Alanine scanning mutational analysis of the ligand binding pocket of the human Vitamin D receptor

We achieved exhaustive alanine scanning mutational analysis of the amino acid residues lining the ligand binding pocket of the Vitamin D receptor to investigate the mechanism of the ligand recognition by the receptor. This is the first exhaustive analysis in the nuclear receptor superfamily. Our results demonstrated the role and importance of all the residues lining the ligand binding pocket. In addition, this analysis was found to indicate ligand-specific ligand-protein interactions, which have key importance in determining the transactivation potency of the individual ligands. Thus, the analysis using 1beta-methyl-1alpha,25-dihydroxyvitamin D(3) revealed the specific van der Waals interactions of 1beta-methyl group with the receptor.     

A critical region in the mineralocorticoid receptor for aldosterone binding and activation by cortisol: evidence for a common mechanism governing ligand binding specificity in steroid hormone receptors

The amino acids that confer aldosterone binding specificity to the mineralocorticoid receptor (MR) remain to be determined. We had previously analyzed a panel of chimeras created between the MR and the glucocorticoid receptor and determined that amino acids 804-874 of the MR ligand binding domain are critical for aldosterone binding. In the present study a further series of chimeras was created within this region. The chimeras were analyzed by a transactivation assay and [(3)H]aldosterone binding, and the critical region was narrowed down to amino acids 820-844. Site-directed mutagenesis was used to create single and multiple amino acid substitutions in this region. These studies identified 12 of the 16 amino acids that differ in the MR and the glucocorticoid receptor in this region as being critical to conferring aldosterone responsivity. The amino acids that differ in the region 820-844 lie on the surface of the molecule and, therefore, it appears that MR ligand binding selectivity is conferred by residues that do not form part of the ligand binding pocket. Other studies have found that the corresponding regions of the androgen and glucocorticoid receptors are critical for the binding of natural and synthetic ligands, suggesting a common mechanism governing ligand binding specificity. The new chimeras also displayed, as previously reported, a dissociation between cortisol binding and transactivation and, intriguingly, only those that bound aldosterone with high affinity were activated by cortisol, suggesting a common mechanism that underlies specificity of aldosterone binding and the ability of cortisol to activate the MR.     

Steric and thermodynamic limits of design for the incorporation of large unnatural amino acids in aminoacyl‐tRNA synthetase enzymes

Orthogonal aminoacyl‐tRNA synthetase/tRNA pairs from archaea have been evolved to facilitate site specific in vivo incorporation of unnatural amino acids into proteins in Escherichia coli. Using this approach, unnatural amino acids have been successfully incorporated with high translational efficiency and fidelity. In this study, CHARMM‐based molecular docking and free energy calculations were used to evaluate rational design of specific protein–ligand interactions for aminoacyl‐tRNA synthetases. A series of novel unnatural amino acid ligands were docked into the p‐benzoyl‐L ‐phenylalanine tRNA synthetase, which revealed that the binding pocket of the enzyme does not provide sufficient space for significantly larger ligands. Specific binding site residues were mutated to alanine to create additional space to accommodate larger target ligands, and then mutations were introduced to improve binding free energy. This approach was used to redesign binding sites for several different target ligands, which were then tested against the standard 20 amino acids to verify target specificity. Only the synthetase designed to bind Man‐α‐O‐Tyr was predicted to be sufficiently selective for the target ligand and also thermodynamically stable. Our study suggests that extensive redesign of the tRNA synthatase binding pocket for large bulky ligands may be quite thermodynamically unfavorable. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

The unique ligand-binding pocket for the human prostacyclin receptor. Site-directed mutagenesis and molecular modeling

The human prostacyclin receptor is a seven-transmembrane alpha-helical G-protein coupled receptor, which plays important roles in both vascular smooth muscle relaxation as well as prevention of blood coagulation. The position of the native ligand-binding pocket for prostacyclin as well as other derivatives of the 20-carbon eicosanoid, arachidonic acid, has yet to be determined. Through the use of prostanoid receptor sequence alignments, site-directed mutagenesis, and the 2.8-A x-ray crystallographic structure of bovine rhodopsin, we have developed a three-dimensional model of the agonist-binding pocket within the seven-transmembrane (TM) domains of the human prostacyclin receptor. Upon mutation to alanine, 11 of 29 candidate residues within TM domains II, III, IV, V, and VII exhibited a marked decrease in agonist binding. Of this group, four amino acids, Arg-279 (TMVII), Phe-278 (TMVII), Tyr-75 (TMII), and Phe-95 (TMIII), were identified (via receptor amino acid sequence alignment, ligand structural comparison, and computer-assisted homology modeling) as having direct molecular interactions with ligand side-chain constituents. This binding pocket is distinct from that of the biogenic amine receptors and rhodopsin where the native ligands (also composed of a carbon ring and a carbon chain) are accommodated in an opposing direction. These findings should assist in the development of novel and highly specific ligands including selective antagonists for further molecular pharmacogenetic studies of the human prostacyclin receptor.     

