Ligand specificity of odorant receptors

Odorant receptors belong to class A of the G protein-coupled receptors (GPCRs) and detect a large number of structurally diverse odorant molecules. A recent structural bioinformatic analysis suggests that structural features are conserved across class A of GPCRs in spite of their low sequence identity. Based on this work, we have aligned the sequences of 29 ORs for which ligand binding data are available. Recent site-directed mutagenesis experiments on one such receptor (MOR174-9) provide information that helped to identify nine amino-acid residues involved in ligand binding. Our modeling provides a rationale for amino acids in equivalent positions in most of the odorant receptors considered and helps to identify other amino acids that could be important for ligand binding. Our findings are consistent with most of the previous models and allow predictions for site-directed mutagenesis experiments, which could also validate our model.     

Location and nature of the residues important for ligand recognition in G-protein coupled receptors

The overall structure of the biogenic amine subclass of the G-protein-coupled receptors, and of their ligand binding sites, is discussed with the aim of highlighting the major structural features of these receptors that are responsible for ligand recognition. A comparison is made between biogenic amine receptors, peptide receptors of the rhodopsin class, and the secretin receptors which all have peptide ligands. The question of where the peptide ligands bind, whether at extracellular sites or within the transmembrane helix bundle, is discussed. The suitability of the rhodopsin crystal structure as a template for construction of homology models is discussed and it is concluded that there are many reasons why a caution should be issued against using it uncritically.     

Ligand binding specificity of the Escherichia coli periplasmic histidine binding protein,HisJ

The HisJ protein from Escherichia coli and related Gram negative bacteria is the periplasmic component of a bacterial ATP‐cassette (ABC) transporter system. Together these proteins form a transmembrane complex that can take up L‐histidine from the environment and translocate it into the cytosol. We have studied the specificity of HisJ for binding L‐His and many related naturally occurring compounds. Our data confirm that L‐His is the preferred ligand, but that 1‐methyl‐L‐His and 3‐methyl‐L‐His can also bind, while the dipeptide carnosine binds weakly and D‐histidine and the histidine degradation products, histamine, urocanic acid and imidazole do not bind. L‐Arg, homo‐L‐Arg, and post‐translationally modified methylated Arg‐analogs also bind with reasonable avidity, with the exception of symmetric dimethylated‐L‐Arg. In contrast, L‐Lys and L‐Orn have considerably weaker interactions with HisJ and methylated and acetylated Lys variants show relatively poor binding. It was also observed that the carboxylate group of these amino acids and their variants was very important for proper recognition of the ligand. Taken together our results are a key step towards designing HisJ as a specific protein‐based reagentless biosensor.     

Ligand binding and homology modelling of insect odorant‐binding proteins

This review describes the main characteristics of odorant‐binding proteins (OBPs) for homology modelling and presents a summary of structure prediction studies on insect OBPs, along with the steps involved and some limitations and improvements. The technique involves a computing approach to model protein structures and is based on a comparison between a target (unknown structure) and one or more templates (experimentally determined structures). As targets for structure prediction, OBPs are considered to play a functional role for recognition, desorption, scavenging, protection and transportation of hydrophobic molecules (odourants) across an aqueous environment (lymph) to olfactory receptor neurones (ORNs) located in sensilla, the main olfactory units of insect antennae. Lepidopteran pheromone‐binding proteins, a subgroup of OBPs, are characterized by remarkable structural features, in which high sequence identities (approximately 30%) among these OBPs and a large number of available templates can facilitate the prediction of precise homology models. Approximately 30 studies have been performed on insect OBPs using homology modelling as a tool to predict their structures. Although some of the studies have assessed ligand‐binding affinity using structural information and biochemical measurements, few have performed docking and molecular dynamic (MD) simulations as a virtual method to predict best ligands. Docking and MD simulations are discussed in the context of discovery of novel semiochemicals (super‐ligands) using homology modelling to conceive further strategies in insect management.     

Ligand binding to proteins: the binding landscape model.

