We developed a general cell-based photocrosslinking approach to investigate the binding interfaces necessary for the formation of G protein-coupled receptor (GPCR) signaling complexes. The two photoactivatable unnatural amino acids p-benzoyl-L-phenylalanine and p-azido-L-phenylalanine were incorporated by amber codon suppression technology into CXC chemokine receptor 4 (CXCR4). We then probed the ligand-binding site for the HIV-1 co-receptor blocker, T140, using a fluorescein-labeled T140 analogue. Among eight amino acid positions tested, we found a unique UV-light dependent crosslink specifically between residue 189 and T140. These results are evaluated with molecular modeling using the crystal structure of CXCR4 bound to CVX15.

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.

Cell surface heptahelical G protein-coupled receptors (GPCRs) mediate critical cellular signaling pathways and are important pharmaceutical drug targets. (1) In addition to traditional small-molecule approaches, lipopeptide-based GPCR-derived pepducins have emerged as a new class of pharmaceutical agents. (2, 3) To better understand how pepducins interact with targeted receptors, we developed a cell-based photo-cross-linking approach to study the interaction between the pepducin agonist ATI-2341 and its target receptor, chemokine C-X-C-type receptor 4 (CXCR4). A pepducin analogue, ATI-2766, formed a specific UV-light-dependent cross-link to CXCR4 and to mutants with truncations of the N-terminus, the known chemokine docking site. These results demonstrate that CXCR4 is the direct binding target of ATI-2341 and suggest a new mechanism for allosteric modulation of GPCR activity. Adaptation and application of our findings should prove useful in further understanding pepducin modulation of GPCRs as well as enable new experimental approaches to better understand GPCR signal transduction.

The inherent instability of heptahelical G protein-coupled receptors (GPCRs) during purification and reconstitution is a primary impediment to biophysical studies and to obtaining high-resolution crystal structures. New approaches to stabilize receptors during purification and to screen reconstitution procedures are needed. Here we report the development of a novel homogeneous time-resolved fluorescence assay (HTRF) to quantify properly folded CC-chemokine receptor 5 (CCR5). The assay permits high-throughput thermal stability measurements of femtomole quantities of CCR5 in detergent and in engineered nanoscale apolipoprotein bound bilayer (NABB) particles. We show that recombinant expressed CCR5 can be incorporated into NABB particles in high yield, resulting in greater thermal stability compared with CCR5 in detergent solution. We also demonstrate that CCR5 binding to the HIV-1 cellular entry inhibitors maraviroc, AD101, CMPD 167, and vicriviroc dramatically increases receptor stability. The HTRF assay technology reported here is applicable to other membrane proteins and could greatly facilitate structural studies of GPCRs. Keywords GPCR; lipoprotein particle; CCR5; NABB; thermal stability; maraviroc; europium cryptate; timeresolved fluorescence; energy transfer G protein-coupled receptors (GPCRs) constitute a large family of heptahelical membrane proteins that recognize a broad array of extracellular ligands and couple to multiple intracellular signaling pathways.(1,2) Their involvement in the molecular pathophysiology of a number of diseases has made GPCRs a major drug target.(3) Recent high-resolution structures of several GPCRs, including rhodopsin, β 2 -adrenergic receptor, β 1 -adrenergic receptor, A 2A adenosine receptor, and opsin have provided insight into the molecular mechanism of receptor activation, but further advances are necessary to understand their physiological function and to enhance drug discovery. Stabilizing these complex polytopic membrane proteins in purified and/or defined systems remains the most significant present challenge for biochemists and structural biologists. Successful approaches to generating stable GPCRs in detergent solution include truncations or deletions of disordered regions,

It has been 50 years since F. H. Westheimer and colleagues reported the first use of a photoactivatable cross-linking reagent to study the active site of chymotrypsin. In studies of seven transmembrane helical receptors, also known as G protein-coupled receptors (GPCRs), recent simultaneous advances in structural biology, molecular dynamics simulations, and amber codon suppression methods have allowed the development of a targeted photo-cross-linking strategy to probe receptor-ligand interactions in cell membranes. We review here recent advances in targeted photo-cross-linking of GPCR-ligand complexes in the context of extensive earlier work that primarily relied upon the use of ligand analogues with photoactivatable constituents.

Background: Unnatural amino acids can be genetically incorporated into 7-transmembrane receptors. Results: A photoreactive amino acid introduced into the neurokinin-1 receptor cross-links substance P to the N-terminal and extracellular loop II domains of the receptor. Conclusion:The extracellular domain of the neurokinin-1 receptor possesses multiple potential binding sites for substance P. Significance: A photocross-linking methodology reveals novel interaction sites in the neurokinin-1-receptor-substance P complex.

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 G␣ i . Treating CD4 ؉ T cells with pertussis toxin to uncouple the G␣ i subunit from CCR5 increased the potency of VVC against the sensitive viruses and revealed that VVC-resistant viruses use the inhibitor-bound form of G␣ 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.

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