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

Grunbeck, Amy; Huber, Thomas; Abrol, Ravinder; Trzaskowski, Bartosz; Goddard, William A.; Sakmar, Thomas P.

doi:10.1021/cb300059z

Cited by 65 publications

(65 citation statements)

References 27 publications

(52 reference statements)

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“…In fact, the position of the C-␤ atom of Y108 suggests that it is indeed located close enough to VVC for an interaction to be possible. In previous mutagenesis-based studies, the residues identified as interacting with VVC and MVC were similar to those predicted by our models, although there were subtle differences in the extent and nature of the interactions of the two inhibitors with individual residues (45)(46)(47). Overall, MVC and VVC are predicted to have a common binding site within the TM bundle and to contact similar residues, albeit with perhaps some differences in their modes of binding.…”

Section: Affinity Of Vvc For G-protein-coupled and Uncoupled Receptorssupporting

confidence: 75%

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

Berro

Yasmeen

Abrol

et al. 2013

J Virol

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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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Section: Affinity Of Vvc For G-protein-coupled and Uncoupled Receptorssupporting

confidence: 75%

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

Berro

Yasmeen

Abrol

et al. 2013

J Virol

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“…In addition to CB1 (7,8), SuperBiHelix has been applied successfully to the adenosine A 3 receptor (16) and other adenosine receptors (22), serotonin 5-HT2B and 5-HT2C receptor (25), the histamine H3 receptor (26), the CCR5 receptor (27,28), TAS2R38 bitter taste receptor (29), and the V2 vasopressin receptor (30). The predicted GPCR structures in these studies were validated by predicting the binding sites and energies for known series of ligands and comparing with experimental mutagenesis, binding, and/or functional data.…”

Section: Discussionmentioning

confidence: 99%

SuperBiHelix method for predicting the pleiotropic ensemble of G-protein–coupled receptor conformations

Bray

Abrol

Goddard

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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There is overwhelming evidence that G-protein-coupled receptors (GPCRs) exhibit several distinct low-energy conformations, each of which might favor binding to different ligands and/or lead to different downstream functions. Understanding the function of such proteins requires knowledge of the ensemble of low-energy configurations that might play a role in this pleiotropic functionality. We earlier reported the BiHelix method for efficiently sampling the (12) 7 = 35 million conformations resulting from 30°rotations about the axis (η) of all seven transmembrane helices (TMHs), showing that the experimental structure is reliably selected as the best conformation from this ensemble. However, various GPCRs differ sufficiently in the tilts of the TMHs that this method need not predict the optimum conformation starting from any other template. In this paper, we introduce the SuperBiHelix method in which the tilt angles (θ, φ) are optimized simultaneously with rotations (η) efficiently enough that it is practical and sufficient to sample (5 × 3 × 5) 7 = 13 trillion configurations. This method can correctly identify the optimum structure of a GPCR starting with the template from a different GPCR. We have validated this method by predicting known crystal structure conformations starting from the template of a different protein structure. We find that the SuperBiHelix conformational ensemble includes the higher energy conformations associated with the active protein in addition to those associated with the more stable inactive protein. This methodology was then applied to design and experimentally confirm structures of three mutants of the CB1 cannabinoid receptor associated with different functions.protein structure prediction | A 2A adenosine receptor | β 2 -adrenergic receptor | constitutive activity | functional selectivity

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“…It is restricted to residues in close vicinity to the reactive moiety of AzF. This prerequisite not only abolishes false-positive hits, it can also be utilized to map interactions, as shown recently for different examples of receptors and ligands in the mammalian system (57,58).…”

Section: Discussionmentioning

confidence: 99%

An Expanded Genetic Code in Candida albicans To Study Protein-Protein Interactions In Vivo

et al. 2013

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cFor novel insights into the pathogenicity of Candida albicans, studies on molecular interactions of central virulence factors are crucial. Since methods for the analysis of direct molecular interactions of proteins in vivo are scarce, we expanded the genetic code of C. albicans with the synthetic photo-cross-linking amino acid p-azido-L-phenylalanine (AzF). Interacting molecules in close proximity of this unnatural amino acid can be covalently linked by UV-induced photo-cross-link, which makes unknown interacting molecules available for downstream identification. Therefore, we applied an aminoacyltRNA synthetase and a suppressor tRNA pair (EcTyrtRNA CUA ) derived from Escherichia coli, which was previously reported to be orthogonal in Saccharomyces cerevisiae. We further optimized the aminoacyl-tRNA synthetase for AzF (AzF-RS) and EcTyrtRNA CUA for C. albicans and identified one AzF-RS with highest charging efficiency. Accordingly, incorporation of AzF into selected model proteins such as Tsa1p or Tup1p could be considerably enhanced. Immunologic detection of C-terminally tagged Tsa1p and Tup1p upon UV irradiation in a strain background containing suppressor tRNA and optimized AzF-RS revealed not only the mutant monomeric forms of these proteins but also higher-molecularweight complexes, strictly depending on the specific position of incorporated AzF and UV excitation. By Western blotting and tandem mass spectrometry, we could identify these higher-molecular-weight complexes as homodimers consisting of one mutant monomer and a differently tagged, wild-type version of Tsa1p or Tup1p, respectively, demonstrating that expanding the genetic code of C. albicans with the unnatural photo-cross-linker amino acid AzF and applying it for in vivo binary protein interaction analyses is feasible.

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

Cited by 65 publications

References 27 publications

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

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

SuperBiHelix method for predicting the pleiotropic ensemble of G-protein–coupled receptor conformations

An Expanded Genetic Code in Candida albicans To Study Protein-Protein Interactions In Vivo

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