Allosteric conversation in the androgen receptor ligand-binding domain surfaces

Androgen receptor (AR) is a major therapeutic target that plays pivotal roles in prostate cancer (PCa) and androgen insensitivity syndromes. We previously proposed that compounds recruited to ligand-binding domain (LBD) surfaces could regulate AR activity in hormone-refractory PCa and discovered several surface modulators of AR function. Surprisingly, the most effective compounds bound preferentially to a surface of unknown function [binding function 3 (BF-3)] instead of the coactivator-binding site [activation function 2 (AF-2)]. Different BF-3 mutations have been identified in PCa or androgen insensitivity syndrome patients, and they can strongly affect AR activity. Further, comparison of AR x-ray structures with and without bound ligands at BF-3 and AF-2 showed structural coupling between both pockets. Here, we combine experimental evidence and molecular dynamic simulations to investigate whether BF-3 mutations affect AR LBD function and dynamics possibly via allosteric conversation between surface sites. Our data indicate that AF-2 conformation is indeed closely coupled to BF-3 and provide mechanistic proof of their structural interconnection. BF-3 mutations may function as allosteric elicitors, probably shifting the AR LBD conformational ensemble toward conformations that alter AF-2 propensity to reorganize into subpockets that accommodate N-terminal domain and coactivator peptides. The induced conformation may result in either increased or decreased AR activity. Activating BF-3 mutations also favor the formation of another pocket (BF-4) in the vicinity of AF-2 and BF-3, which we also previously identified as a hot spot for a small compound. We discuss the possibility that BF-3 may be a protein-docking site that binds to the N-terminal domain and corepressors. AR surface sites are attractive pharmacological targets to develop allosteric modulators that might be alternative lead compounds for drug design.     

Characterization of high affinity binding motifs for the discoidin domain receptor DDR2 in collagen

The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that are activated by native triple-helical collagen. Here we have located three specific DDR2 binding sites by screening the entire triple-helical domain of collagen II, using the Collagen II Toolkit, a set of overlapping triple-helical peptides. The peptide sequence that bound DDR2 with highest affinity interestingly contained the sequence for the high affinity binding site for von Willebrand factor in collagen III. Focusing on this sequence, we used a set of truncated and alanine-substituted peptides to characterize the sequence GVMGFO (O is hydroxyproline) as the minimal collagen sequence required for DDR2 binding. Based on a recent NMR analysis of the DDR2 collagen binding domain, we generated a model of the DDR2-collagen interaction that explains why a triple-helical conformation is required for binding. Triple-helical peptides comprising the DDR2 binding motif not only inhibited DDR2 binding to collagen II but also activated DDR2 transmembrane signaling. Thus, DDR2 activation may be effected by single triple-helices rather than fibrillar collagen.     

Functional characterisation of mutations in the ligand-binding domain of the androgen receptor gene in patients with androgen insensitivity syndrome

Five mutations in the ligand-binding domain of the androgen receptor gene were identified in patients with complete (A765T, C784Y, R831X and M895T) or partial (R840G) androgen insensitivity. A765T and R831X have been reported previously whereas the other three mutations are novel. Receptors carrying these mutations were transiently expressed in COS-1 cells, and androgen binding and capacity to transactivate an androgen-responsive reporter gene were assayed. C784Y led to abolished androgen binding and transactivating capacity, R840G and M895T showed reduced specific binding and partial transactivation. The in vitro functions of the R840G and M895T mutants were improved with supraphysiological concentrations of steroid. Received: 10 June 1998 / Accepted: 10 September 1998     

Comparison of crystal structures of human androgen receptor ligand-binding domain complexed with various agonists reveals molecular determinants responsible for binding affinity

Androgens exert their effects by binding to the highly specific androgen receptor (AR). In addition to natural potent androgens, AR binds a variety of synthetic agonist or antagonist molecules with different affinities. To identify molecular determinants responsible for this selectivity, we have determined the crystal structure of the human androgen receptor ligand-binding domain (hARLBD) in complex with two natural androgens, testosterone (Testo) and dihydrotestosterone (DHT), and with an androgenic steroid used in sport doping, tetrahydrogestrinone (THG), at 1.64, 1.90, and 1.75 A resolution, respectively. Comparison of these structures first highlights the flexibility of several residues buried in the ligand-binding pocket that can accommodate a variety of ligand structures. As expected, the ligand structure itself (dimension, presence, and position of unsaturated bonds that influence the geometry of the steroidal nucleus or the electronic properties of the neighboring atoms, etc.) determines the number of interactions it can make with the hARLBD. Indeed, THG--which possesses the highest affinity--establishes more van der Waals contacts with the receptor than the other steroids, whereas the geometry of the atoms forming electrostatic interactions at both extremities of the steroid nucleus seems mainly responsible for the higher affinity measured experimentally for DHT over Testo. Moreover, estimation of the ligand-receptor interaction energy through modeling confirms that even minor modifications in ligand structure have a great impact on the strength of these interactions. Our crystallographic data combined with those obtained by modeling will be helpful in the design of novel molecules with stronger affinity for the AR.     

