Biomedical subjects
Publications and source records attributed to R E Hubbard.
We have determined the structures of the oestrogen receptor ligand-binding domain in complex with a range of ligands and with fragments of co-regulator proteins. These structures provide insights into the structural mechanisms underlying the receptor's complex pharmacological properties and how the conformation of the receptor modulates its ability to recruit co-regulators that are necessary for transcriptional activation.
BACKGROUND: Estrogens exert their effects on target tissues by binding to a nuclear transcription factor termed the estrogen receptor (ER). Previous structural studies have demonstrated that each class of ER ligand (agonist, partial agonist, and SERM antagonist) induces distinctive orientations in the receptor's carboxy-terminal transactivation helix. The conformation of this portion of the receptor determines whether ER can recruit and interact with the components of the transcriptional machinery, thereby facilitating target gene expression. RESULTS: We have determined the structure of rat ERbeta ligand binding domain (LBD) in complex with the pure antiestrogen ICI 164,384 at 2.3 A resolution. The binding of this compound to the receptor completely abolishes the association between the transactivation helix (H12) and the rest of the LBD. The structure reveals that the terminal portion of ICI's bulky side chain substituent protrudes from the hormone binding pocket, binds along the coactivator recruitment site, and physically prevents H12 from adopting either its characteristic agonist or AF2 antagonist orientation. CONCLUSIONS: The binding mode adopted by the pure antiestrogen is similar to that seen for other ER antagonists. However, the size and resultant positioning of the ligand's side chain substituent produces a receptor conformation that is distinct from that adopted in the presence of other classes of ER ligands. The novel observation that binding of ICI results in the complete destabilization of H12 provides some indications as to a possible mechanism for pure receptor antagonism.
Type III antifreeze proteins (AFPs) are present in the body fluids of some polar fishes where they inhibit ice growth at subzero temperatures. Previous studies of the structure of type III AFP by NMR and X-ray identified a remarkably flat surface on the protein containing amino acids that were demonstrated to be important for interaction with ice by mutational studies. It was proposed that this protein surface binds onto the (1 0 [\bar 1] 0) plane of ice with the key amino acids interacting directly with the water molecules in the ice crystal. Here, we show that the mechanism of type III AFP interaction with ice crystals is more complex than that proposed previously. We report a high-resolution X-ray structure of type III AFP refined at 1.15 A resolution with individual anisotropic temperature factors. We report the results of ice-etching experiments that show a broad surface coverage, suggesting that type III AFP binds to a set of planes that are parallel with or inclined at a small angle to the crystallographic c-axis of the ice crystal. Our modelling studies, performed with the refined structure, confirm that type III AFP can make energetically favourable interactions with several ice surfaces.
Multiple Solvent Crystal Structures (MSCS) is a crystallographic technique to identify energetically favorable positions and orientations of small organic molecules on the surface of proteins. We determined the high-resolution crystal structures of thermolysin (TLN), generated from crystals soaked in 50--70% acetone, 50--80% acetonitrile and 50 mM phenol. The structures of the protein in the aqueous-organic mixtures are essentially the same as the native enzyme and a number of solvent interaction sites were identified. The distribution of probe molecules shows clusters in the main specificity pocket of the active site and a buried subsite. Within the active site, we compared the experimentally determined solvent positions with predictions from two computational functional group mapping techniques, GRID and Multiple Copy Simultaneous Search (MCSS). The experimentally determined small molecule positions are consistent with the structures of known protein--ligand complexes of TLN.
Here we review the results that have emerged from our structural studies on the oestrogen receptor ligand-binding domain (ER-LBD). The effects of agonists and antagonists on the structure of ERalpha- and ERbeta-LBDs are examined. In addition, the findings from structural studies of ER-LBD in complex with peptide fragments corresponding to the NR-box II and III modules of the p160 coactivator TIF2 are discussed in the context of the assembly of ER:coactivator complexes. Together these studies have broadened our understanding of ER function by providing a unique insight into ER's ligand specificity, it's ability to interact with coactivators and the structural changes that underlie receptor agonism and antagonism.
Here we summarise the results that have emerged from our structural studies on the oestrogen receptor (ER) ligand-binding domain. We have investigated the conformational effects of a variety of ligands on the structures of both ER isoforms. Each class of ligand (agonists, partial agonists and selective oestrogen receptor modulators) induces a unique conformation in the receptor's ligand-dependent transcriptional activation function. Together these studies have broadened our understanding of ER function by providing a unique insight into ER's ligand specificity and the structural changes that underlie receptor agonism and antagonism.
