Molecular recognition events involved in the activation of the growth hormone receptor by growth hormone

Binding of growth hormone (GH) to its receptor (GHR) is a well-studied example of molecular recognition between a cytokine and its receptor. Extensive mutagenesis studies and several crystal structures have defined the key interactive amino acid residues that are involved in binding and subsequent receptor dimerization. This review encompasses each of the three molecular recognition events involved in GHR activation, namely binding of GH to its two receptors and the interactions that occur between these receptors. Particular attention is given to species and ligand specificity of hormone binding and to the molecular recognition events involved in receptor activation, including the possibility that a conformational change in the receptor is required.     

Efficient incorporation of protein flexibility and dynamics into molecular docking simulations

Flexibility and dynamics are protein characteristics that are essential for the process of molecular recognition. Conformational changes in the protein that are coupled to ligand binding are described by the biophysical models of induced fit and conformational selection. Different concepts that incorporate protein flexibility into protein-ligand docking within the context of these two models are reviewed. Several computational studies that discuss the validity and possible limitations of such approaches will be presented. Finally, different approaches that incorporate protein dynamics, e.g., configurational entropy, and solvation effects into docking will be highlighted.     

Binding Affinities Controlled by Shifting Conformational Equilibria: Opportunities and Limitations

Conformational selection is an established mechanism in molecular recognition. Despite its power to explain binding events, it is hardly used in protein/ligand design to modulate molecular recognition. Here, we explore the opportunities and limitations of design by conformational selection. Using appropriate thermodynamic cycles, our approach predicts the effects of a conformational shift on binding affinity and also allows one to disentangle the effects induced by a conformational shift from other effects influencing the binding affinity. The method is assessed and applied to explain the contribution of a conformational shift on the binding affinity of six ubiquitin mutants showing different conformational shifts in six different complexes.     

The nicotinic receptor ligand binding domain

The ligand binding domain (LBD) of the nicotinic acetylcholine receptor has served as a prototype for understanding molecular recognition in the family of neurotransmitter-gated ion channels. During the past fifty years, studies progressed from fundamental electrophysiological analyses of ACh-evoked ion flow, to biochemical purification of the receptor protein, pharmacological measurements of ligand binding, molecular cloning of receptor subunits, site-directed mutagenesis combined with functional analysis and recently, atomic structural determination. The emerging picture of the nicotinic receptor LBD is a specialized pocket of aromatic and hydrophobic residues formed at interfaces between protein subunits that changes conformation to convert agonist binding into gating of an intrinsic ion channel.     

Evolutionarily conserved paired immunoglobulin-like receptor α (PILRα) domain mediates its interaction with diverse sialylated ligands

Paired immunoglobulin-like receptor (PILR) α is an inhibitory receptor that recognizes several ligands, including mouse CD99, PILR-associating neural protein, and Herpes simplex virus-1 glycoprotein B. The physiological function(s) of interactions between PILRα and its cellular ligands are not well understood, as are the molecular determinants of PILRα/ligand interactions. To address these uncertainties, we sought to identify additional PILRα ligands and further define the molecular basis for PILRα/ligand interactions. Here, we identify two novel PILRα binding partners, neuronal differentiation and proliferation factor-1 (NPDC1), and collectin-12 (COLEC12). We find that sialylated O-glycans on these novel PILRα ligands, and on known PILRα ligands, are compulsory for PILRα binding. Sialylation-dependent ligand recognition is also a property of SIGLEC1, a member of the sialic acid-binding Ig-like lectins. SIGLEC1 Ig domain shares ～22% sequence identity with PILRα, an identity that includes a conserved arginine localized to position 97 in mouse and human SIGLEC1, position 133 in mouse PILRα and position 126 in human PILRα. We observe that PILRα/ligand interactions require conserved PILRα Arg-133 (mouse) and Arg-126 (human), in correspondence with a previously reported requirement for SIGLEC1 Arg-197 in SIGLEC1/ligand interactions. Homology modeling identifies striking similarities between PILRα and SIGLEC1 ligand binding pockets as well as at least one set of distinctive interactions in the galactoxyl-binding site. Binding studies suggest that PILRα recognizes a complex ligand domain involving both sialic acid and protein motif(s). Thus, PILRα is evolved to engage multiple ligands with common molecular determinants to modulate myeloid cell functions in anatomical settings where PILRα ligands are expressed.     