Local motifs involved in the canonical structure of the ligand-binding domain in the nuclear receptor superfamily

Structural and sequence alignment analyses have revealed the existence of class-dependent and -independent local motifs involved in the overall fold of the ligand-binding domain (LBD) in the nuclear receptor (NR) superfamily. Of these local motifs, three local motifs, i.e., AF-2 fixed motifs, were involved in the agonist conformation of the activation function-2 (AF-2) region of the LBD. Receptor–agonist interactions increased the stability of these AF-2 fixed motifs in the agonist conformation. In contrast, perturbation of the AF-2 fixed motifs by a ligand or another protein molecule led the AF-2 architecture to adopt an antagonist conformation. Knowledge of this process should provide us with novel insights into the ‘agonism’ and ‘antagonism’ of NRs.     

Mass spectrometric characterization of the human androgen receptor ligand-binding domain expressed in Escherichia coli

The ligand-binding domain (LBD) of the human androgen receptor (hAR LBD), encompassing amino acids (AAs) 647-919, was expressed in Escherichia coli with an N-terminal polyhistidine tag (His(10)-hAR LBD) from a pET-16b vector. The overexpressed protein was initially insoluble in inclusion bodies, and was subsequently solubilized in 8 M guanidine hydrochloride (GdnHCl). The solubilized His(10)-hAR LBD was purified to apparent homogeneity by metal ion affinity chromatography in the presence of 6 M GdnHCl. The isolated protein migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular mass of 33-34 kDa, as expected from the plasmid construct. Immunoblot analysis with C-terminal antibodies raised against a peptide corresponding to the last 19 AAs (AAs 901-919) of hAR revealed that the purified protein contained an immunoreactive epitope present within the AR and was of the appropriate size. Further characterization, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS), showed a single protein species of average mass 34 580 Da, confirming the size and purity of the purified His(10)-hAR LBD. Detailed tryptic peptide mapping analysis, using MALDI/TOF-MS, identified a total of eight peptides with a 30% coverage of the LBD, including the last tryptic peptide in the hAR sequence. These data confirm that the purified protein was the intact hAR LBD. AA sequencing of these tryptic peptides, using an HPLC-coupled electrospray ionization ion trap mass spectrometer (LC/ESI-ITMS and MS/MS), unambiguously confirmed that the peptides were from the hAR LBD. The purified His(10)-hAR LBD in 6 M GdnHCl could be renatured as determined by ligand-binding activity, with a similar equilibrium dissociation constant (K(d)) for [(3)H]-mibolerone and a similar steroid specificity to the AR isolated from rat ventral prostate.     

Structural plasticity in the oestrogen receptor ligand-binding domain

The steroid hormone receptors are characterized by binding to relatively rigid, inflexible endogenous steroid ligands. Other members of the nuclear receptor superfamily bind to conformationally flexible lipids and show a corresponding degree of elasticity in the ligand-binding pocket. Here, we report the X-ray crystal structure of the oestrogen receptor alpha (ERalpha) bound to an oestradiol derivative with a prosthetic group, ortho- trifluoromethlyphenylvinyl, which binds in a novel extended pocket in the ligand-binding domain. Unlike ER antagonists with bulky side groups, this derivative is enclosed in the ligand-binding pocket, and acts as a potent agonist. This work shows that steroid hormone receptors can interact with a wider array of pharmacophores than previously thought through structural plasticity in the ligand-binding pocket.     

