust subsequent validations. In an attempt to evaluate the quality of Melanin-concentrating hormone 1 receptor (MCH1R) homology models, a number of homology structures were scanned for potential binding cavities. Subsequently, a group of 35 benzylpiperidines' MCH1R inhibitors were docked into each of the proposed binding sites via four different scoring functions. The docked structures were utilized to construct corresponding protein-aligned comparative molecular field analysis (CoMFA) models by employing probe-based (H +, OH, CH 3) energy grids and genetic partial least squares (G/PLS) statistical analysis. The docking-based alignment succeeded in accessing self-consistent CoMFA models upon employing JAIN scoring function in one of the proposed binding pockets in a particular homology model. Furthermore, a ligand-based pharmacophore model was developed for the same set of inhibitors and was found to agree with the successful docking configuration. Therefore, we proved that the overall procedure of docking, scoring, and CoMFA evaluation can be a useful tool to validate homology models, which can be of value for structure-based design, in-silico screening, and in understanding the structural basis of ligand binding to MCH1R.

Binding of ADP in the Mitochondrial ADP/ATP Carrier Is Driven by an Electrostatic Funnel

The ADP/ATP carrier (AAC) is a membrane protein of paramount importance for the energy-fueling function of the mitochondria, transporting ADP from the intermembrane space to the matrix and ATP in the opposite direction. On the basis of the high-resolution, 2.2-A structure of the bovine carrier, a total of 0.53 micros of classical molecular dynamics simulations were conducted in a realistic membrane environment to decipher the early events of ADP (3-) translocation across the inner membrane of the mitochondria. Examination of apo-AAC underscores the impermeable nature of the carrier, impeding passive transport of permeants toward the matrix. The electrostatic funnel illuminated from three-dimensional mapping of the electrostatic potential forms a privileged passageway anticipated to drive the diphosphate nucleotide rapidly toward the bottom of the internal cavity. This conjecture is verified in the light of repeated, independent numerical experiments, whereby the permeant is dropped near the mouth of the mitochondrial carrier. Systematic association of ADP (3-) to the crevice of the AAC, an early event in its transport across the inner membrane, is accompanied by the formation of an intricate network of noncovalent bonds. Simulations relying on the use of an adaptive biasing force reveal for the first time that the proposed binding site corresponds to a minimum of the free energy landscape delineating the translocation of ADP (3-) in the carrier. The present work paves the way to the design of novel nucleotides and new experiments aimed at unveiling key structural features in the chronology of ADP/ATP transport across the mitochondrial membrane.

Peptides Inhibitors of F11R/JAM‐A Adhesion Molecules

F11‐R receptor was characterized as an adhesion protein, called also aka JAM‐A (or JAM‐1), which under normal physiological conditions is expressed constitutively on the surface of the platelets and localized within tight junctions of endothelial cells (EC). The utilization of specific F11R/JAM‐A peptide antagonists and recombinant proteins supported the role of F11R/JAM‐A in the process of platelets adhesion to inflamed endothelial cells and identified plaque formation leading to inflammatory thrombosis and atherosclerosis where the platelets have a critical influence in the progression and development of the cardiovascular disease. Thus, the development of new drugs antagonizing the F11R/JAM‐A function could evolve as an effective strategy for the treatment of atherosclerosis, heart attacks and stroke. We present one of the first trials toward development of peptide‐based inhibitors of F11R/JAM‐A function. Among many trials, the peptide D‐Lys‐Ser‐Val‐Ser‐D‐Arg‐Glu‐Asp‐Thr‐Gly‐Thr‐Tyr‐Thr‐Cys‐CONH2 proved to be a potent inhibitor of human platelets aggregation in vitro. Further molecular docking experiments showed that this peptide makes favorable hydrophobic and electrostatic interactions within the proposed binding site of JAM‐1 (X‐Ray structure 1nbq.pdb was used as template).

PepA and ArgR do not regulate Cre recombination at the bacteriophage P1 loxP site

In the lysogenic state, bacteriophage P1 is maintained as a low copy-number circular plasmid. Site-specific recombination at loxP by the phage-encoded Cre protein keeps P1 monomeric, thus helping to ensure stable plasmid inheritance. Two Escherichia coli DNA-binding proteins, PepA and ArgR, were recently reported to be necessary for maintenance or establishment of P1 lysogeny. PepA and ArgR bind to regulatory DNA sequences upstream of the ColE1 cer recombination site to regulate site-specific recombination by the XerCD recombinases. This recombination keeps ColE1 in a monomeric state and helps to ensure stable plasmid maintenance. It has been suggested that ArgR and PepA play a similar role in P1 maintenance, regulating Cre recombination by binding to DNA sequences upstream of loxP. Here, we show that ArgR does not bind to its proposed binding site upstream of loxP, and that Cre recombination at loxP in its natural P1 context is not affected by PepA and ArgR in vitro. When sequences upstream of loxP were mutated to allow ArgR binding, PepA and ArgR still had no effect on Cre recombination. Our results demonstrate that PepA requires specific DNA sequences for binding, and that PepA and ArgR have no direct role in Cre recombination at P1 loxP.