A method for biomolecular structural recognition and docking allowing conformational flexibility.

In this work, we present an algorithm developed to handle biomolecular structural recognition problems, as part of an interdisciplinary research endeavor of the Computer Vision and Molecular Biology fields. A key problem in rational drug design and in biomolecular structural recognition is the generation of binding modes between two molecules, also known as molecular docking. Geometrical fitness is a necessary condition for molecular interaction. Hence, docking a ligand (e.g., a drug molecule or a protein molecule), to a protein receptor (e.g., enzyme), involves recognition of molecular surfaces. Conformational transitions by "hinge-bending" involves rotational movements of relatively rigid parts with respect to each other. The generation of docked binding modes between two associating molecules depends on their three dimensional structures (3-D) and their conformational flexibility. In comparison to the particular case of rigid-body docking, the computational difficulty grows considerably when taking into account the additional degrees of freedom intrinsic to the flexible molecular docking problem. Previous docking techniques have enabled hinge movements only within small ligands. Partial flexibility in the receptor molecule is enabled by a few techniques. Hinge-bending motions of protein receptors domains are not addressed by these methods, although these types of transitions are significant, e.g., in enzymes activity. Our approach allows hinge induced motions to exist in either the receptor or the ligand molecules of diverse sizes. We allow domains/subdomains/group of atoms movements in either of the associating molecules. We achieve this by adapting a technique developed in Computer Vision and Robotics for the efficient recognition of partially occluded articulated objects. These types of objects consist of rigid parts which are connected by rotary joints (hinges). Our method is based on an extension and generalization of the Hough transform and the Geometric Hashing paradigms for rigid object recognition. We show experimental results obtained by the successful application of the algorithm to cases of bound and unbound molecular complexes, yielding fast matching times. While the "correct" molecular conformations of the known complexes are obtained with small RMS distances, additional, predictive good-fitting binding modes are generated as well. We conclude by discussing the algorithm's implications and extensions, as well as its application to investigations of protein structures in Molecular Biology and recognition problems in Computer Vision.     

Biochemistry and Function of Nematode Steroids

Abstract

Elucidation of the molecular mechanisms that govern ligand-receptor recognition is essential to the rational design of specific pharmacological reagents. Whereas often the receptor and its binding site are the target of investigation, study of the ligand in its free and bound state can also reveal important information regarding this recognition process. Nuclear magnetic resonance (NMR) spectroscopy can be extremely useful for such studies. In this review, we discuss the attributes of NMR in the study of ligand receptor interactions. The cholinergic receptor and its binding to the neurotransmitter, acetylcholine, and cholinergic antagonists serve as a model system, illustrating the power of ligand analysis by NMR. The results discussed prove that the region of residues a 180–205 of the nicotinic acetylcholine receptor are an essential component of the cholinergic binding site and that ligand binding involves a positively charged hydrophobic motif.     

Self-assembly of HEK cell-secreted ApoE particles resembles ApoE enrichment of lipoproteins as a ligand for the LDL receptor-related protein

Recent studies have shown that the lipidation and assembly state of apolipoprotein E (apoE) determine receptor recognition and amyloid-beta peptide (Abeta) binding. We previously demonstrated that apoE secreted by HEK cells stably expressing apoE3 or apoE4 (HEK-apoE) binds Abeta and inhibits Abeta-induced neurotoxicity by an isoform-specific process that requires apoE receptors. Here we characterized the structure of HEK-apoE assemblies and determined their receptor binding specificity. By chromatography, HEK-apoE elutes in high molecular mass fractions and is the size of plasma HDL, consistent with a multiprotein assembly. No lipid was associated with these apoE assemblies. Several methods for analyzing receptor binding indicate that HEK-apoE is a ligand for low-density lipoprotein (LDL) receptor-related protein (LRP) but not the LDL receptor. This suggests that self-assembly of apoE may induce a functional conformation necessary for binding to LRP. Our results indicate that, in addition to lipid content, the assembly state of apoE influences Abeta binding and receptor recognition.     

