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J A Ballesteros

Publications and source records attributed to J A Ballesteros.

34 records · Page 2Linked to original sources
PubMed1995

Related contribution of specific helix 2 and 7 residues to conformational activation of the serotonin 5-HT2A receptor.

A conserved helix 2 Asp is required for the proper function of many G-protein-coupled receptors. To reveal the structural basis for the role of this residue, the additive effects of mutations at this locus and at a conserved helix 7 locus were investigated in the 5-HT2A receptor. All mutant receptors studied retained high affinity agonist and antagonist binding. Whereas an Asp-->Asn mutation in helix 2 eliminated coupling, interchanging the residues at the two positions by a second mutation of Asn-->Asp in helix 7 restored receptor function. These data suggest that these residues are adjacent in space and interact. The loss of function observed with Ala at either position is consistent with each side chain forming hydrogen bonds. Molecular dynamics simulations were performed on three-dimensional computational models of agonist-receptor complexes of both the wild-type receptor and the Asp-->Asn mutant receptor. Consonant with the lack of coupling observed for the mutant construct, introducing the mutation into the computational model produced a conformational change in a direction opposite to that seen from computational simulations of activation of the wild-type receptor model. These results implicate both loci in a common hydrogen-bonding network underlying receptor activation by agonist.

Mutation↗
PubMed1995

Construction of a 3D model of the cannabinoid CB1 receptor: determination of helix ends and helix orientation.

The goal of this study was to determine the ends and orientations of the seven transmembrane helices of the cannabinoid (CB1) receptor, a G-protein coupled receptor (GPCR). After initial sequence alignment, Fourier transform methods were used with the nPRIFT hydrophobicity scale and with a variability profile to calculate the alpha-helical periodicity (AP) in the primary amino acid sequence of the human CB1 receptor and of its alignment. AP plots were used to identify the amino acids which comprise each of the seven CB1 transmembrane helices. An intracellular alpha helix extension of Helix 7 was characterized by analyzing the relative direction of variability and hydrophobic moment vectors. Variability moment vectors were then used to delineate the orientation of each helix in the membrane. Based upon these vector calculations, a tentative helix bundle arrangement was obtained. This arrangement is largely consistent with the proposed transmembrane helix bundle arrangement in rhodopsin, a GPCR.

Amino Acid Sequence↗
PubMed1994

A reciprocal mutation supports helix 2 and helix 7 proximity in the gonadotropin-releasing hormone receptor.

Activation of the pituitary gonadotropin-releasing hormone receptor, a member of the seven-transmembrane G protein-coupled receptor (GPCR) family, triggers a cascade of events leading to gonadotropin release and stimulation of the reproductive system. An unusual feature of this receptor, observed in mice, rats, and humans, is the presence of Asn87 in the second putative transmembrane helix at the location of a highly conserved aspartate in the GPCR family and of Asp318 in the putative seventh transmembrane helix where nearly all other GPCRs have asparagine. The possibility that these residues interact was suggested by this reciprocal pattern and by a three-dimensional model of the gonadotropin-releasing hormone receptor and was investigated by site-directed mutagenesis. Replacing Asn87 in the second transmembrane domain by aspartate eliminated detectable ligand binding. A second mutation, generating the double-mutant receptor Asp87Asn318, recreated the arrangement found in other GPCRs and re-established high affinity agonist and antagonist binding. The restoration of binding by a reciprocal mutation indicates that these two specific residues in helices 2 and 7 are adjacent in space and provides an empirical basis to refine the model of the transmembrane helix bundle of the receptor.

Amino Acid Sequence↗
PubMed1992

On the use of the transmembrane domain of bacteriorhodopsin as a template for modeling the three-dimensional structure of guanine nucleotide-binding regulatory protein-coupled receptors.

The molecular architecture of bacteriorhodopsin (BR) is commonly regarded as a structural template for the three-dimensional structure of membrane receptors that are functionally coupled to guanine nucleotide-binding regulatory proteins (GPCR). More recently, specific molecular models of such GPCR were constructed on the basis of the functional and structural relation of rhodopsin to BR as well as the sequence homology between rhodopsin and the GPCR. Such models of GPCR leave unresolved the difficulty caused by the apparent lack of any significant degree of sequence homology between the seven transmembrane helices (TMH) of BR and the portions in the sequence of the various GPCR that are considered to constitute their transmembrane domains. Evolutionary arguments offered in favor of the structural relation between BR and the opsins, and hence the GPCR, prompted our investigation of the possibility that the sequence homology, including any similarity in the distribution of kink-inducing proline residues among the helices, might have been obscured by the assumption that the TMH maintained their sequential order from BR in the evolution of the mammalian proteins. With a definition of the TMH in the neurotransmitter GPCR guided by hydropathicity predictions, and additional criteria used to define the span of each helix, optimal alignment of each pair of sequences was determined with no gaps allowed in the matching. The resulting alignment proposed here reveals considerable homology between the TMH in BR and those in GPCR, if the sequential order of the helices is ignored. These findings suggest the possibility that exon shuffling could have occurred in the proposed evolution of the GPCR gene from BR and point to a modification of the BR template to account for the correct packing of the helices in the tertiary structures of GPCR. These findings could guide the construction of three-dimensional models of the neurotransmitter GPCR on the basis of specific interhelical interactions observed in BR.

Amino Acid Sequence↗
PubMed1992

Analysis and refinement of criteria for predicting the structure and relative orientations of transmembranal helical domains.

We are interested in modeling the membrane-spanning domain of the serotonin 5-HT1A G-protein coupled receptor. This superfamily of proteins is predicted to share the topology of the seven transmembrane helices of bacteriorhodopsin (BR), even though no significant sequence homology had been identified. We found significant homologies by allowing for helix shuffling corresponding to minimal exon shuffling during evolution. Consequently, our strategy for building the model for the 5-HT1A receptor has been to construct hypotheses concerning helix-helix interactions, their orientations, and arrangement in bundles surrounded by lipid, based on the 3.5 A resolution structure of BR. Inferences resulting from such models were tested against the 2.3 A resolution structure of the photosynthetic reaction center (PRC) from Rhodobacter Viridis. These comparisons led us to a reevaluation of current methods for the identification and topological orientation of membrane-embedded alpha-helices. We find that methods used currently in the construction of helical transmembrane domains could be misleading if used indiscriminately. These methods include the hydrophobicity profile, the hydrophobic moment, helix amphiphilicity, and charge neutralization. A refinement is proposed here, based on empirical observations, molecular modeling, and physicochemical considerations designed to overcome some of the shortcomings inherent in the use of the above mentioned methods. Here we present the analysis of two of the motifs identified in our study that led to the proposed refinements: the distribution of acidic and basic residues in the transmembranal domains, and the kink induced by a Pro residue in an alpha-helix.

Amino Acids, Diamino↗
PubMed1989

[Disaster medicine].

The authors have reviewed the concept of catastrophe from the health care stand-point as well as, that concerning medicine in catastrophies. The basic outlines for planning adequate health care when a massive number of victims have to be attended are pointed-out as well as, the prevention means for possible flare ups of transmissible diseases.

Accidents↗