Models of ligand binding are often based on four assumptions: (1) steric fit: that binding is determined mainly by shape complementarity; (2) native binding: that ligands mainly bind to native states; (3) locality: that ligands perturb protein structures mainly at the binding site; and (4) continuity: that small changes in ligand or protein structure lead to small changes in binding affinity. Using a generalization of the 2D HP lattice model, we study ligand binding and explore these assumptions. We first validate the model by showing that it reproduces typical binding behaviors. We observe ligand-induced denaturation, ANS and heme-like binding, and "lock-and-key" and "induced-fit" specific binding behaviors characterized by Michaelis-Menten or more cooperative types of binding isotherms. We then explore cases where the model predicts violations of the standard assumptions. For example, very different binding modes can result from two ligands of identical shape. Ligands can sometimes bind highly denatured states more tightly than native states and yet have Michaelis-Menten isotherms. Even low-population binding to denatured states can cause changes in global stability, hydrogen-exchange rates, and thermal B-factors, contrary to expectations, but in agreement with experiments. We conclude that ligand binding, similar to protein folding, may be better described in terms of energy landscapes than in terms of simpler mass-action models.     

Ligand binding to a high‐energy partially unfolded protein

The conformational energy landscape of a protein determines populations of all possible conformations of the protein and also determines the kinetics of the conversion between the conformations. Interaction with ligands influences the conformational energy landscapes of proteins and shifts populations of proteins in different conformational states. To investigate the effect of ligand binding on partial unfolding of a protein, we use Escherichia coli dihydrofolate reductase (DHFR) and its functional ligand NADP⁺ as a model system. We previously identified a partially unfolded form of DHFR that is populated under native conditions. In this report, we determined the free energy for partial unfolding of DHFR at varying concentrations of NADP⁺ and found that NADP⁺ binds to the partially unfolded form as well as the native form. DHFR unfolds partially without releasing the ligand, though the binding affinity for NADP⁺ is diminished upon partial unfolding. Based on known crystallographic structures of NADP⁺‐bound DHFR and the model of the partially unfolded protein we previously determined, we propose that the adenosine‐binding domain of DHFR remains folded in the partially unfolded form and interacts with the adenosine moiety of NADP⁺. Our result demonstrates that ligand binding may affect the conformational free energy of not only native forms but also high‐energy non‐native forms.     

Ligand binding characteristics of two molecular forms,one equipped with a hydrophobic glycosyl phosphatidylinositol tail,of the folate binding protein purified from human milk

Two molecular forms of the folate binding protein were isolated and purified from human milk by a combination of cation exchange- and affinity chromatography. One protein (27 kDa) was a cleavage product of the other 100 kDa protein as evidenced by N-terminal amino acid sequence homology and a reduction in the molecular size of the latter protein to 27 kDa after cleavage of its hydrophobic glycosylphosphatidylinositol tail by phosphatidylinositol-specific phospholipase C. High-affinity binding of [³H]folate was characterized by upward convex Scatchard plots and increasing ligand binding affinity with decreasing concentrations of both proteins. Downward convex Scatchard plots and binding affinities showing no dependence on the protein concentration were, however, observed in highly diluted solutions of both proteins. Radioligand binding was inhibited by folate analogs, and dissociation of radioligand was slow at pH 7.4 but rapid and complete at pH 5.0 and 3.5. Ligand binding quenched the tryptophan fluorescence of the 27 kDa protein suggesting that tryptophan is present at the binding site and/or ligand binding induces a conformation change that affects tryptophan environment in the protein. The 27 kDa protein representing soluble folate binding protein exhibited a greater affinity for ligand binding than the 100 kDa protein which possesses a hydrophobic tail identical to the one that anchors the folate receptor to the cell membrane.     