A profile of the residues in the second extracellular loop that are critical for ligand recognition of human prostacyclin receptor

The residues in the second extracellular loop (eLP2) of the prostanoid receptors, which are important for specific ligand recognition, were previously predicted in our earlier studies of the thromboxane A2 receptor (TP) using a combination of NMR spectroscopy and recombinant protein approaches. To further test this hypothesis, another prostanoid receptor, the prostacyclin receptor (IP), which has opposite biological characteristics to that of TP, was used as a model for these studies. A set of recombinant human IPs with site-directed mutations at the nonconserved eLP2 residues were constructed using an Ala-scanning approach, and then expressed in HEK293 and COS-7 cells. The expression levels of the recombinant receptors were six-fold higher in HEK293 cells than in COS-7 cells. The residues important for ligand recognition and binding within the N-terminal segment (G159, Q162, and C165) and the C-terminal segment (L172, R173, M174, and P179) of IP eLP2 were identified by mutagenesis analyses. The molecular mechanisms for the specific ligand recognition of IP were further demonstrated by specific site-directed mutagenesis using different amino acid residues with unique chemical properties for the key residues Q162, L172, R173, and M174. A comparison with the corresponding functional residues identified in TP eLP2 revealed that three (Q162, R173, and M174) of the four residues are nonconserved, and these are proposed to be involved in specific ligand recognition. We discuss the importance of G159 and P179 in ligand recognition through configuration of the loop conformation is discussed. These studies have further indicated that characterization of the residues in the eLP2 regions for all eight prostanoid receptors could be an effective approach for uncovering the molecular mechanisms of the ligand selectivities of the G-protein-coupled receptors.     

Computational simulations of stem-cell factor/c-Kit receptor interaction

Stem-cell factor (SCF) is a noncovalent homodimeric cytokine that exhibits profound biological function in the early stages of hematopoiesis by binding to a cell surface tyrosine kinase receptor that is encoded by the c-Kit proto-oncogene. The results obtained from a combined implementation of homology-based molecular modeling and computational simulations in the study of species-specific SCF/c-Kit interactions are reported. The structural models of the human and rat SCF ligands are based on the close structural similarity to the cytokine M-CSF, whose Cα structure has recently become available. The constant domains of the human Fc fragment are used as a template for the ligand binding domains of the c-Kit receptor. The factors responsible for the stabilization of the SCF quaternary structure and the molecular determinants for ligand recognition and ligand specificity have been identified by assessing the conformational, topographical, and dynamic features of the isolated ligands and of the ligand-receptor complexes. © 1996 Wiley-Liss, Inc.     

Structure activity studies of the melanocortin-4 receptor by in vitro mutagenesis: identification of agouti-related protein (AGRP), melanocortin agonist and synthetic peptide antagonist interaction determinants

In vitro mutagenesis of the mouse melanocortin-4 receptor (mMC4R) has been performed, based upon homology molecular modeling and previous melanocortin receptor mutagenesis studies that identified putative ligand-receptor interactions. Twenty-three mMC4 receptor mutants were generated and pharmacologically characterized using several melanocortin-based ligands [alpha-MSH, NDP-MSH, MTII, DNal (1')(7)-MTII, Nal(2')(7)-MTII, SHU9119, and SHU9005]. Selected mutant receptors possessing significant differences in the melanocortin-based peptide agonist and/or antagonist pharmacology were further evaluated using the endogenous antagonist agouti-related protein fragment hAGRP(83-132) and hAGRP(109-118) molecules. These studies of the mouse MC4R provide further experimental data suggesting that the conserved melanocortin receptor residues Glu92 (TM2), Asp114 (TM3), and Asp118 (TM3) (mouse MC4R numbering) are important for melanocortin-based peptide molecular recognition. Additionally, the Glu92 and Asp118 mMC4R residues are important for molecular recognition and binding of AGRP(83-132). We have identified the Phe176 (TM4), Tyr179 (TM4), Phe254 (TM6), and Phe259 (TM6) receptor residues as putatively interacting with the melanocortin-based ligand Phe(7) by differences between alpha-MSH and NDP-MSH agonist potencies. The Glu92, Asp118, and Phe253 mMC4R receptor residues appear to be critical for hAGRP(83-132) molecular recognition and binding while Phe176 appears to be important for functional antagonism of AGRP(83-132) and AGRP(109-118) but not molecular recognition. The Phe253 mMC4R residue appears to be important for AGRP(83-132) molecular recognition and general mMC4 receptor stimulation. The Phe254 and Phe259 mMC4R amino acids may participate in the differentiation of agonist versus antagonist activity of the melanocortin-based peptide antagonists SHU9119 and SHU9005, but not AGRP(83-132) or AGRP(109-118). The Met192 side chain when mutated to a Phe results in a constitutively active mMC4R that does not effect agonist ligand binding or potency. Melanocortin-based peptides modified at the 7 position of MTII with DPhe, DNal(1'), Nal(2'), and DNal(2') have been pharmacologically characterized at these mutant mouse MC4Rs. These data suggest a revised hypothesis for the mechanism of SHU9119 antagonism at the MC4R which may be attributed to the presence of a "bulky" naphthyl moiety at the 7 position (original hypothesis), and additionally that both the stereochemistry and naphthyl ring position (2' versus 1') are important for positioning of the ligand Arg(8) residue with the corresponding mMC4R amino acids.     