The monoclonal antibody hGR-2 F6 has been raised against the human glucagon receptor and shown to act as a competitive antagonist. As a first step in the structural characterization of the receptor, the crystal structure of the Fab fragment from this antibody is reported at 2.1 A resolution. The hGR-2 F6 Fab crystallizes in the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 76.14, b = 133.74, c = 37.46 A. A model generated by homology modelling was used as an aid in the chain-tracing and the Fab fragment structure was subsequently refined (final R factor = 21.7%). The structure obtained exhibits the typical immunoglobulin fold. Complementarity-determining regions (CDRs) L1, L2, L3, H1 and H2 could be superposed onto standard canonical CDR loops. The H3 loop could be classified according to recently published rules regarding loop length, sequence and conformation. This loop is 14 residues long, with an approximate beta-hairpin geometry, which is distorted somewhat by the presence of two trans proline residues at the beginning of the loop. It is expected that this H3 loop will facilitate the design of synthetic probes for the glucagon receptor that may be used to investigate receptor activity.
We have determined the three-dimensional structures of both alpha- and beta-forms of the ligand-binding domain of the oestrogen receptor (ER) in complexes with a range of receptor agonists and antagonists. Here, we summarize how these structures provide both an understanding of the ER's distinctive pharmacophore and a rationale for its ability to bind a diverse range of chemically distinct compounds. In addition, these studies provide a unique insight into the mechanisms that underlie receptor activation, as well as providing a structural basis for the antagonist action of molecules, such as raloxifene.
Multiple-solvent crystal structure determination (MSCS) allows the position and orientation of bound solvent fragments to be identified by determining the structure of protein crystals soaked in organic solvents. We have extended this technique by the determination of high-resolution crystal structures of thermolysin (TLN), generated from crystals soaked in 2% to 100% isopropanol. The procedure causes only minor changes to the conformation of the protein, and an increasing number of isopropanol interaction sites could be identified as the solvent concentration is increased. Isopropanol occupies all four of the main subsites in the active site, although this was only observed at very high concentrations of isopropanol for three of the four subsites. Analysis of the isopropanol positions shows little correlation with interaction energy computed using a molecular mechanics force field, but the experimentally determined positions of isopropanol are consistent with the structures of known protein-ligand complexes of TLN.
Chemokines selectively recruit and activate a variety of cells during inflammation. Interactions between cell surface glycosaminoglycans (GAGs) and chemokines drive the formation of haptotactic or immobilized gradients of chemokines at the site of inflammation, directing this recruitment. Chemokines bind to glycosaminoglycans on human umbilical vein endothelial cells (HUVECs) with affinities in the micromolar range: RANTES > MCP-1 > IL-8 > MIP-1alpha. This binding can be competed with by soluble glycosaminoglycans: heparin, heparin sulfate, chondroitin sulfate, and dermatan sulfate. RANTES binding showed the widest discrimination between glycosaminoglycans (700-fold), whereas MIP-1alpha was the least selective. Almost identical results were obtained in an assay using heparin sulfate beads as the source of immobilized glycosaminoglycan. The binding of chemokines to glycosaminoglycan fragments has a strong length dependence, and optimally requires both N- and O-sulfation. Isothermal titration calorimetry data confirm these results; IL-8 binds heparin fragments with a K(d) of 0.39-2.63 microM, and requires five saccharide units to bind each monomer of chemokine. In membranes from cells expressing the G-protein-coupled chemokine receptors CXCR1, CXCR2, and CCR1, soluble GAGs inhibit the binding of chemokine ligands to their receptors. Consistent with this, heparin and heparin sulfate could inhibit IL-8-induced neutrophil calcium flux. Chemokines can therefore form complexes with both cell surface and soluble GAGs; these interactions have different functions. Soluble GAG chemokines complexes are unable to bind the receptor, resulting in a block of the biological activity. Previously, we have shown that cell surface GAGs present chemokines to the G-protein-coupled receptors, by increasing the local concentration of protein. A model is presented which brings together all of these data. The selectivity in the chemokine-GAG interaction suggests selective disruption of the haptotactic gradient may be an achievable therapeutic approach in inflammatory disease.