Biochemical and spectroscopic studies of human melanotransferrin (MTf): Electron-paramagnetic resonance evidence for a difference between the iron-binding site of MTf and other transferrins

Melanotransferrin (MTf) is a member of the transferrin (Tf) family of iron (Fe)-binding proteins that was first identified as a cell-surface marker of melanoma. Although MTf has a high-affinity Fe-binding site that is practically identical to that of serum Tf, the protein does not play an essential role in Fe homeostasis and its precise molecular function remains unclear. A Zn(II)-binding motif, distinct from the Fe-binding site, has been proposed in human MTf based on computer modelling studies. However, little is known concerning the interaction of its proposed binding site(s) with metals and the consequences in terms of MTf conformation. For the first time, biochemical and spectroscopic techniques have been used in this study to characterise metal ion-binding to recombinant MTf. Initially, the binding of Fe to MTf was examined using 6 M urea gel electrophoresis. Although four different iron-loaded forms were observed with serum Tf, only two forms were found with MTf, the apo-form and the N-monoferric holo-protein, suggesting a single high-affinity site. The presence of a single Fe(III)-binding site was also supported by EPR results which indicated that the Fe(III)-binding characteristics of MTf were unique, but somewhat comparable to the N-lobes of human serum Tf and chicken ovo-Tf. Circular dichroism (CD) analysis indicated that, as for Tf, no changes in secondary structure could be observed upon Fe(III)-binding. The ability of MTf to bind Zn(II) was also investigated using CD which demonstrated that the single high-affinity Fe-binding site was distinct from a potential Zn(II)-binding site.

BSAlign: a rapid graph-based algorithm for detecting ligand-binding sites in protein structures.

Detection of ligand-binding sites in protein structures is a crucial task in structural bioinformatics, and has applications in important areas like drug discovery. Given the knowledge of the site in a particular protein structure that binds to a specific ligand, we can search for similar sites in the other protein structures that the same ligand is likely to bind. In this paper, we propose a new method named "BSAlign" (Binding Site Aligner) for rapid detection of potential binding site(s) in the target protein(s) that is/are similar to the query protein's ligand-binding site. We represent both the binding site and the protein structure as graphs, and employ a subgraph isomorphism algorithm to detect the similarities of the binding sites in a very time-efficient manner. Preliminary experimental results show that the proposed BSAlign binding site detection method is about 14 times faster than a well-known method called SiteEngine, while offering the same level of accuracy. Both BSAlign and SiteEngine achieve 60% search accuracy in finding adenine-binding sites from a data set of 126 proteins. The proposed method can be a useful contribution towards speed-critical applications such as drug discovery in which a large number of proteins are needed to be processed. The program is available for download at: http://www1.i2r.a-star.edu.sg/~azeyar/BSAlign/.

Tropomyosin's Periods Are Quasi-Equivalent for Actin Binding but Have Specific Regulatory Functions

Tropomyosin is a coiled coil that associates N-terminus to C-terminus to form a continuous strand along both sides of the actin filament and regulates its function. One long, high molecular weight tropomyosin molecule spans the length of seven actin subunits. In these forms there is a 7-fold periodicity in noninterface residues that have been proposed to correspond to seven quasi-equivalent actin binding sites. Interruption of the stable, canonical coiled coil by residues that destabilize the interhelical interface, such as Ala clusters, is required for actin binding. Previous studies have shown that the N-terminal half of period 5 (residues 165-188) is critical for actin binding and regulatory function and that both the surface "consensus" residues and the embedded, destabilizing Ala cluster are required for function. In the present work we test the hypothesis of quasi-equivalence of tropomyosin's periodic sites by replacing the proposed binding sites by substituting the crucial period 5 region with regions of period 1 or 2. Replacement mutants were designed to test the importance of the coincidence of the consensus residues and a destabilizing interface. The results show that generic (interface instability) and specific periodic surface residues are essential for function and that the periods tested (periods 1, 2, and 5) are quasi-equivalent for actin binding. However, regulatory functions are period-specific: periods 1 and 5 for binding to actin in the force-producing state and period 5 for Ca2+-dependent regulation with troponin.