Differential helical orientations among related G protein-coupled receptors provide a novel mechanism for selectivity. Studies with salvinorin A and the kappa-opioid receptor

Salvinorin A, the active component of the hallucinogenic sage Salvia divinorum, is an apparently selective and highly potent kappa-opioid receptor (KOR) agonist. Salvinorin A is unique among ligands for peptidergic G protein-coupled receptors in being nonnitrogenous and lipid-like in character. To examine the molecular basis for the subtype-selective binding of salvinorin A, we utilized an integrated approach using chimeric opioid receptors, site-directed mutagenesis, the substituted cysteine accessibility method, and molecular modeling and dynamics studies. We discovered that helix 2 is required for salvinorin A binding to KOR and that two residues (Val-108(2.53) and Val-118(2.63)) confer subtype selectivity. Intriguingly, molecular modeling studies predicted that these loci exhibit an indirect effect on salvinorin A binding, presumably through rotation of helix 2. Significantly, and in agreement with our in silico predictions, substituted cysteine accessibility method analysis of helix 2 comparing KOR and the delta-opioid receptor, which has negligible affinity for salvinorin A, revealed that residues known to be important for salvinorin A binding exhibit a differential pattern of water accessibility. These findings imply that differences in the helical orientation of helix 2 are critical for the selectivity of salvinorin A binding to KOR and provide a structurally novel basis for ligand selectivity.     

Impact of polyphenols on receptor–ligand interactions by NMR: the case of neurotensin (NT)–neurotensin receptor fragment (NTS1) complex

Abstract

Ligand–receptor interactions can be implicated in many pathological events such as chronic neurodegenerative diseases. Thus, the discovery of molecules disrupting this type of interactions could be an interesting therapeutic approach. Polyphenols are well known for their affinity for proteins and several studies have characterized these direct interactions. But studying the direct influence of multi-therapeutic drugs on a ligand–receptor complex relevant to a neurodegenerative disorder is a challenging issue. Solution NMR, molecular modeling and iterative calculations were used to obtain information about the interaction between a phenolic compound, α-glucogallin (α-2) and a ligand/fragment receptor complex neurotensin (NT) and its receptor NTS1. The α-2 was shown to bind to NT and a peptidic fragment of its NTS1 receptor, independently. Although the formation of the corresponding ligand–receptor complex did not seem to be affected, this experimental modeling protocol will enable the evaluation of other anti-amyloidogenic compounds such as blockers of NT–NTS1 binding. These types of studies help in understanding the specificity and influence in binding and can provide information to develop new molecules with a putative pharmacological interest.

Communicated by Ramaswamy H. Sarma     

Identification of Edg1 receptor residues that recognize sphingosine 1-phosphate

Originating from its DNA sequence, a computational model of the Edg1 receptor has been developed that predicts critical interactions with its ligand, sphingosine 1-phosphate. The basic amino acids Arg(120) and Arg(292) ion pair with the phosphate, whereas the acidic Glu(121) residue ion pairs with the ammonium moiety of sphingosine 1-phosphate. The requirement of these interactions for specific ligand recognition has been confirmed through examination of site-directed mutants by radioligand binding, ligand-induced [(35)S]GTPgammaS binding, and receptor internalization assays. These ion-pairing interactions explain the ligand specificity of the Edg1 receptor and provide insight into ligand specificity differences within the Edg receptor family. This computational map of the ligand binding pocket provides information necessary for understanding the molecular pharmacology of this receptor, thus underlining the potential of the computational method in predicting ligand-receptor interactions.     