Vibrational spectroscopic investigation of the ligand binding domain of kainate receptors

Fourier transform infrared spectroscopy has been used to probe the agonist‐protein interactions in the ligand binding domain of the GluR6 subunit, one subunit of the kainate subtype of glutamate receptors. In order to study the changes in the interactions over a range of activations the investigations were performed using the wild type, N690S, and T661E mutations. These studies show that the strength of the interactions at the α‐amine group of the agonist, as probed by studying the environment of the nondisulphide bonded Cys 432, acts as a switch with weaker interactions at lower activations and stronger interactions at higher activations. The α‐carboxylate interactions of the agonist, however, are not significantly different over the wide range of activations, as measured by the maximum currents mediated by the receptors at saturating concentrations of agonists. Previous investigations of AMPA receptors show a similar dependence of the α‐amine interactions on activation indicating that the roles of the α‐amine interactions in mediating receptor activation are similar for both subtypes of receptors; however, in the case of the AMPA receptors a tug of war type of change was observed between the α‐amine and α‐carboxylate interactions and this is not observed in kainate receptors. This decoupling of the two interactions could arise due to the larger cleft observed in kainate receptors, which allows for a more flexible interaction for the α‐amine and α‐carboxylate groups of the agonists.     

Expression, binding, and signaling properties of CRF2(a) receptors endogenously expressed in human retinoblastoma Y79 cells: passage-dependent regulation of functional receptors

Endogenous expression of the corticotropin-releasing factor type 2a receptor [CRF2(a)] but not CRF2(b) and CRF2(c) was observed in higher passage cultures of human Y79 retinoblastoma cells. Functional studies further demonstrated an increase in CRF2(a) mRNA and protein levels with higher passage numbers (> 20 passages). Although the CRF1 receptor was expressed at higher levels than the CRF2(a) receptor, both receptors were easily distinguishable from one another by selective receptor ligands. CRF(1)-preferring or non-selective agonists such as CRF, urocortin 1 (UCN1), and sauvagine stimulated cAMP production in Y79 to maximal responses of approximately 100 pmoles/10(5) cells, whereas the exclusive CRF2 receptor-selective agonists UCN2 and 3 stimulated cAMP production to maximal responses of approximately 25-30 pmoles/10(5) cells. UCN2 and 3-mediated cAMP stimulation was potently blocked by the approximately 300-fold selective CRF2 antagonist antisauvagine (IC50 = 6.5 +/- 1.6 nmol/L), whereas the CRF(1)-selective antagonist NBI27914 only blocked cAMP responses at concentrations > 10 microL. When the CRF(1)-preferring agonist ovine CRF was used to activate cAMP signaling, NBI27914 (IC50 = 38.4 +/- 3.6 nmol/L) was a more potent inhibitor than antisauvagine (IC50 = 2.04 +/- 0.2 microL). Finally, UCN2 and 3 treatment potently and rapidly desensitized the CRF2 receptor responses in Y79 cells. These data demonstrate that Y79 cells express functional CRF1 and CRF2a receptors and that the CRF2(a) receptor protein is up-regulated during prolonged culture.     

Ligand binding by TPR domains

Tetratricopeptide repeat (TPR) domains bind specific peptide ligands and are thought to mediate protein-protein interactions in a variety of biological systems. Here we compare peptide ligand-binding by several different TPR domains. We present specific examples that demonstrate that TPR domains typically undergo little or no structural rearrangement upon ligand binding. Our data suggest that, contrary to a recent proposal, coupled folding and binding is not the common mechanism of ligand recognition by TPR domains.     