Beryllium binding at neutral pH: the importance of the Be-O-Be motif

Beryllium speciation at physiological conditions is critical to understanding chronic beryllium disease (CBD). The MHC-class II receptor alleles that have been linked to CBD have more than six carboxylates in a short 20 amino acid segment of the binding pocket and it has been suggested that beryllium may bind within the MHC-class II receptor via the carboxylates. Previous reports also show that citric acid binds beryllium significantly stronger than similar carboxylate ligands such as tartaric acid and is one of the few ligands that can compete with hydrolysis to solubilize beryllium across the entire pH range at molar concentrations. We have characterized the binding of Be to citric acid and shown using a combination of NMR, mass spectrometry and ligand competition studies that Be2L and Be4L2 species dominate. A Be-O-Be linkage with the bridging oxygen coming from the aliphatic alcohol is critical to the stability of the complex. We show through competition experiments that the most stable Be-O-Be arrangement has one Be in a five-member ring and the other Be in a six-member ring. The unusual deprotonation of an aliphatic alcohol (pK(a) = 18) at neutral pH has significant ramifications on the potential interactions of Be with biological ligands such as carbohydrates and Ser and Thr residues.     

Structure-activity relationship studies of Bz amide-containing α-GalCer derivatives as natural killer T cell modulators

Abstract

CD1d is a non-polymorphic antigen-presenting glycoprotein that recognizes glycolipids as ligands. Ligands bind to the hydrophobic grooves of CD1d, and the resulting ligand-CD1d complexes activate natural killer T (NKT) cells by means of T cell receptor recognition, leading to the secretion of various cytokines. However, details of the ligand recognition mechanism of a large hydrophobic ligand binding pocket and the relationship between cytokine induction and ligand structure are unclear. We report the synthesis of α-GalCer derivatives containing a Bz amide group having various substituting groups in the ceramide moiety, and the analysis of the structure-activity relationships. The assays reveal that the Bz amide-containing CD1d ligands function as NKT cell modulators displaying Th2 cytokine biasing responses. Furthermore, molecular dynamics simulation studies suggest that the phenyl groups can interact with the aromatic amino acid residues in the lipid binding pocket of CD1d.     

Binding modes of 6,7 di-substituted 4-anilinoquinoline-3-carbonitriles to EGFR

4-Anilino-3-cyanoquinolines were reported to have irreversible binding to epidermal growth factor receptor kinase (EGFRK) by forming a covalent linkage to C773. Our initial docking studies gave results inconsistent with the in vitro data and showed two different binding modes. To perceive the exact mode of binding of these ligands, two models of the ligand-EGFR complexes were considered: (1) reversible binding mode in which the ligand had hydrogen bond interactions at the binding site and (2) irreversible binding mode wherein the ligand's Michael acceptor side chain has proximity to the sulfhydryl group of C773 of EGFR, thereby enabling a covalent interaction. The irreversible binding mode correlated better than reversible binding mode with respect to in vitro data. However, our results indicate that both modes are being adopted by the ligands and could be utilized to design more potent EGFRK inhibitors.     

Zn-alpha2-glycoprotein, an MHC class I-related glycoprotein regulator of adipose tissues: modification or abrogation of ligand binding by site-directed mutagenesis

Zn-alpha(2)-glycoprotein (ZAG) is a soluble lipid-mobilizing factor associated with cancer cachexia and is a novel adipokine. Its X-ray crystal structure reveals a poly(ethylene glycol) molecule, presumably substituting for a higher affinity natural ligand, occupying an apolar groove between its alpha(1) and alpha(2) domain helices that corresponds to the peptide binding groove in class I MHC proteins. We previously provided evidence that the groove is a binding site for hydrophobic ligands that may relate to the protein's signaling function and that the natural ligands are probably (polyunsaturated) fatty acid-like. Using fluorescence-based binding assays and site-directed mutagenesis, we now demonstrate formally that the groove is indeed the binding site for hydrophobic ligands. We also identify amino acid positions that are involved in ligand binding and those that control the shape and exposure to solvent of the binding site itself. Some of the mutants showed minimal effects on their binding potential, one showed enhanced binding, and several were completely nonbinding. Particularly notable is Arg-73, which projects into one end of the binding groove and is the sole charged amino acid adjacent to the ligand. Replacing this amino acid with alanine abolished ligand binding and closed the groove to solvent. Arg-73 may therefore have an unexpected dual role in binding site access and anchor for an amphiphilic ligand. These data add weight to the distinctiveness of ZAG among MHC class I-like proteins in addition to providing defined binding-altered mutants for cellular signaling studies and potential medical applications.     