An Intracellular Allosteric Site for a Specific Class of Antagonists of the CC Chemokine G Protein-Coupled Receptors CCR4 and CCR5

A novel mechanism for antagonism of the human chemokine receptors CCR4 and CCR5 has been discovered with a series of small-molecule compounds that seems to interact with an allosteric, intracellular site on the receptor. The existence of this site is supported by a series of observations: 1) intracellular access of these antagonists is required for their activity; 2) specific, saturable binding of a radiolabeled antagonist requires the presence of CCR4; and 3) through engineering receptor chimeras by reciprocal transfer of C-terminal domains between CCR4 and CCR5, compound binding and the selective structure-activity relationships for antagonism of these receptors seem to be associated with the integrity of that intracellular region. Published antagonists from other chemical series do not seem to bind to the novel site, and their interaction with either CCR4 or CCR5 is not affected by alteration of the C-terminal domain. The precise location of the proposed binding site remains to be determined, but the known close association of the C-terminal domain, including helix 8, as a proposed intracellular region that interacts with transduction proteins (e.g., G proteins and beta-arrestin) suggests that this could be a generic allosteric site for chemokine receptors and perhaps more broadly for class A G protein-coupled receptors. The existence of such a site that can be targeted for drug discovery has implications for screening assays for receptor antagonists, which would need, therefore, to consider compound properties for access to this intracellular site.

Mapping the Binding Site on Small Ankyrin 1 for Obscurin

Small ankyrin 1 (sAnk1), an integral protein of the sarcoplasmic reticulum encoded by the ANK1 gene, binds with nanomolar affinity to the C terminus of obscurin, a giant protein surrounding the contractile apparatus in striated muscle. We used site-directed mutagenesis to characterize the binding site on sAnk1, specifically addressing the role of two putative amphipathic, positively charged helices. We measured binding qualitatively by blot overlay assays and quantitatively by surface plasmon resonance and showed that both positively charged sequences are required for activity. We showed further that substitution of a lysine or arginine with an alanine or glutamate located at the same position along either of the two putative helices has similar inhibitory or stimulatory effects on binding and that the effects of a particular mutation depended on the position of the mutated amino acid in each helix. We modeled the structure of the binding region of sAnk1 by homology with ankyrin repeats of human Notch1, which have a similar pattern of charged and hydrophobic residues. Our modeling suggested that each of the two positively charged sequences forms pairs of amphipathic, anti-parallel alpha-helices flanked by beta-hairpin-like turns. Most of the residues in homologous positions along each helical unit have similar, though not identical, orientations. CD spectroscopy confirmed the alpha-helical content of sAnk1, approximately 33%, predicted by the model. Thus, structural and mutational studies of the binding region on sAnk1 for obscurin suggest that it consists of two ankyrin repeats with very similar structures.

Identification of the Substrate Binding Sites within the Yeast Mitochondrial Citrate Transport Protein

The objective of the present investigation was to identify the substrate binding site(s) within the yeast mitochondrial citrate transport protein (CTP). Our strategy involved kinetically characterizing 30 single-Cys CTP mutants that we had previously constructed based on their hypothesized importance in the structure-based mechanism of this carrier. As part of these studies, a modified transport assay was developed that permitted, for the first time, the accurate determination of K(m) values that were elevated >100-fold compared with the Cys-less control value. We identified 10 single-Cys CTP mutants that displayed sharply elevated K(m) values (i.e. 5 to >300-fold). Each of these mutants displayed V(max) values that were reduced by > or = 98% and resultant catalytic efficiencies that were reduced by > or = 99.9%. Importantly, superposition of this functional data onto the three-dimensional homology-modeled CTP structure, which we previously had developed, revealed that nine of these ten residues form two topographically distinct clusters. Additional modeling showed that: (i) each cluster is capable of forming numerous hydrogen bonds with citrate and (ii) the two clusters are sufficiently distant from one another such that citrate is unlikely to interact with all of these residues at the same time. We deduced from these findings that the CTP contains at least two citrate binding sites per monomer, which are located at increasing depths within the translocation pathway. The identification of these sites, combined with an initial assessment of the citrate-amino acid side-chain interactions that may occur at these sites, substantially extends our understanding of CTP functioning at the molecular level.