Different binding modes of tropeines mediating inhibition and potentiation of α1 glycine receptors

Tropeines are bidirectional modulators of native and recombinant glycine receptors (GlyRs) and promising leads for the development of novel modulatory agents. Tropisetron potentiates and inhibits agonist-triggered GlyR currents at femto- to nanomolar and micromolar concentrations respectively. Here, the potentiating and inhibitory effects of another tropeine, 3α-(3'-methoxy-benzoyloxy)nortropane (MBN) were examined by voltage-clamp electrophysiology at wild type and mutant α1 GlyRs expressed in Xenopus laevis oocytes. Several substitutions around the agonist-binding cavity of the α1 subunit interface (N46C, F63A, N102A, R119K, R131A, E157C, K200A, Y202L and F207A) were found to reduce or eliminate MBN inhibition of glycine activation. In contrast, the binding site mutations Q67A, R119A and S129A which did not affect MBN inhibition abolished the potentiation of chloride currents elicited by low concentrations of the partial agonist taurine following pre-incubation with MBN. Thus, potentiation and inhibition involve distinct binding modes of MBN in the inter-subunit agonist-binding pocket of α1 GlyRs. Homology modelling and molecular dynamics simulations disclosed two distinct docking modes for MBN, which are consistent with the differential effects of individual binding site substitutions on MBN inhibition and potentiation respectively. Together these results suggest that distinct binding modes at adjacent binding sites located within the agonist-binding pocket of the GlyR mediate the bidirectional modulatory effects of tropeines.     

Three dimensional modeling of N-terminal region of galanin and its interaction with the galanin receptor

The neuropeptide galanin comes under the powerful and versatile modulators of classical neurotransmitters and is present in brain tissues, which are intimately involved in epileptogenesis. It acts as appealing targets for studying basic mechanisms of seizure initiation and arrest, and for the development of novel approaches for various neurodegenerative diseases. Galanin is widely distributed in the mammalian brain which controls various processes such as sensation of pain, learning, feeding, sexual behaviour, carcinogenesis, pathophysiology of neuroendocrine tumors and others. The function of galanin can be exploited through its interaction with three G-protein coupled receptors subtypes such as GalR1, GalR2 and GalR3. The N-terminal region of galanin comprises about highly conserved 15 amino acid residues, which act as the crucial region for agonist-receptor binding. We have constructed a theoretical structural model for the N-terminal region of galanin from Homo sapiens by homology modeling. The stereochemistry of the model was checked using PROCHECK. The functionally conserved regions were identified by surface mapping of phylogenetic information generated by online web algorithm ConSurf. The docking studies on the pharmacologically important galanin receptors with the theoretical model of N-terminal region of galanin predicted crucial residues for binding which would be useful in the development of novel leads for neurodegenerative disorders.     

G蛋白偶联受体计算研究的进展和前瞻

G蛋白偶联受体(G protein-coupled receptor,GPCR)是含有七个跨膜螺旋的一类重要蛋白,是迄今为止发现的最大的多药物靶标受体超蛋白家族。例如,目前上市药物中有超过30%是以GPCR为靶点的。然而,与GPCR重要性形成强烈反差的是科学界对于其结构与功能的了解非常贫乏,主要原因是通过实验手段来获得GPCR的结构与功能信息极其困难。利用生物信息学方法从基因组规模的数据中识别GPCR并预测三维结构是可行途径之一。基于生物信息学的GPCR研究将为新型药物靶标的筛选和药物的开发提供一定的帮助。本文论述了几种较为典型的GPCR计算方法,并基于已有研究提出可能的创新性研究策略来解决GPCR蛋白识别、跨膜区定位、以及结构和功能预测等问题。     

An assessment of protein-ligand binding site polarizability

Electronic polarizability, an important physical property of biomolecules, is currently ignored in most biomolecular calculations. Yet, it is widely believed that polarization could account for a substantial fraction of the total nonbonded energy of a system. This belief is supported by studies of small complexes in vacuum. This perception is driving the development of a new class of polarizable force fields for biomolecular calculations. However, the quantification of this term for protein-ligand complexes has never been attempted. Here we explore the polarizable nature of protein-ligand complexes in order to evaluate the importance of this effect. We introduce two indexes describing the polarizability of protein binding sites. These we apply to a large range of pharmaceutically relevant complexes. We offer a recommendation of particular complexes as test systems with which to determine the effects of polarizability and as test cases with which to test the new generation of force fields. Additionally, we provide a tabulation of the amino acid composition of these binding sites and show that composition can be specific for certain classes of proteins. We also show that the relative abundance of some amino acids is different in binding sites than elsewhere in a protein's structure.     

