Attractant- and Disulfide-Induced Conformational Changes in the Ligand Binding Domain of the Chemotaxis Aspartate Receptor: A 19F NMR Study

The mineralocorticoid receptor (MR) is highly conserved across vertebrate evolution. In terrestrial vertebrates, the MR mediates sodium homeostasis by aldosterone and also acts as a receptor for cortisol. Although the MR is present in fish, they lack aldosterone. The MR binds progesterone and spironolactone as antagonists in human MR but as agonists in zebrafish MR. We have defined the molecular basis of these divergent responses using MR chimeras between the zebrafish and human MR coupled with reciprocal site-directed mutagenesis and molecular dynamic (MD) simulation based on the crystal structures of the MR ligand-binding domain. Substitution of a leucine by threonine in helix 8 of the ligand-binding domain of the zebrafish MR confers the antagonist response. This leucine is conserved across fish species, whereas threonine (serine in rodents) is conserved in terrestrial vertebrate MR. MD identified an interaction of the leucine in helix 8 with a highly conserved leucine in helix 1 that stabilizes the agonist conformation including the interaction between helices 3 and 5, an interaction which has previously been characterized. This switch in the MR coincides with the evolution of terrestrial vertebrates and of aldosterone synthesis. It was perhaps mandatory if the appearance of aldosterone as a specific mediator of the homeostatic salt retention was to be tolerated. The conformational changes also provide insights into the structural basis of agonism versus antagonism in steroid receptors with potential implications for drug design in this important therapeutic target.

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In Silico Mutation of Cysteine Residues in the Ligand-Binding Domain of anN-Methyl-d-aspartate Receptor†

Biochemistry ◽

10.1021/bi061462d ◽

2007 ◽

Vol 46 (8) ◽

pp. 2136-2145 ◽

Cited By ~ 3

Author(s):

Samantha L. Kaye ◽

Mark S. P. Sansom ◽

Philip C. Biggin

Keyword(s):

Ligand Binding ◽

In Silico ◽

Binding Domain ◽

Ligand Binding Domain ◽

Cysteine Residues ◽

Aspartate Receptor

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Kinetic and Thermodynamic Characterization of Dihydrotestosterone-Induced Conformational Perturbations in Androgen Receptor Ligand-Binding Domain

Molecular Endocrinology ◽

10.1210/me.2008-0304 ◽

2009 ◽

Vol 23 (8) ◽

pp. 1231-1241 ◽

Cited By ~ 15

Author(s):

Ravi Jasuja ◽

Jagadish Ulloor ◽

Christopher M. Yengo ◽

Karen Choong ◽

Andrei Y. Istomin ◽

...

Keyword(s):

Androgen Receptor ◽

Ligand Binding ◽

Conformational Changes ◽

Solvent Accessibility ◽

Molten Globule ◽

Binding Pocket ◽

Binding Domain ◽

Ligand Binding Domain ◽

Second Derivative ◽

Ligand Binding Pocket

Abstract Ligand-induced conformational perturbations in androgen receptor (AR) are important in coactivator recruitment and transactivation. However, molecular rearrangements in AR ligand-binding domain (AR-LBD) associated with agonist binding and their kinetic and thermodynamic parameters are poorly understood. We used steady-state second-derivative absorption and emission spectroscopy, pressure and temperature perturbations, and 4,4′-bis-anilinonaphthalene 8-sulfonate (bis-ANS) partitioning to determine the kinetics and thermodynamics of the conformational changes in AR-LBD after dihydrotestosterone (DHT) binding. In presence of DHT, the second-derivative absorption spectrum showed a red shift and a change in peak-to-peak distance. Emission intensity increased upon DHT binding, and center of spectral mass was blue shifted, denoting conformational changes resulting in more hydrophobic environment for tyrosines and tryptophans within a more compact DHT-bound receptor. In pressure perturbation calorimetry, DHT-induced energetic stabilization increased the Gibbs free energy of unfolding to 8.4 ± 1.3 kcal/mol from 3.5 ± 1.6 kcal/mol. Bis-ANS partitioning studies revealed that upon DHT binding, AR-LBD underwent biphasic rearrangement with a high activation energy (13.4 kcal/mol). An initial, molten globule-like burst phase (k ∼30 sec−1) with greater solvent accessibility was followed by rearrangement (k ∼0.01 sec−1), leading to a more compact conformation than apo-AR-LBD. Molecular simulations demonstrated unique sensitivity of tyrosine and tryptophan residues during pressure unfolding with rearrangement of residues in the coactivator recruitment surfaces distant from the ligand-binding pocket. In conclusion, DHT binding leads to energetic stabilization of AR-LBD domain and substantial rearrangement of residues distant from the ligand-binding pocket. DHT binding to AR-LBD involves biphasic receptor rearrangement including population of a molten globule-like intermediate state.

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Correlating efficacy and desensitization with GluK2 ligand-binding domain movements

Open Biology ◽

10.1098/rsob.130051 ◽

2013 ◽

Vol 3 (5) ◽

pp. 130051 ◽

Cited By ~ 4

Author(s):

Naushaba Nayeem ◽

Olga Mayans ◽

Tim Green

Keyword(s):

Ligand Binding ◽

Conformational Changes ◽

Binding Domain ◽

Ligand Binding Domain ◽

Anion Binding ◽

Charge Balance ◽

Dimer Interface ◽

Ligand Affinity ◽

Cleft Closure ◽

Binding Cleft

Gating of AMPA- and kainate-selective ionotropic glutamate receptors can be defined in terms of ligand affinity, efficacy and the rate and extent of desensitization. Crucial insights into all three elements have come from structural studies of the ligand-binding domain (LBD). In particular, binding-cleft closure is associated with efficacy, whereas dissociation of the dimer formed by neighbouring LBDs is linked with desensitization. We have explored these relationships in the kainate-selective subunit GluK2 by studying the effects of mutating two residues (K531 and R775) that form key contacts within the LBD dimer interface, but whose truncation unexpectedly attenuates desensitization. One mutation (K531A) also switches the relative efficacies of glutamate and kainate. LBD crystal structures incorporating these mutations revealed several conformational changes that together explain their phenotypes. K531 truncation results in new dimer contacts, consistent with slower desensitization and sideways movement in the ligand-binding cleft correlating with efficacy. The tested mutants also disrupted anion binding; no chloride was detected in the dimer-interface site, including in R775A where absence of chloride was the only structural change evident. From this, we propose that the charge balance in the GluK2 LBD dimer interface maintains a degree of instability, necessary for rapid and complete desensitization.

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