A structural perspective of the sequence variability within botulinum neurotoxin subtypes A1-A4

Botulinum neurotoxin (BoNT) is a category A toxin that has been classified within seven serotypes, designated A-G. Recently, it has been discovered that sequence variability occurs in BoNTs produced by serotype A (BoNT/A) variant strains, designated as subtypes A1 and A2, which have significantly different antibody-binding properties. We have therefore made efforts to understand at the molecular level the diversity and its effects on the biological actions of the toxin, including receptor binding, substrate recognition, and catalysis. We provide the results of these studies, including the analysis of two newly sequenced BoNT/A variants, Loch Maree (A3) and 657Ba (A4), and their comparison to A1 and A2. Using sequence analysis, available functional data, molecular modeling, and comparison of models with the crystal structures of BoNT/A1 and the light chain of BoNT/A2, we conclude that these sequence differences within subtypes will impact development of broad-spectrum antibody and small ligand therapeutics, and suggest dissimilarities in binding affinity and cleavage efficiency of the SNAP-25 substrate. In particular, sequence variation in subtypes BoNT/A3 and BoNT/A4 will likely effect alpha-exosite and S1' subsite recognition, respectively.     

Computational modeling study of functional microdomains in cannabinoid receptor type 1

The seven transmembrane helices (TMH) G-protein-coupled receptors (GPCRs) constitute one of the largest superfamily of signaling proteins found in mammals. Some of its members, in which the cannabinoid (CB) receptors are included, stand out because their functional states can be modulated by a broad spectrum of effector molecules. The relative ligand promiscuity exhibited by these receptors could be related with particular attributes conferred by their molecular architecture and represents a motivating issue to be explored. In this regard, this study represents an effort to investigate the cannabinoid receptor type 1 (CB1) ligand recognition plasticity, using comparative modeling, molecular dynamics (MD) simulations and docking. Our results suggest that a cooperative set of subtle structural rearrangements within the TMHs provide to the CB1 protein the plasticity to reach alternate configurations. These changes include the relaxation of intramolecular constraints, the rotations, translations and kinks of the majority of TMHs and the reorganization of the ligand binding cavities.     

Long-Timescale Molecular-Dynamics Simulations of the Major Urinary Protein Provide Atomistic Interpretations of the Unusual Thermodynamics of Ligand Binding

The mouse major urinary protein (MUP) has proved to be an intriguing test bed for detailed studies on protein-ligand recognition. NMR, calorimetric, and modeling investigations have revealed that the thermodynamics of ligand binding involve a complex interplay between competing enthalpic and entropic terms. We performed six independent, 1.2 μs molecular-dynamics simulations on MUP—three replicates on the apo-protein, and three on the complex with the pheromone isobutylmethoxypyrazine. Our findings provide the most comprehensive picture to date of the structure and dynamics of MUP, and how they are modulated by ligand binding. The mechanical pathways by which amino acid side chains can transmit information regarding ligand binding to surface loops and either increase or decrease their flexibility (entropy-entropy compensation) are identified. Dewetting of the highly hydrophobic binding cavity is confirmed, and the results reveal an aspect of ligand binding that was not observed in earlier, shorter simulations: bound ligand retains extensive rotational freedom. Both of these features have significant implications for interpretations of the entropic component of binding. More generally, these simulations test the ability of current molecular simulation methods to produce a reliable and reproducible picture of protein dynamics on the microsecond timescale.     

Conversion of human-selective PPARalpha agonists to human/mouse dual agonists: a molecular modeling analysis

To understand the species selectivity in a series of alpha-methyl-alpha-phenoxy carboxylic acid PPARalpha/gamma dual agonists (1-11), structure-based molecular modeling was carried out in the ligand binding pockets of both human and mouse PPARalpha. This study suggested that interaction of both 4-phenoxy and phenyloxazole substituents of these ligands with F272 and M279 in mouse PPARalpha leads to the species-specific divergence in ligand binding. Insights obtained in the molecular modeling studies of these key interactions resulted in the ability to convert a human-selective PPARalpha agonist to a human and mouse dual agonist within the same platform.