Structural determinants of odorant recognition by the human olfactory receptors OR1A1 and OR1A2

An interaction of odorants with olfactory receptors is thought to be the initial step in odorant detection. However, ligands have been reported for only 6 out of 380 human olfactory receptors, with their structural determinants of odorant recognition just beginning to emerge. Guided by the notion that amino acid positions that interact with specific odorants would be conserved in orthologs, but variable in paralogs, and based on the prediction of a set of 22 of such amino acid positions, we have combined site-directed mutagenesis, rhodopsin-based homology modelling, and functional expression in HeLa/Olf cells of receptors OR1A1 and OR1A2. We found that (i) their odorant profiles are centred around citronellic terpenoid structures, (ii) two evolutionary conserved amino acid residues in transmembrane domain 3 are necessary for the responsiveness of OR1A1 and the mouse ortholog Olfr43 to (S)-(-)-citronellol, (iii) changes at these two positions are sufficient to account for the differential (S)-(-)-citronellol responsiveness of the paralogs OR1A1 and OR1A2, and (iv) the interaction sites for (S)-(-)-citronellal and (S)-(-)-citronellol differ in both human receptors. Our results show that the orientation of odorants within a homology modelling-derived binding pocket of olfactory receptor orthologs is defined by evolutionary conserved amino acid positions.     

Molecular basis for the subtype discrimination of the estrogen receptor-beta-selective ligand,diarylpropionitrile

Although the two subtypes of the human estrogen receptor (ER), ERalpha and ERbeta, share only 56% amino acid sequence identity in their ligand binding domain (LBD), the residues that surround the ligand are nearly identical; nevertheless, subtype-selective ligands are known. To understand the molecular basis by which diarylpropionitrile (DPN), an ERbeta-selective ligand, is able to discriminate between the two ERs, we examined its activity on ER mutants and chimeric constructs generated by DNA shuffling. The N-terminal region of the ERbeta LBD (through helix 6) appears to be fully responsible for the ERbeta selectivity of DPN. In fact, a single ERalpha point mutation (L384M) was largely sufficient to switch the DPN response of this ER to that of the ERbeta type, but residues in helix 3 are also important in achieving the full ERbeta selectivity of DPN. Using molecular modeling, we found an energetically favorable fit for the S-DPN enantiomer in ERbeta, in which the proximal phenol mimics the A ring of estradiol, and the nitrile engages in stabilizing interactions with residues in the ligand-binding pocket of ERbeta. Our findings highlight that a limited number of critical interactions of DPN with the ERbeta ligand-binding pocket underlie its ER subtype-selective character.     

Screening and docking chemical ligands onto pocket cavities of a protease for designing a biocatalyst

A detailed study of the trypsin surface has been carried out to gain insight into its biological functions and interactions which helped to determine the binding specificity. Twenty-four cavity pockets were automatically identified on trypsin from PDB file entry 1AUJ using CASTp (Computed Atlas of Surface Topography of proteins). Molecular docking was exploited as an efficient in silico screening tool for studying protein–ligand interactions. A systematic docking study using Autodock 3.05 has been performed on the five largest binding pockets in trypsin. A set of ten putative chemical ligands was used to dock into selected binding pockets. Docking of ligands into the five largest pockets in trypsin showed that 1,10-phenanthroline and ethanolamine preferentially bound at pocket 24 and benzamidine at pocket 22. Thermodynamically, we also found that ethanol, propanol, propandiol and phosphoethanolamine preferentially bound at pocket 21 whereas p-aminobenzamidine, phenylacetic acid and phenylalanine interacted mainly at pocket 20 based on their lowest interaction free energy.     