cAMP binds with high affinity and specificity to the androgen receptor from murine skeletal muscle

cAMP binding of the androgen receptor (AR) from murine skeletal muscle was studied. Testosterone affinity chromatography yielded androgen receptor with about 4000-fold purification. Determination of the cAMP binding in the affinity eluate, by adsorption of protein-cAMP complexes to cellulose ester filters or removal of unbound cAMP by dextran-coated charcoal, was not possible, as the observed binding was not stable during the assays. Displacement studies suggest that this is due to a very fast dissociation kinetics of the binding. The problem could be solved by assaying the components of affinity eluate immobilized to a testosterone affinity resin that stabilizes the cAMP-protein complexes. The cAMP binding found in the affinity eluate shows an upward concave Scatchard plot and is compatible with a model containing two independent binding sites with dissociation constants of 7 and 58 nM. However, a larger number of binding sites or negative cooperativity cannot be excluded. Sixteen cAMP binding sites were observed per testosterone binding site. The binding affinity of cAMP exceeds that of cGMP 200-fold, that of cCMP 2000-fold, and that of AMP and 2',3'-cAMP more than 10,000-fold. Results indicate that cAMP is bound by the AR, although it only represents about 1% of the total protein in the affinity eluate: (i) Specific testosterone and cAMP binding of affinity eluate was copurified by affinity chromatography, density gradient centrifugation, and gel filtration. The ratio of cAMP to testosterone binding in each peak was about 16:1, identical with that found in the total affinity eluate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular modeling of the GABAC receptor ligand-binding domain

We have constructed a molecular model of the ligand-binding domain of the GABA(C) receptor, which is a member of the Cys-loop ligand-gated ion channel family. The extracellular domains of these receptors share similar sequence homology (20%) with Limnaea acetylcholine-binding protein for which an X-ray crystal structure is available. We used this structure as a template for homology modeling of the GABA(C) receptor extracellular domain using FUGUE and MODELLER software. FlexX was then used to dock GABA into the receptor ligand-binding site, resulting in three alternative energetically favorable orientations. Residues located no more than 5 A from the docked GABA were identified for each model; of these, three were found to be common to all models with 14 others present only in certain models. Using data from experimental studies, we propose that the most likely orientation of GABA is with its amine close to Y198, and its carboxylate close to R104. These studies have therefore provided a model of the ligand-binding domain, which will be useful for both GABA(C) and GABA(A) receptor studies, and have also yielded an experimentally testable hypothesis of the location of GABA in the binding pocket. [Figure: see text].     

Androgen receptor ligand-binding domain interaction and nuclear receptor specificity of FXXLF and LXXLL motifs as determined by L/F swapping

The androgen receptor (AR) ligand-binding domain (LBD) binds FXXLF motifs, present in the AR N-terminal domain and AR-specific cofactors, and some LXXLL motifs of nuclear receptor coactivators. We demonstrated that in the context of the AR FXXLF motif many different amino acid residues at positions +2 and +3 are compatible with strong AR LBD interaction, although a preference for E at +2 and K or R at +3 was found. Pairwise systematic analysis of F/L swaps at +1 and +5 in FXXLF and LXXLL motifs showed: 1) F to L substitutions in natural FXXLF motifs abolished AR LBD interaction; 2) binding of interacting LXXLL motifs was unchanged or increased upon L to F substitutions; 3) certain noninteracting LXXLL motifs became strongly AR-interacting FXXLF motifs; whereas 4) other nonbinders remained unaffected by L to F substitutions. All FXXLF motifs, but not the corresponding LXXLL motifs, displayed a strong preference for AR LBD. Progesterone receptor LBD interacted with some FXXLF motifs, albeit always less efficiently than corresponding LXXLL motifs. AR LBD interaction of most FXXLF and LXXLL peptides depended on classical charge clamp residue K720, whereas E897 was less important. Other charged residues lining the AR coactivator-binding groove, K717 and R726, modulated optimal peptide binding. Interestingly, these four charged residues affected binding of individual peptides independent of an F or L at +1 and +5 in swap experiments. In conclusion, F residues determine strong and selective peptide interactions with AR. Sequences flanking the core motif determine the specific mode of FXXLF and LXXLL interactions.     

Novel 1,2,3,4-tetrahydroisoquinolines with high affinity and selectivity for the dopamine D3 receptor

Using clearance and brain penetration studies as a screen, tetrahydroisoquinoline 3 was identified as a lead having low clearance in rats (CLb 20 ml/min/kg). Introduction of a 7-CF3SO2O- substituent into the tetrahydroisoquinoline, followed by replacement of the biphenylamido group of 3 by a 3-indolylpropenamido group gave 31, having high D3 receptor affinity (pKi 8.4) and 150 fold selectivity over the D2 receptor.