Publications by authors named "Darren L Beene"

7 Publications

  • Page 1 of 1

Multiple Tyrosine Residues Contribute to GABA Binding in the GABA(C) Receptor Binding Pocket.

ACS Chem Neurosci 2012 Mar 15;3(3):186-192. Epub 2011 Dec 15.

The ligand binding site of Cys-loop receptors is dominated by aromatic amino acids. In GABA(C) receptors, these are predominantly tyrosine residues, with a number of other aromatic residues located in or close to the binding pocket. Here we examine the roles of these residues using substitution with both natural and unnatural amino acids followed by functional characterization. Tyr198 (loop B) has previously been shown to form a cation-π interaction with GABA; the current data indicate that none of the other aromatic residues form such an interaction, although the data indicate that both Tyr102 and Phe138 may contribute to stabilization of the positively charged amine of GABA. Tyr247 (loop C) was very sensitive to substitution and, combined with data from a model of the receptor, suggest a π-π interaction with Tyr241 (loop C); here again functional data show aromaticity is important. In addition the hydroxyl group of Tyr241 is important, supporting the presence of a hydrogen bond with Arg104 suggested by the model. At position Tyr102 (loop D) size and aromaticity are important; this residue may play a role in receptor gating and/or ligand binding. The data also suggest that Tyr167, Tyr200, and Tyr208 have a structural role while Tyr106, Trp246, and Tyr251 are not critical. Comparison of the agonist binding site "aromatic box" across the superfamily of Cys-loop receptors reveals some interesting parallels and divergences.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/cn200103nDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3309607PMC
March 2012

A-kinase anchoring proteins take shape.

Curr Opin Cell Biol 2007 Apr 20;19(2):192-8. Epub 2007 Feb 20.

Howard Hughes Medical Institute, Oregon Health & Science University, 3181 SW Sam Jackson Park Rd., Portland, OR 97239, USA.

A-kinase anchoring proteins (AKAPs) are signaling scaffolds that contribute to various aspects of cAMP signaling. They do this by tethering protein kinase-A to specific subcellular sites, thereby focusing its activity toward relevant substrates. Recently the structural basis for these protein-protein interactions has been elucidated by x-ray crystallography. Recent reports have identified AKAPs that bind to adenylyl cyclases to regulate cAMP synthesis and that sequester phosphodiesterases to break down this second messenger locally. Another emerging aspect of AKAP function is their role in integrating cAMP signaling with other signaling pathways. For example, molecular and genetic approaches have been used to show that the neuronal anchoring protein WAVE1 integrates signaling from PKA and Cdk5 to regulate actin polymerization and cytoskeletal events.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ceb.2007.02.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3521038PMC
April 2007

Cis-trans isomerization at a proline opens the pore of a neurotransmitter-gated ion channel.

Nature 2005 Nov;438(7065):248-52

Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK.

5-hydroxytryptamine type 3 (5-HT3) receptors are members of the Cys-loop receptor superfamily. Neurotransmitter binding in these proteins triggers the opening (gating) of an ion channel by means of an as-yet-uncharacterized conformational change. Here we show that a specific proline (Pro 8*), located at the apex of the loop between the second and third transmembrane helices (M2-M3), can link binding to gating through a cis-trans isomerization of the protein backbone. Using unnatural amino acid mutagenesis, a series of proline analogues with varying preference for the cis conformer was incorporated at the 8* position. Proline analogues that strongly favour the trans conformer produced non-functional channels. Among the functional mutants there was a strong correlation between the intrinsic cis-trans energy gap of the proline analogue and the activation of the channel, suggesting that cis-trans isomerization of this single proline provides the switch that interconverts the open and closed states of the channel. Consistent with this proposal, nuclear magnetic resonance studies on an M2-M3 loop peptide reveal two distinct, structured forms. Our results thus confirm the structure of the M2-M3 loop and the critical role of Pro 8* in the 5-HT3 receptor. In addition, they suggest that a molecular rearrangement at Pro 8* is the structural mechanism that opens the receptor pore.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature04130DOI Listing
November 2005

A cation-pi binding interaction with a tyrosine in the binding site of the GABAC receptor.

