The evolutionary trace (ET) method, a data mining approach for determining significant levels of amino acid conservation, has been applied to over 700 aligned G-protein-coupled receptor (GPCR) sequences. The method predicted the occurrence of functionally important clusters of residues on the external faces of helices 5 and 6 for each family or subfamily of receptors; similar clusters were observed on helices 2 and 3. The probability that these clusters are not random was determined using Monte Carlo techniques. The cluster on helices 5 and 6 is consistent with both 5,6-contact and 5,6-domain swapped dimer formation; the possible equivalence of these two types of dimer is discussed because this relates to activation by homo- and heterodimers. The observation of a functionally important cluster of residues on helices 2 and 3 is novel, and some possible interpretations are given, including heterodimerization and oligomerization. The application of the evolutionary trace method to 113 aligned G-protein sequences resulted in the identification of two functional sites. One large, well-defined site is clearly identified with adenyl cyclase, beta/gamma and regulator of G-protein signaling (RGS) binding. The other G-protein functional site, which extends from the ras-like domain onto the helical domain, has the correct size and electrostatic properties for GPCR dimer binding. The implications of these results are discussed in terms of the conformational changes required in the G-protein for activation by a receptor dimer. Further, the implications of GPCR dimerization for medicinal chemistry are discussed in the context of these ET results.

We have characterized an assay measuring CD8 T cell-mediated inhibition of human immunodeficiency virus (HIV) type 1 replication, demonstrating specificity and reproducibility and employing a panel of primary HIV-1 isolates. The assay uses relatively simple autologous cell culture and enzyme-linked immunosorbent assay, avoids generation of T cell clones, and can be performed with <2 million peripheral blood mononuclear cells. Efficient CD8 T cell-mediated cross-clade inhibition of HIV-1 replication in vitro was demonstrated in antiretroviral therapy-naive HIV-1-infected subjects with controlled viral replication in vivo but not in viremic subjects. An HIV-1 vaccine candidate, consisting of DNA and recombinant adenovirus 5 vectors tested in a phase I clinical trial, induced CD8 T cells that efficiently inhibited HIV-1 in a HLA-I-dependent manner. Assessment of direct antiviral T cell function by this assay provides additional information to guide vaccine design and the prioritizing of candidates for further clinical trials.

A series of triazolylpurine analogues show interesting and unintuitive structure-activity relationships against the A2A adenosine receptor. As the 2-substituted aliphatic group is initially increased to methyl and isopropyl, there is a decrease in potency; however, extending the substituent to n-butyl and n-pentyl results in a significant gain in potency. This trend cannot be readily explained by ligand-receptor interactions, steric effects, or differences in ligand desolvation. Here, we show that a novel method for characterizing solvent thermodynamics in protein binding sites correctly predicts the trend in binding affinity for this series based on the differential water displacement patterns. In brief, small unfavorable substituents occupy a region in the A2A adenosine receptor binding site predicted to contain stable waters, while the longer favorable substituents extend to a region that contains several unstable waters. The predicted binding energies associated with displacing water within these hydration sites correlate well with the experimental activities.

Computer simulations were performed on models of the beta2-adrenergic receptor dimer, including 5,6-domain swapped dimers which have been proposed as the active, high affinity form (here the dimer interface lies between helices 5 and 6). The calculations suggest that the domain swapped dimer is a high energy structure in both the apo dimer and in the presence of propranolol. In the presence of agonist the energy of the domain swapped dimer is significantly lowered. Analysis of the dimer structure suggests that the agonist-induced conformational change optimizes the helix-helix interactions at the 5-6 interface. An antagonist on the other hand has little effect on these interactions. These observations are consistent with the hypothesis that the agonist functions by shifting the equilibrium in favour of the domain swapped dimer. Indirect support for the domain swapping hypothesis was obtained from the correlated mutations amongst the external residues of the known beta2-adrenergic receptors. These occur mainly at the 5-6 interface at precisely the locations predicted by the simulations; site-directed mutagenesis data in support of a functional role for these lipid-facing correlated residues is presented. The article includes a review of the experimental evidence for G-protein coupled receptor dimerization. Many other aspects of G-protein coupled receptor activation are discussed in terms of this domain swapping hypothesis