Browsing by Subject "Protein Structure, Tertiary"

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    Asn 102 of the Gonadotropin-releasing Hormone Receptor Is a Critical Determinant of Potency for Agonists Containing C-terminal Glycinamide
    (1996) Davidson, James S; McArdle, Craig A; Davies, Peter; Elario, Ricardo; Flanagan, Colleen A; Millar, Robert P
    We demonstrate a critical role for Asn102 of the human gonadotropin-releasing hormone (GnRH) receptor in the binding of GnRH. Mutation of Asn102, located at the top of the second transmembrane helix, to Ala resulted in a 225-fold loss of potency for GnRH. Eight GnRH analogs, all containing glycinamide C termini like GnRH, showed similar losses of potency between 95- and 750-fold for the [Ala102]GnRHR, compared with wild-type receptor. In contrast, four GnRH analogs that had ethylamide in place of the C-terminal glycinamide residue, showed much smaller decreases in potency between 2.4- and 11-fold. In comparisons of three agonist pairs, differing only at the C terminus, glycinamide derivatives showed an 11-20-fold greater loss of potency for the mutant receptor than their respective ethylamide derivatives. Thus Asn102 is a critical determinant of potency specifically for ligands with C-terminal glycinamide, while ligands with C-terminal ethylamide are less dependent on Asn102. These findings indicate a role for Asn102 in the docking of the glycinamide C terminus and are consistent with hydrogen bonding of the Asn102 side chain with the C-terminal amide moiety. Taken with previous data, they suggest a region of the GnRH receptor formed by the top of helices 2 and 7 as a binding pocket for the C-terminal part of the ligand.
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    Critical Interaction of Actuator Domain Residues Arginine 174, Isoleucine 188, and Lysine 205 with Modulatory Nucleotide in Sarcoplasmic Reticulum Ca 2+ -ATPase
    (2008) Clausen, Johannes D; McIntosh, David B; Woolley, David G; Andersen, Jens Peter
    ATP plays dual roles in the reaction cycle of the sarcoplasmic reticulum Ca2+-ATPase by acting as the phosphorylating substrate as well as in nonphosphorylating (modulatory) modes accelerating conformational transitions of the enzyme cycle. Here we have examined the involvement of actuator domain residues Arg174, Ile188, Lys204, and Lys205 by mutagenesis. Alanine mutations to these residues had little effect on the interaction of the Ca2E1 state with nucleotide or on the HnE 2 to Ca2E1 transition of the dephosphoenzyme. The phosphoenzyme processing steps, Ca2E1P to E2P and E2P dephosphorylation, and their stimulation by MgATP/ATP were markedly affected by mutations to Arg174, Ile188, and Lys205. Replacement of Ile188 with alanine abolished nucleotide modulation of dephosphorylation but not the modulation of the Ca2E1P to E2P transition. Mutation to Arg174 interfered with nucleotide modulation of either of the phosphoenzyme processing steps, indicating a significant overlap between the modulatory nucleotide-binding sites involved. Mutation to Lys205 enhanced the rates of the phosphoenzyme processing steps in the absence of nucleotide and disrupted the nucleotide modulation of the Ca2E1P to E2P transition. Remarkably, the mutants with alterations to Lys205 showed an anomalous inhibition by ATP of the dephosphorylation, and in the alanine mutant the affinity for the inhibition by ATP was indistinguishable from that for stimulation by ATP of the wild type. Hence, the actuator domain is an important player in the function of ATP as modulator of phosphoenzyme processing, with Arg174, Ile188, and Lys205 all being critically involved, although in different ways. The data support a variable site model for the modulatory effects with the nucleotide binding somewhat differently in each of the conformational states occurring during the transport cycle.
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    Structural Determinants for Ligand-Receptor Conformational Selection in a Peptide G Protein-coupled Receptor
    (2007) Lu, Zhi-Liang; Coetsee, Marla; White, Colin D; Millar, Robert P
    G protein coupled receptors (GPCRs) modulate the majority of physiological processes through specific intermolecular interactions with structurally diverse ligands and activation of differential intracellular signaling. A key issue yet to be resolved is how GPCRs developed selectivity and diversity of ligand binding and intracellular signaling during evolution. We have explored the structural basis of selectivity of naturally occurring gonadotropin-releasing hormones (GnRHs) from different species in the single functional human GnRH receptor. We found that the highly variable amino acids in position 8 of the naturally occurring isoforms of GnRH play a discriminating role in selecting receptor conformational states. The human GnRH receptor has a higher affinity for the cognate GnRH I but a lower affinity for GnRH II and GnRHs from other species possessing substitutions for Arg(8). The latter were partial agonists in the human GnRH receptor. Mutation of Asn(7.45) in transmembrane domain (TM) 7 had no effect on GnRH I affinity but specifically increased affinity for other GnRHs and converted them to full agonists. Using molecular modeling and site-directed mutagenesis, we demonstrated that the highly conserved Asn(7.45) makes intramolecular interactions with a highly conserved Cys(6.47) in TM 6, suggesting that disruption of this intramolecular interaction induces a receptor conformational change which allosterically alters ligand specific binding sites and changes ligand selectivity and signaling efficacy. These results reveal GnRH ligand and receptor structural elements for conformational selection, and support co-evolution of GnRH ligand and receptor conformations.