Modelling the restoration of wild-type dynamic behaviour in ΔF508-CFTR NBD1 by 8-cyclopentyl-1,3-dipropylxanthine

Cystic fibrosis (CF) is the most frequently occurring severe, genetic disease in western populations with an incidence as high as 1 in 2500. The principal biochemical defect in CF is a mutation in a membrane transport protein, namely the cystic fibrosis transmembrane conductance regulator (CFTR), which is responsible for the conductance of chloride ions across cell membranes. In 70% of cases a single mutation in CFTR, namely the deletion of amino acid 508 (called ΔF508) is sufficient to cause severe disease. This mutation manifests as a failure of the protein to be effectively targeted to the membrane. Recently, it has been shown that small molecule drug therapy can restore the membrane-targeting of ΔF508-CFTR, where the mutant channel functions adequately. We have created models of the first nucleotide-binding domain (NBD1) region (which houses the proposed binding site of these restorative drugs) of the wild-type and mutant forms of human CFTR. We have simulated the dynamical behaviour of these proteins in the presence of drugs that restore trafficking of the protein. Our results indicate that there are particular modes of dynamic motion that are distinguishable between wild-type and mutant CFTR. These regions of motion are localized in the regions of the ΔF508 mutation and the drug-binding regions. The simulations of drug binding indicate that wild-type dynamic motions are restored in these regions. We conclude therefore that these drugs are able to alter the dynamic properties of ΔF508-CFTR such that the drug-bound mutant protein more closely resembles the wild-type protein dynamic behaviour, and hence we hypothesize that it is this that allows for correct targeting to the membrane.

In silico approach of azadirachtin binding with actins

In silico docking analysis reported here suggests that insect cellular cytoskeletal β-actin could be the target of Azadirachtin A (Aza—the principle bioactive compound of neem seeds). The best docking energy of −40.09 kcal/mol at 8.73 Å RMSD and predicted hydrogen bond between Arg210 and carboxymethyl group of Aza accompanied with seven hydrophobic interactions in the proposed binding site strongly support this hypothesis. This is of specific interest due to the non-affinity of Aza to mammalian β-actins under the same set of conditions, although β-actins across the species are highly conserved. Our results show that few scattered amino acid changes have caused significant steric hindrance in the binding pocket for mammalian β-actin, causing a reverse orientation of Aza. These results suggest a model to support the recently observed biological effects caused by Aza in Drosophila cytoskeletal elements and explain why Aza is highly specific to insects than mammals.

Defining the nucleotide binding sites of P2Y receptors using rhodopsin-based homology modeling

Ongoing efforts to model P2Y receptors for extracellular nucleotides, i.e., endogenous ADP, ATP, UDP, UTP, and UDP-glucose, were summarized and correlated for the eight known subtypes. The rhodopsin-based homology modeling of the P2Y receptors is supported by a growing body of site-directed mutagenesis data, mainly for P2Y(1) receptors. By comparing molecular models of the P2Y receptors, it was concluded that nucleotide binding could occur in the upper part of the helical bundle, with the ribose moiety accommodated between transmembrane domain (TM) 3 and TM7. The nucleobase was oriented towards TM1, TM2, and TM7, in the direction of the extracellular side of the receptor. The phosphate chain was oriented towards TM6, in the direction of the extracellular loops (ELs), and was coordinated by three critical cationic residues. In particular, in the P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptors the nucleotide ligands had very similar positions. ADP in the P2Y(12) receptor was located deeper inside the receptor in comparison to other subtypes, and the uridine moiety of UDP-glucose in the P2Y(14) receptor was located even deeper and shifted toward TM7. In general, these findings are in agreement with the proposed binding site of small molecules to other class A GPCRs.

Comparison of the Activities of the Truncated Halichondrin B Analog NSC 707389 (E7389) with Those of the Parent Compound and a Proposed Binding Site on Tubulin

The complex marine natural product halichondrin B was compared with NSC 707389 (E7389), a structurally simplified, synthetic macrocyclic ketone analog, which has been selected for clinical trials in human patients. NSC 707389 was invariably more potent than halichondrin B in its interactions with tubulin. Both compounds inhibited tubulin assembly, inhibited nucleotide exchange on beta-tubulin, and were noncompetitive inhibitors of the binding of radiolabeled vinblastine and dolastatin 10 to tubulin. Neither compound seemed to induce an aberrant tubulin assembly reaction, as occurs with vinblastine (tight spirals) or dolastatin 10 (aggregated rings and spirals). We modeled the two compounds into a shared binding site on tubulin consistent with their biochemical properties. Of the two tubulin structures available, we selected for modeling the complex of a stathmin fragment with two tubulin heterodimers with two bound colchicinoid molecules and a single bound vinblastine between the two heterodimers (Nature (Lond) 435:519-522, 2005). Halichondrin B and NSC 707389 fit snugly between the two heterodimers adjacent to the exchangeable site nucleotide. Fitting the compounds into this site, which was also close to the vinblastine site, resulted in enough movement of amino acid residues at the vinblastine site to cause the latter compound to bind less well to tubulin. The model suggests that halichondrin B and NSC 707389 most likely form highly unstable, small aberrant tubulin polymers rather than the massive stable structures observed with vinca alkaloids and antimitotic peptides.