Application of Genetically-Encoded Photoactivatable Crosslinkers to Map Ligand-Binding Sites on G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) are dynamic membrane proteins that bind extracellular molecules to transduce biological signals. Although GPCRs represent the largest class of targets for therapeutic agents, ligand-binding sites have been precisely defined for only a small percentage of the receptors in the human genome. A general cellbased photocrosslinking approach was developed to investigate the binding interfaces necessary for the formation of GPCR signaling complexes. Amber codon suppression was extended to facilitate the incorporation of photoactivatable unnatural amino acids, pbenzoyl- L-phenylalanine and p-azido-L-phenylalanine, into engineered GPCRs expressed in mammalian cells in culture. Proof-of-concept studies were carried out in chemokine receptors C-X-C chemokine receptor 4 (CXCR4) and C-C chemokine receptor 5 (CCR5), which are known HIV-1 co-receptors required for HIV-1 cellular entry in CD4+ cells. A cyclic peptide CXCR4-specific inhibitor, T140, photocrosslinked primarily to a specific site on CXCR4 and the result was reconciled with existing structural biology data. A small molecule drug, maraviroc, photocrosslinked to multiple sites on CCR5 and the results were extended to develop a computer homology model of the CCR5-maraviroc complex. In summary, the application of a novel targeted cellbased photocrosslinking strategy provided detailed information about receptor-ligand complexes in two chemokine receptors. The general approach described here using genetically-encoded photoreactive molecules to study the binding interactions between GPCRs and ligands represents a significant advance in the application of photocrosslinking reagents to address problems in biochemistry and pharmacology

Genetically Encoded Photo-cross-linkers Map the Binding Site of an Allosteric Drug on a G Protein-Coupled Receptor

G protein-coupled receptors (GPCRs) are dynamic membrane proteins that bind extracellular molecules to transduce signals. Although GPCRs represent the largest class of therapeutic targets, only a small percentage of their ligand-binding sites are precisely defined. Here we describe the novel application of targeted photo-cross-linking using unnatural amino acids to obtain structural information about the allosteric binding site of a small molecule drug, the CCR5-targeted HIV-1 co-receptor blocker maraviroc

Use of G-Protein-Coupled and -Uncoupled CCR5 Receptors by CCR5 Inhibitor-Resistant and -Sensitive Human Immunodeficiency Virus Type 1 Variants

Small-molecule CCR5 inhibitors such as vicriviroc (VVC) and maraviroc (MVC) are allosteric modulators that impair HIV-1 entry by stabilizing a CCR5 conformation that the virus recognizes inefficiently. Viruses resistant to these compounds are able to bind the inhibitor-CCR5 complex while also interacting with the free coreceptor. CCR5 also interacts intracellularly with G proteins, as part of its signal transduction functions, and this process alters its conformation. Here we investigated whether the action of VVC against inhibitor-sensitive and -resistant viruses is affected by whether or not CCR5 is coupled to G proteins such as Gαi. Treating CD4^(+)T cells with pertussis toxin to uncouple the Gαi subunit from CCR5 increased the potency of VVC against the sensitive viruses and revealed that VVC-resistant viruses use the inhibitor-bound form of Gα_(i)-coupled CCR5 more efficiently than they use uncoupled CCR5. Supportive evidence was obtained by expressing a signaling-deficient CCR5 mutant with an impaired ability to bind to G proteins, as well as two constitutively active mutants that activate G proteins in the absence of external stimuli. The implication of these various studies is that the association of intracellular domains of CCR5 with the signaling machinery affects the conformation of the external and transmembrane domains and how they interact with small-molecule inhibitors of HIV-1 entry

Mapping Substance P Binding Sites on the Neurokinin-1 Receptor Using Genetic Incorporation of a Photoreactive Amino Acid

Substance P (SP) is a neuropeptide that mediates numerous physiological responses, including transmission of pain and inflammation through the neurokinin-1 (NK1) receptor, a G protein-coupled receptor. Previous mutagenesis studies and photoaffinity labeling using ligand analogues suggested that the binding site for SP includes multiple domains in the N-terminal (Nt) segment and the second extracellular loop (ECLII) of NK1. To map precisely the NK1 residues that interact with SP, we applied a novel receptor-based targeted photocross-linking approach. We used amber codon suppression to introduce the photoreactive unnatural amino acid p-benzoyl-l-phenylalanine (BzF) at 11 selected individual positions in the Nt tail (residues 11–21) and 23 positions in the ECLII (residues 170(C-10)–193(C+13)) of NK1. The 34 NK1 variants were expressed in mammalian HEK293 cells and retained the ability to interact with a fluorescently labeled SP analog. Notably, 10 of the receptor variants with BzF in the Nt tail and 4 of those with BzF in ECLII cross-linked efficiently to SP, indicating that these 14 sites are juxtaposed to SP in the ligand-bound receptor. These results show that two distinct regions of the NK1 receptor possess multiple determinants for SP binding and demonstrate the utility of genetically encoded photocross-linking to map complex multitopic binding sites on G protein-coupled receptors in a cell-based assay format

Genetically Encoded Photo-cross-linkers Map the Binding Site of an Allosteric Drug on a G Protein-Coupled Receptor

G protein-coupled receptors (GPCRs) are dynamic membrane proteins that bind extracellular molecules to transduce signals. Although GPCRs represent the largest class of therapeutic targets, only a small percentage of their ligand-binding sites are precisely defined. Here we describe the novel application of targeted photo-cross-linking using unnatural amino acids to obtain structural information about the allosteric binding site of a small molecule drug, the CCR5-targeted HIV-1 co-receptor blocker maraviroc