Muscarinic receptors: A comparative analysis of structural features and binding modes through homology modelling and molecular docking

Three-dimensional models of the five human muscarinic receptors were obtained from their known sequences. Homology modelling based on the crystallographic structure of bovine rhodopsin yielded models compatible with known results from site-directed mutagenesis studies. The only exceptions were the cytoplasmic loop 3 (CL3) in the five receptors, and the large C-terminal domain in M(1). Here, homology modelling with other closely related proteins allowed to solve these gaps. A detailed comparative discussion of the five models is given. The second part of the work involved docking experiments with the physiological ligand acetylcholine, again yielding results entirely compatible with results from mutagenesis experiments. The study revealed analogies and differences between the five receptors in the residues, and interactions leading to the recognition and binding of acetylcholine.     

Variations in binding characteristics of glycosylated human C-reactive proteins in different pathological conditions

C-reactive protein (CRP) is a clinically important classic acute phase pentameric protein. It is thought to play an important role in immunomodulation. Earlier reports convincingly demonstrated that human CRP is differentially glycosylated in different pathological conditions. Although CRP is considered to be a clinically important molecule, changes in binding characteristics with appropriate ligands with respect to glycosylation remain unexplored. In an effort to demonstrate that these glycosylated molecular variants are capable of modulating their binding activity with different ligands, CRPs were affinity purified from six different clinical samples. Variable amounts of linkage-specific sialic acid derivatives were found in these CRPs with varying tryptophan contents. Differential binding patterns with antibodies against human CRP, human IgG, and other ligands like fibronectin, fetuin, and asialofetuin indicated that the purified CRPs differed significantly in their lectin-like interactions. Thus, we have convincingly demonstrated that differentially induced CRPs exhibited variable binding characteristics. These results may have far reaching practical applications for understanding acute phase responses. Published in 2004..     

Crystal structure of the human odorant binding protein,OBPIIa

Human odorant‐binding protein, OBP_IIa, is expressed by nasal epithelia to facilitate transport of hydrophobic odorant molecules across the aqueous mucus. Here, we report its crystallographic analysis at 2.6 Å resolution. OBP_IIa is a monomeric protein that exhibits the classical lipocalin fold with a conserved eight‐stranded β‐barrel harboring a remarkably large hydrophobic pocket. Basic residues within the four loops that shape the entrance to this ligand‐binding site evoke a positive electrostatic potential. Human OBP_IIa shows distinct features compared with other mammalian OBPs, including a potentially reactive Cys side chain within its pocket similar to human tear lipocalin. Proteins 2015; 83:1180–1184. © 2015 Wiley Periodicals, Inc.     

How inaccuracies in protein structure models affect estimates of protein-ligand interactions: computational analysis of HIV-I protease inhibitor binding

The influence of possible inaccuracies that can arise during homology modeling of protein structures used for ligand binding studies were investigated with the molecular mechanics generalized Born surface area (MM-GBSA) method. For this, a family of well-characterized HIV-I protease-inhibitor complexes was used. Validation of MM-GBSA led to a correlation coefficient ranging from 0.72 to 0.93 between calculated and experimental binding free energies DeltaG. All calculated DeltaG values were based on molecular dynamics simulations with explicit solvent. Errors introduced into the protein structure through misplacement of side-chains during rotamer modeling led to a correlation coefficient between DeltaG(calc) and DeltaG(exp) of 0.75 compared with 0.90 for the correctly placed side chains. This is in contrast to homology models for members of the retroviral protease family with template structures ranging in sequence identity between 32% and 51%. For these protein models, the correlation coefficients vary between 0.84 and 0.87, which is considerably closer to the original protein (0.90). It is concluded that HIV-I low sequence identity with the template structure still allows creating sufficiently reliable homology models to be used for ligand-binding studies, although placement of the rotamers is a critical step during the modeling.