Oestrogens exert their physiological effects through two receptor subtypes. Here we report the three-dimensional structure of the oestrogen receptor beta isoform (ERbeta) ligand-binding domain (LBD) in the presence of the phyto-oestrogen genistein and the antagonist raloxifene. The overall structure of ERbeta-LBD is very similar to that previously reported for ERalpha. Each ligand interacts with a unique set of residues within the hormone-binding cavity and induces a distinct orientation in the AF-2 helix (H12). The bulky side chain of raloxifene protrudes from the cavity and physically prevents the alignment of H12 over the bound ligand. In contrast, genistein is completely buried within the hydrophobic core of the protein and binds in a manner similar to that observed for ER's endogenous hormone, 17beta-oestradiol. However, in the ERbeta-genistein complex, H12 does not adopt the distinctive 'agonist' position but, instead, lies in a similar orientation to that induced by ER antagonists. Such a sub-optimal alignment of the transactivation helix is consistent with genistein's partial agonist character in ERbeta and demonstrates how ER's transcriptional response to certain bound ligands is attenuated.
The crystal structure of the kinase domain of C-terminal Src kinase (CSK) has been determined by molecular replacement, co-complexed with the protein kinase inhibitor staurosporine (crystals belong to the space group P21212 with a=44.5 A, b=120.6 A, c=48.3 A). The final model of CSK has been refined to an R-factor of 19.9 % (Rfree=28.7 %) at 2.4 A resolution. The structure consists of a small, N-terminal lobe made up mostly of a beta-sheet, and a larger C-terminal lobe made up mostly of alpha-helices. The structure reveals atomic details of interactions with staurosporine, which binds in a deep cleft between the lobes. The polypeptide chain fold of CSK is most similar to c-Src, Hck and fibroblast growth factor receptor 1 kinase (FGFR1K) and most dissimilar to insulin receptor kinase (IRK). Interactions between the N and C-terminal lobe are mediated by the bound staurosporine molecule and by hydrogen bonds. In addition, there are several water molecules forming lobe-bridging hydrogen bonds, which may be important for maintaining the catalytic integrity of the kinase. Furthermore, the conserved Lys328 and Glu267 residues utilise water in the formation of a molecular pivot which is essential in allowing relative movement of the N and C-terminal lobes. An analysis of the residues around the ATP-binding site reveals structural differences with other protein tyrosine kinases. Most notable of these are different orientations of the conserved residues Asp332 and Phe333, suggesting that inhibitor binding proceeds via an induced fit. These structural observations have implications for understanding protein tyrosine kinase catalytic mechanisms and for the design of ATP-mimicking inhibitors of protein kinases.
The oligopeptide-binding protein OppA provides a useful model system for studying the physical chemistry underlying noncovalent interactions since it binds a variety of readily synthesized ligands. We have studied the binding of eight closely related tripeptides of the type Lysine-X-Lysine, where X is an abnormal amino acid, by isothermal titration calorimetry (ITC) and X-ray crystallography. The tripeptides fall into three series of ligands, which have been designed to examine the effects of small changes to the central side chain. Three ligands have a primary amine as the second side chain, two have a straight alkane chain, and three have ring systems. The results have revealed a definite preference for the binding of hydrophobic residues over the positively charged side chains, the latter binding only weakly due to unfavorable enthalpic effects. Within the series of positively charged groups, a point of lowest affinity has been identified and this is proposed to arise from unfavorable electrostatic interactions in the pocket, including the disruption of a key salt bridge. Marked entropy-enthalpy compensation is found across the series, and some of the difficulties in designing tightly binding ligands have been highlighted.
The enzyme penicillin acylase (penicillin amidohydrolase EC 3.5.1. 11) catalyses the cleavage of the amide bond in the benzylpenicillin (penicillin G) side-chain to produce phenylacetic acid and 6-aminopenicillanic acid (6-APA). The enzyme is of great pharmaceutical importance, as the product 6-APA is the starting point for the synthesis of many semi-synthetic penicillin antibiotics. Studies have shown that the enzyme is specific for hydrolysis of phenylacetamide derivatives, but is more tolerant of features in the rest of the substrate. It is this property that has led to many other applications for the enzyme, and greater knowledge of the enzyme's structure and specificity could facilitate engineering of the enzyme, enhancing its potential for chemical and industrial applications. An extensive study of the binding of a series of phenylacetic acid derivatives has been carried out. A measure of the relative degree of inhibition of the enzyme by each of the compounds has been obtained using a competitive inhibition assay, and the structures of a number of these complexes have been determined by X-ray crystallography. The structures reveal a clear rationale for the observed kinetic results, but show also that some of the ligands cause a conformational change within the binding pocket. This change can generally be understood in terms of the size and orientation of the ligand within the active site.The results reveal that ligand binding in penicillin acylase is facilitated by certain amino acid residues that can adopt two distinct, energetically favourable positions in order to accommodate a variety of compounds within the active site. The structures of these complexes provide evidence for conformational changes in the substrate-binding region that may act as a switch in the mechanism of autocatalytic processing of this enzyme.