Structural mechanism of specific ligand recognition by a lipocalin tailored for the complexation of digoxigenin

DigA16 is an artificial digoxigenin-binding protein, which was derived from the bilin-binding protein, a lipocalin of Pieris brassicae, via reshaping of its natural ligand pocket. Here we report the crystal structures of DigA16 in the presence of either digoxigenin or digitoxigenin and for the apo-protein at resolutions below 1.9A. As a consequence of the altogether 17 amino acid substitutions within the binding site significant structural changes have occurred in the four loops that form the entrance to the ligand pocket on top of the structurally conserved beta-barrel framework. For example, one loop adopts a new alpha-helical backbone structure, which seems to be induced by few critical side-chain contacts. Digoxigenin becomes almost fully buried (by 95%) upon complexation, whereby specificity for the hydrophilic steroid is maintained through hydrogen-bonding networks and shape complementarity. The differential binding of the related steroid digitoxigenin is mainly governed by an internal histidine residue, whose side-chain undergoes significant induced fit. Among those amino acids that line the ligand pocket two tyrosine and one tryptophan residue provide the largest contacts. Interestingly, corresponding three side-chains are found with the same mutual orientation in the anti-digoxigenin antibody 26-10, even though the hapten orientation is quite different there and only 66% of the steroid surface is buried in the combining site. Hence, in the case of the engineered lipocalin DigA16 an example of convergent in vitro evolution is observed. Generally, the remarkable structural plasticity of the loop region and the role of polar residues in the binding site illustrate the potential of the lipocalin scaffold for the generation of specific receptor proteins towards a variety of ligands.     

Mutation-induced quisqualic acid and ibotenic acid affinity at the metabotropic glutamate receptor subtype 4: ligand selectivity results from a synergy of several amino acid residues

The metabotropic glutamate receptors (mGluRs) are key modulators of excitatory neurotransmission in the central nervous system. The eight mGluR subtypes are seven trans-membrane-spanning proteins that possess a large extracellular amino-terminal domain in which the endogenous ligand binding pocket resides. In this study, we have identified four non-conserved amino acid residues that are essential for differentiating mGluR1 from mGluR4. Our approach has been to increase the affinity of the classic mGluR1 agonists, quisqualic acid and ibotenic acid, at mGluR4 by making various point mutations that mimicked mGluR1 residues. Based on ligand docking to homology models, the non-conserved residues, Lys-74, Glu-287, Ser-313, and Lys-317, were chosen for the mutational studies and all of the mutations proved capable of partially or completely restoring the affinities of the ligands. In particular, the mutations K74Y and K317R induced dramatic triple-order-of-magnitude increases in the affinity of ibotenic acid at mGluR4, making the affinity equivalent to that of mGluR1. Furthermore, the affinity of quisqualic acid at mGluR4 was increased to the same level as mGluR1 by the two double mutations, K74Y/K317R and K74Y/E287G. Advanced analysis of ligand conformation and docking procedures were used for the interpretation of these results. The study shows that mGluR subtype selectivity results from a complex interplay of residues shaping the binding pocket, rather than being attributable to a single specific ligand-receptor interaction.     

Side-chain flexibility in proteins upon ligand binding

Ligand binding may involve a wide range of structural changes in the receptor protein, from hinge movement of entire domains to small side-chain rearrangements in the binding pocket residues. The analysis of side chain flexibility gives insights valuable to improve docking algorithms and can provide an index of amino-acid side-chain flexibility potentially useful in molecular biology and protein engineering studies. In this study we analyzed side-chain rearrangements upon ligand binding. We constructed two non-redundant databases (980 and 353 entries) of "paired" protein structures in complexed (holo-protein) and uncomplexed (apo-protein) forms from the PDB macromolecular structural database. The number and identity of binding pocket residues that undergo side-chain conformational changes were determined. We show that, in general, only a small number of residues in the pocket undergo such changes (e.g., approximately 85% of cases show changes in three residues or less). The flexibility scale has the following order: Lys > Arg, Gln, Met > Glu, Ile, Leu > Asn, Thr, Val, Tyr, Ser, His, Asp > Cys, Trp, Phe; thus, Lys side chains in binding pockets flex 25 times more often then do the Phe side chains. Normalizing for the number of flexible dihedral bonds in each amino acid attenuates the scale somewhat, however, the clear trend of large, polar amino acids being more flexible in the pocket than aromatic ones remains. We found no correlation between backbone movement of a residue upon ligand binding and the flexibility of its side chain. These results are relevant to 1. Reduction of search space in docking algorithms by inclusion of side-chain flexibility for a limited number of binding pocket residues; and 2. Utilization of the amino acid flexibility scale in protein engineering studies to alter the flexibility of binding pockets.     