Chem Biol 2005 Sep;12(9):993-7

Department of Biochemistry, University of Cambridge, UK.

GABA(C) (rho) receptors are members of the Cys-loop superfamily of neurotransmitter receptors, which includes nicotinic acetylcholine (nACh), 5-HT(3), and glycine receptors. As in other members of this family, the agonist binding site of GABA(C) receptors is rich in aromatic amino acids, but while other receptors bind agonist through a cation-pi interaction to a tryptophan, the GABA(C) binding site has tyrosine at the aligning positions. Incorporating a series of tyrosine derivatives at position 198 using unnatural amino acid mutagenesis reveals a clear correlation between the cation-pi binding ability of the side chain and EC(50) for receptor activation, thus demonstrating a cation-pi interaction between a tyrosine side chain and a neurotransmitter. Comparisons among four homologous receptors show variations in cation-pi binding energies that reflect the nature of the cationic center of the agonist.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chembiol.2005.06.012DOI Listing
September 2005

Tyrosine residues that control binding and gating in the 5-hydroxytryptamine3 receptor revealed by unnatural amino acid mutagenesis.

J Neurosci 2004 Oct;24(41):9097-104

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.

The mechanism by which agonist binding triggers pore opening in ligand-gated ion channels is poorly understood. Here, we used unnatural amino acid mutagenesis to introduce subtle changes to the side chains of tyrosine residues (Tyr141, Tyr143, Tyr153, and Tyr234), which dominate the 5-HT3 receptor binding site. Heterologous expression in oocytes, combined with radioligand binding data and a model of 5-HT (serotonin) computationally docked into the binding site, has allowed us to determine which of these residues are responsible for binding and/or gating. We have shown that Tyr 143 forms a hydrogen bond that is essential for receptor gating but does not affect binding, whereas a hydrogen bond formed by Tyr153 is involved in both binding and gating of the receptor. The aromatic group of Tyr234 is essential for binding and gating, whereas its hydroxyl does not affect binding but plays a steric role in receptor gating. Tyr141 is not involved in agonist binding or receptor gating but is important for antagonist interactions. These data, combined with a new model of the nonliganded 5-HT3 receptor, lead to a mechanistic explanation of the interactions that initiate the conformational change leading to channel opening. Thus, we suggest that agonist entry into the binding pocket may displace Tyr143 and Tyr153 and results in their forming new hydrogen bonds. These bonds may form part of the network of bond rearrangements that trigger the conformational change leading to channel opening. Similar rearrangements may initiate gating in all Cys-loop receptors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.2429-04.2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6730062PMC
October 2004

Unnatural amino acid mutagenesis in mapping ion channel function.

Curr Opin Neurobiol 2003 Jun;13(3):264-70

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.

Unnatural amino acid mutagenesis makes possible the site-specific incorporation of synthetic amino acids, enabling detailed structure-function studies as well as the incorporation of biophysical probes. This method has been adapted for use with heterologous expression in Xenopus oocytes, allowing experiments on ion channels.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/s0959-4388(03)00068-0DOI Listing
June 2003

Cation-pi interactions in ligand recognition by serotonergic (5-HT3A) and nicotinic acetylcholine receptors: the anomalous binding properties of nicotine.

Biochemistry 2002 Aug;41(32):10262-9

Divisions of Chemistry and Chemical Engineering and Biology, California Institute of Technology, Pasadena, California 91125, USA.

A series of tryptophan analogues has been introduced into the binding site regions of two ion channels, the ligand-gated nicotinic acetylcholine and serotonin 5-HT(3A) receptors, using unnatural amino acid mutagenesis and heterologous expression in Xenopus oocytes. A cation-pi interaction between serotonin and Trp183 of the serotonin channel 5-HT(3A)R is identified for the first time, precisely locating the ligand-binding site of this receptor. The energetic contribution of the observed cation-pi interaction between a tryptophan and the primary ammonium ion of serotonin is estimated to be approximately 4 kcal/mol, while the comparable interaction with the quaternary ammonium of acetylcholine is approximately 2 kcal/mol. The binding mode of nicotine to the nicotinic receptor of mouse muscle is examined by the same technique and found to differ significantly from that of the natural agonist, acetylcholine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi020266dDOI Listing
August 2002