The electron density projection map of frog rhodopsin at 6 A resolution had been until recently the most direct evidence for the three-dimensional structure of a transmembrane domain of any G-protein-coupled receptor. Only three out of seven transmembrane helices are clearly defined, whilst the other four are hidden in a patch of unresolved electron density. A model of the seven-helix bundle has been created by generating positions and orientations for the four unresolved helices through performing a conformational search directed by structural restraints derived from other experimental data. These four helices are significantly tilted with respect to the membrane normal, and the cytosolic end of helix C is inserted between helices D and E. These calculations produce positions and orientations for these additional helices that are consistent with the recently published low-resolution three-dimensional map, and provide a template for more detailed modelling of rhodopsin structure and function.
The activation of leukocytes by chemokines is believed to be mediated via binding of chemokines to glycosaminoglycan chains of the extracellular matrix. The binding site on the chemokine interleukin-8 (IL-8) for the glycosaminoglycan heparin has been characterized using a systematic series of site-directed mutants of IL-8 in which the basic residues of the protein have been replaced by alanine. Mutation of K64 and R68 caused the largest decrease in affinity for a heparin Sepharose matrix, with smaller effects seen with mutations of K20, R60, and K67. Heparin-derived disaccharides that could disrupt the IL-8-heparin Sepharose interaction were identified by a competitive binding assay. Heteronuclear NMR spectroscopic titration of 15N-labeled IL-8 with a trisulfated disaccharide revealed a cluster of residues on IL-8 which were perturbed by disaccharide binding. These data identify a heparin-binding surface on IL-8 that includes the C-terminal alpha-helix and the proximal loop around residues 18-23. The heparin-binding site is spatially distinct from the residues involved in receptor binding.
The interfacial activation of Rhizomucor miehei lipase (RmL) involves the motion of an alpha-helical region (residues 82-96) which acts as a "lid" over the active site of the enzyme, undergoing a displacement from a "closed" to an "open" conformation upon binding of substrate. Normal mode analyses performed in both low and high dielectric media reveal that low-frequency vibrational modes contribute significantly to the conformational transition between the closed and open conformations. In these modes, the lid displacement is coupled to local motions of active site loops as well as global breathing motions. Atomic fluctuations of the first hinge of the lid (residues 83-84) are substantially larger in the low dielectric medium than in the high dielectric medium. Our results also suggest that electrostatic interactions of Arg86 play an important role in terms of both the intrinsic stability of the lid and its displacement, through enhancement of hinge mobility in a high dielectric medium. Additional calculations demonstrate that the observed patterns of atomic fluctuations are an intrinsic feature of the protein structure and not dependent on the nature of specific energy minima.
Molecular models of the transmembrane domain of the phospholamban pentamer have been generated by a computational method that uses the experimentally measured effects of systematic single-site mutations as a guiding force in the modeling procedure. This method makes the assumptions that 1) the phospholamban transmembrane domain is a parallel five-helix bundle, and 2) nondisruptive mutation positions are lipid exposed, whereas 3) disruptive or partially disruptive mutations are not. Our procedure requires substantially less computer time than systematic search methods, allowing rapid assessment of the effects of different experimental results on the helix arrangement. The effectiveness of the approach is investigated in test calculations on two helix-dimer systems of known structure. Two independently derived sets of mutagenesis data were used to define the restraints for generating models of phospholamban. Both resulting models are left-handed, highly symmetrical pentamers. Although the overall bundle geometry is very similar in the two models, the orientation of individual helices differs by approximately 50 degrees, resulting in different sets of residues facing the pore. This demonstrates how differences in restraints can have an effect on the model structures generated, and how the violation of these restraints can identify inconsistent experimental data.