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.     

Ligand specificity and evolution of liver X receptors

Liver X receptors (LXRs) are key regulators of lipid and cholesterol metabolism in mammals. Little is known, however, about the function and evolution of LXRs in non-mammalian species. The present study reports the cloning of LXRs from African clawed frog (Xenopus laevis), Western clawed frog (Xenopus tropicalis), and zebrafish (Danio rerio), and their functional characterization and comparison with human and mouse LXRs. Additionally, an ortholog of LXR in the chordate invertebrate Ciona intestinalis was cloned and functionally characterized. Ligand specificities of the frog and zebrafish LXRs were very similar to LXRalpha and LXRbeta from human and mouse. All vertebrate LXRs studied were activated robustly by the synthetic ligands T-0901317 and GW3965 and by a variety of oxysterols. In contrast, Ciona LXR was not activated by T-0901317 or GW3965 but was activated by a limited number of oxysterols, as well as some androstane and pregnane steroids. Pharmacophore analysis, homology modeling, and docking studies of Ciona LXR predict a receptor with a more restricted ligand-binding pocket and less intrinsic disorder in the ligand-binding domain compared to vertebrate LXRs. The results suggest that LXRs have a long evolutionary history, with vertebrate LXRs diverging from invertebrate LXRs in ligand specificity.     

Ligand promiscuity through the eyes of the aminoglycoside N3 acetyltransferase IIa

Aminoglycoside‐modifying enzymes (AGMEs) are expressed in many pathogenic bacteria and cause resistance to aminoglycoside (AG) antibiotics. Remarkably, the substrate promiscuity of AGMEs is quite variable. The molecular basis for such ligand promiscuity is largely unknown as there is not an obvious link between amino acid sequence or structure and the antibiotic profiles of AGMEs. To address this issue, this article presents the first kinetic and thermodynamic characterization of one of the least promiscuous AGMEs, the AG N3 acetyltransferase‐IIa (AAC‐IIa) and its comparison to two highly promiscuous AGMEs, the AG N3‐acetyltransferase‐IIIb (AAC‐IIIb) and the AG phosphotransferase(3′)‐IIIa (APH). Despite having similar antibiotic selectivities, AAC‐IIIb and APH catalyze different reactions and share no homology to one another. AAC‐IIa and AAC‐IIIb catalyze the same reaction and are very similar in both amino acid sequence and structure. However, they demonstrate strong differences in their substrate profiles and kinetic and thermodynamic properties. AAC‐IIa and APH are also polar opposites in terms of ligand promiscuity but share no sequence or apparent structural homology. However, they both are highly dynamic and may even contain disordered segments and both adopt well‐defined conformations when AGs are bound. Contrary to this AAC‐IIIb maintains a well‐defined structure even in apo form. Data presented herein suggest that the antibiotic promiscuity of AGMEs may be determined neither by the flexibility of the protein nor the size of the active site cavity alone but strongly modulated or controlled by the effects of the cosubstrate on the dynamic and thermodynamic properties of the enzyme.     

Laminin receptors: achieving specificity through cooperation

The laminins are a large family of extracellular matrix proteins that can profoundly influence development, differentiation and disease progression. The biological effects of the laminins are mediated by surface receptors that link laminin matrices to intracellular signalling pathways. Several classes of receptors, including integrins and other molecules, may cooperate to provide the specificity apparent in the diverse array of laminin-mediated phenomena. This review assesses our current understanding of laminin receptors and discusses how such receptors could recognize structural differences among the laminins and relay these differences to the cell.     

Signalling through nuclear receptors

A century ago, secretions from the pancreas were described as 'hormones', which we now know are secreted from all ductless glands. The development of various technologies has already contributed a great deal -- and will undoubtedly offer more -- to our understanding of their mode of action.     

Identification of specificity and promiscuity of PDZ domain interactions through their dynamic behavior

PDZ domains (PDZs), the most common interaction domain proteins, play critical roles in many cellular processes. PDZs perform their job by binding specific protein partners. However, they are very promiscuous, binding to more than one protein, yet selective at the same time. We examined the binding related dynamics of various PDZs to have insight about their specificity and promiscuity. We used full atomic normal mode analysis and a modified coarse‐grained elastic network model to compute the binding related dynamics. In the latter model, we introduced specificity for each single parameter constant and included the solvation effect implicitly. The modified model, referred to as specific‐Gaussian Network Model (s‐GNM), highlights some interesting differences in the conformational changes of PDZs upon binding to Class I or Class II type peptides. By clustering the residue fluctuation profiles of PDZs, we have shown: (i) binding selectivities can be discriminated from their dynamics, and (ii) the dynamics of different structural regions play critical roles for Class I and Class II specificity. s‐GNM is further tested on a dual‐specific PDZ which showed only Class I specificity when a point mutation exists on the βA‐βB loop. We observe that the binding dynamics change consistently in the mutated and wild type structures. In addition, we found that the binding induced fluctuation profiles can be used to discriminate the binding selectivity of homolog structures. These results indicate that s‐GNM can be a powerful method to study the changes in binding selectivities for mutant or homolog PDZs. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Antibody against nuclear thyroid hormone receptors

Rat liver nuclear thyroid hormone receptor was purified to 700-1600 pmol T3 binding capacity/mg protein by sequentially using hydroxylapatite column, ammonium sulfate precipitation, Sephadex G-150 gel filtration, DNA-cellulose column, DEAE-Sephadex A-50 column, and heparin-Sepharose column. Serum from a mouse immunized using this purified receptor preparation caused a shift of [125I]T3-receptor peak on glycerol density gradient sedimentation from 3.4 S to approximately 7 S. [125I]T3-receptor complex was immunoprecipitated using this serum and goat anti-mouse IgG. The serum showed reduced ability to immunoprecipitate the globular T3 binding fragment with Stokes radius of 22 A produced by trypsin digestion, a receptor fragment which has core histone and hormone binding but not DNA binding activity. These data indicate the production of anti-nuclear thyroid hormone receptor antibody which mainly recognized epitopes unrelated to hormone and core histone binding domain.     