New pyrazolyl and thienyl aminohydantoins as potent BACE1 inhibitors: exploring the S2' region

The proteolytic enzyme β-secretase (BACE1) plays a central role in the synthesis of the pathogenic β-amyloid in Alzheimer's disease. SAR studies of the S2' region of the BACE1 ligand binding pocket with pyrazolyl and thienyl P2' side chains are reported. These analogs exhibit low nanomolar potency for BACE1, and demonstrate >50- to 100-fold selectivity for the structurally related aspartyl proteases BACE2 and cathepsin D. Small groups attached at the nitrogen of the P2' pyrazolyl moiety, together with the P3 pyrimidine nucleus projecting into the S3 region of the binding pocket, are critical components to ligand's potency and selectivity. P2' thiophene side chain analogs are highly potent BACE1 inhibitors with excellent selectivity against cathepsin D, but only modest selectivity against BACE2. The cell-based activity of these new analogs tracked well with their increased molecular binding with EC(50) values of 0.07-0.2 μM in the ELISA assay for the most potent analogs.     

Structures of liganded and unliganded RsrI N6-adenine DNA methyltransferase: a distinct orientation for active cofactor binding

The structures of RsrI DNA methyltransferase (M.RsrI) bound to the substrate S-adenosyl-l-methionine (AdoMet), the product S-adenosyl-l-homocysteine (AdoHcy), the inhibitor sinefungin, as well as a mutant apo-enzyme have been determined by x-ray crystallography. Two distinct binding configurations were observed for the three ligands. The substrate AdoMet adopts a bent shape that directs the activated methyl group toward the active site near the catalytic DPPY motif. The product AdoHcy and the competitive inhibitor sinefungin bind with a straight conformation in which the amino acid moiety occupies a position near the activated methyl group in the AdoMet complex. Analysis of ligand binding in comparison with other DNA methyltransferases reveals a small, common subset of available conformations for the ligand. The structures of M.RsrI with the non-substrate ligands contained a bound chloride ion in the AdoMet carboxylate-binding pocket, explaining its inhibition by chloride salts. The L72P mutant of M.RsrI is the first DNA methyltransferase structure without bound ligand. With respect to the wild-type protein, it had a larger ligand-binding pocket and displayed movement of a loop (223-227) that is responsible for binding the ligand, which may account for the weaker affinity of the L72P mutant for AdoMet. These studies show the subtle changes in the tight specific interactions of substrate, product, and an inhibitor with M.RsrI and help explain how each displays its unique effect on the activity of the enzyme.     

Computational Biology of Olfactory Receptors

Olfactory receptors, in addition to being involved in first step of the physiological processes that leads to olfaction, occupy an important place in mammalian genomes. ORs constitute super families in these genomes. Elucidating ol-factory receptor function at a molecular level can be aided by a computationally derived structure and an understanding of its interactions with odor molecules. Experimental functional analyses of olfactory receptors in conjunction with computational studies serve to validate findings and generate hypotheses. We present here a review of the research efforts in: creating computational models of olfactory receptors, identifying binding strategies for these receptors with odorant molecules, performing medium to long range simulation studies of odor ligands in the receptor binding region, and identifying amino acid positions within the receptor that are responsible for ligand-binding and olfactory receptor activation. Written as a primer and a teaching tool, this review will help researchers extend the methodologies described herein to other GPCRs.     

Structural probing and damage selection of citrulline- and arginine-specific RNA aptamers identify base positions required for binding.

In a recent study, an RNA aptamer for the specific recognition of the amino acid L-arginine was evolved from an in vitro selected L-citrulline binding parent sequence [M. Famulok (1994) J. Am. Chem. Soc. 116, 1698-1706]. We have now carried out a structural analysis of these aptamers by using chemical modification experiments. Footprinting experiments and a damage selection approach were performed to identify those positions protected from modification in the presence of the amino acids and modifications that interfere with the binding of the ligand. It is shown that of the two bulged regions present in both aptamers one can be modified without loss of binding activity whereas in the other bulge nearly every position is shown to be involved in the recognition of the ligands. This might be indicative for non-canonical base pairing to occur within the non-Watson-Crick paired regions which might be stabilized by the complexed amino acid. Binding to the cognate amino acid significantly enhances the conformational stability of the RNA. We also tested the sensitivity of both aptamers towards lead (II) ion induced cleavage and identified a hypersensitive cleavage site within the invariant bulged region. Lead cleavage is inhibited by the complexed amino acid, indicating a conformational change of the aptamer upon ligand binding. NMR titration data obtained with both aptamers and their cognate ligands confirm the proposed conformational changes and indicate the formation of a 1:1 complex of RNA:amino acid.