Intrinsic disorder in nuclear hormone receptors

Many proteins possess intrinsic disorder (ID) and lack a rigid three-dimensional structure in at least part of their sequence. ID has been hypothesized to influence protein-protein and protein-ligand interactions. We calculated ID for nearly 400 vertebrate and invertebrate members of the biomedically important nuclear hormone receptor (NHR) superfamily, including all 48 known human NHRs. The predictions correctly identified regions in 20 of the 23 NHRs suggested as disordered based on published X-ray and NMR structures. Of the four major NHR domains (N-terminal domain, DNA-binding domain, D-domain, and ligand-binding domain), we found ID to be highest in the D-domain, a region of NHRs critical in DNA recognition and heterodimerization, coactivator/corepressor interactions and protein-protein interactions. ID in the D-domain and LBD was significantly higher in "hub" human NHRs that have 10 or more downstream proteins in their interaction networks compared to "non-hub" NHRs that interact with fewer than 10 downstream proteins. ID in the D-domain and LBD was also higher in classic, ligand-activated NHRs than in orphan, ligand-independent NHRs in human. The correlation between ID in human and mouse NHRs was high. Less correlation was found for ID between mammalian and non-mammalian vertebrate NHRs. For some invertebrate species, particularly sea squirts ( Ciona), marked differences were observed in ID between invertebrate NHRs and their vertebrate orthologs. Our results indicate that variability of ID within NHRs, particularly in the D-domain and LBD, is likely an important evolutionary force in shaping protein-protein interactions and NHR function. This information enables further understanding of these therapeutic targets.     

Genome-wide histone modifications: gaining specificity by preventing promiscuity

More than 20 residues within the four core histone proteins of the nucleosome are potential sites of post-translational modifications, such as methylation, acetylation, ubiquitination and phosphorylation. It has been hypothesized that specific patterns of these modifications on the nucleosome facilitate recruitment of non-histone proteins to chromatin. When such modifications are restricted to particular regions of the genome, they seem to play an important role in creating specific chromatin domains. However, more recent results suggest that some histone modifications, particularly those that exist on a genome-wide scale, act to reduce nonspecific binding by chromatin proteins involved in silencing. This decrease of promiscuous binding ensures that the silent chromatin proteins are not titrated away from their normal locations on chromosomes. We suggest that preventing such promiscuous binding of chromatin proteins is an important part of generating specificity to create chromatin domains and overall chromosome organization.     

Origins of specificity and promiscuity in metabolic networks

How enzymes have evolved to their present form is linked to the question of how pathways emerged and evolved into extant metabolic networks. To investigate this mechanism, we have explored the chemical diversity present in a largely unbiased data set of catalytic reactions processed by modern enzymes across the tree of life. In order to get a quantitative estimate of enzyme chemical diversity, we measure enzyme multispecificity or promiscuity using the reaction molecular signatures. Our main finding is that reactions that are catalyzed by a highly specific enzyme are shared by poorly divergent species, suggesting a later emergence of this function during evolution. In contrast, reactions that are catalyzed by highly promiscuous enzymes are more likely to appear uniformly distributed across species in the tree of life. From a functional point of view, promiscuous enzymes are mainly involved in amino acid and lipid metabolisms, which might be associated with the earliest form of biochemical reactions. In this way, results presented in this paper might assist us with the identification of primeval promiscuous catalytic functions contributing to life's minimal metabolism.     

Glycoprotein hormone receptors: determinants in leucine-rich repeats responsible for ligand specificity

Glycoprotein hormone receptors [thyrotropin (TSHr), luteinizing hormone/chorionic gonadotropin (LH/CGr), follicle stimulating hormone (FSHr)] are rhodopsin-like G protein-coupled receptors with a large extracellular N-terminal portion responsible for hormone recognition and binding. In structural models, this ectodomain is composed of two cysteine clusters flanking nine leucine-rich repeats (LRRs). The LRRs form a succession of beta-strands and alpha-helices organized into a horseshoe-shaped structure. It has been proposed that glycoprotein hormones interact with residues of the beta-strands making the concave surface of the horseshoe. Gain-of-function homology scanning of the beta-strands of glycoprotein hormone receptors allowed identification of the critical residues responsible for the specificity towards human chorionic gonadotropin (hCG). Substitution of eight or two residues of the LH/CGr into the TSHr or FSHr, respectively, resulted in constructs displaying almost the same affinity and sensitivity for hCG as wild-type LH/CGr. Molecular dynamics simulations and additional site-directed mutagenesis provided a structural rationale for the evolution of binding specificity in this duplicated gene family.