Xavier Deupi

Molecular signatures of G-protein-coupled receptors

G-protein-coupled receptors (GPCRs) are physiologically important membrane proteins that sense signalling molecules such as hormones and neurotransmitters, and are the targets of several prescribed drugs. Recent exciting developments are providing unprecedented insights into the structure and function of several medically important GPCRs. Here, through a systematic analysis of high-resolution GPCR structures, we uncover a conserved network of non-covalent contacts that defines the GPCR fold. Furthermore, our comparative analysis reveals characteristic features of ligand binding and conformational changes during receptor activation. A holistic understanding that integrates molecular and systems biology of GPCRs holds promise for new therapeutics and personalized medicine.

Energy Landscapes as a Tool to Integrate GPCR Structure, Dynamics, and Function

G protein-coupled receptors (GPCRs) are versatile signaling molecules that mediate the majority of physiological responses to hormones and neurotransmitters. Recent high-resolution structural insights into GPCR structure and dynamics are beginning to shed light on the molecular basis of this versatility. We use energy landscapes to conceptualize the link between structure and function.

Tracking G-protein-coupled receptor activation using genetically encoded infrared probes

Rhodopsin is a prototypical heptahelical family A G-protein-coupled receptor (GPCR) responsible for dim-light vision. Light isomerizes rhodopsins retinal chromophore and triggers concerted movements of transmembrane helices, including an outward tilting of helix 6 (H6) and a smaller movement of H5, to create a site for G-protein binding and activation. However, the precise temporal sequence and mechanism underlying these helix rearrangements is unclear. We used site-directed non-natural amino acid mutagenesis to engineer rhodopsin with p-azido-l-phenylalanine residues incorporated at selected sites, and monitored the azido vibrational signatures using infrared spectroscopy as rhodopsin proceeded along its activation pathway. Here we report significant changes in electrostatic environments of the azido probes even in the inactive photoproduct Meta I, well before the active receptor state was formed. These early changes suggest a significant rotation of H6 and movement of the cytoplasmic part of H5 away from H3. Subsequently, a large outward tilt of H6 leads to opening of the cytoplasmic surface to form the active receptor photoproduct Meta II. Thus, our results reveal early conformational changes that precede larger rigid-body helix movements, and provide a basis to interpret recent GPCR crystal structures and to understand conformational sub-states observed during the activation of other GPCRs.

The effect of ligand efficacy on the formation and stability of a GPCR-G protein complex

G protein-coupled receptors (GPCRs) mediate the majority of physiologic responses to hormones and neurotransmitters. However, many GPCRs exhibit varying degrees of agonist-independent G protein activation. This phenomenon is referred to as basal or constitutive activity. For many of these GPCRs, drugs classified as inverse agonists can suppress basal activity. There is a growing body of evidence that basal activity is physiologically relevant, and the ability of a drug to inhibit basal activity may influence its therapeutic properties. However, the molecular mechanism for basal activation and inhibition of basal activity by inverse agonists is poorly understood and difficult to study, because the basally active state is short-lived and represents a minor fraction of receptor conformations. Here, we investigate basal activation of the G protein Gs by the β2 adrenergic receptor (β2AR) by using purified receptor reconstituted into recombinant HDL particles with a stoichiometric excess of Gs. The β2AR is site-specifically labeled with a small, environmentally sensitive fluorophore enabling direct monitoring of agonist- and Gs-induced conformational changes. In the absence of an agonist, the β2AR and Gs can be trapped in a complex by enzymatic depletion of guanine nucleotides. Formation of the complex is enhanced by the agonist isoproterenol, and it rapidly dissociates on exposure to concentrations of GTP and GDP found in the cytoplasm. The inverse agonist ICI prevents formation of the β2AR-Gs complex, but has little effect on preformed complexes. These results provide insights into G protein-induced conformational changes in the β2AR and the structural basis for ligand efficacy.

Ligand-regulated oligomerization of β2-adrenoceptors in a model lipid bilayer

The β2‐adrenoceptor (β2AR) was one of the first Family A G protein‐coupled receptors (GPCRs) shown to form oligomers in cellular membranes, yet we still know little about the number and arrangement of protomers in oligomers, the influence of ligands on the organization or stability of oligomers, or the requirement for other proteins to promote oligomerization. We used fluorescence resonance energy transfer (FRET) to characterize the oligomerization of purified β2AR site‐specifically labelled at three different positions with fluorophores and reconstituted into a model lipid bilayer. Our results suggest that the β2AR is predominantly tetrameric following reconstitution into phospholipid vesicles. Agonists and antagonists have little effect on the relative orientation of protomers in oligomeric complexes. In contrast, binding of inverse agonists leads to significant increases in FRET efficiencies for most labelling pairs, suggesting that this class of ligand promotes tighter packing of protomers and/or the formation of more complex oligomers by reducing conformational fluctuations in individual protomers. The results provide new structural insights into β2AR oligomerization and suggest a possible mechanism for the functional effects of inverse agonists.

Stabilized G protein binding site in the structure of constitutively active metarhodopsin-II

G protein-coupled receptors (GPCR) are seven transmembrane helix proteins that couple binding of extracellular ligands to conformational changes and activation of intracellular G proteins, GPCR kinases, and arrestins. Constitutively active mutants are ubiquitously found among GPCRs and increase the inherent basal activity of the receptor, which often correlates with a pathological outcome. Here, we have used the M257Y6.40 constitutively active mutant of the photoreceptor rhodopsin in combination with the specific binding of a C-terminal fragment from the G protein alpha subunit (GαCT) to trap a light activated state for crystallization. The structure of the M257Y/GαCT complex contains the agonist all-trans-retinal covalently bound to the native binding pocket and resembles the G protein binding metarhodopsin-II conformation obtained by the natural activation mechanism; i.e., illumination of the prebound chromophore 11-cis-retinal. The structure further suggests a molecular basis for the constitutive activity of 6.40 substitutions and the strong effect of the introduced tyrosine based on specific interactions with Y2235.58 in helix 5, Y3067.53 of the NPxxY motif and R1353.50 of the E(D)RY motif, highly conserved residues of the G protein binding site.

Structural insights into agonist-induced activation of G-protein-coupled receptors

Recent years have seen tremendous breakthroughs in structure determination of G-protein-coupled receptors (GPCRs). In 2011, two agonist-bound active-state structures of rhodopsin have been published. Together with structures of several rhodopsin activation intermediates and a wealth of biochemical and spectroscopic information, they provide a unique structural framework on which to understand GPCR activation. Here we use this framework to compare the recent crystal structures of the agonist-bound active states of the β(2) adrenergic receptor (β(2)AR) and the A(2A) adenosine receptor (A(2A)AR). While activation of these three GPCRs results in rearrangements of TM5 and TM6, the extent of this conformational change varies considerably. Displacements of the cytoplasmic side of TM6 ranges between 3 and 8Å depending on whether selective stabilizers of the active conformation are used (i.e. a G-protein peptide in the case of rhodopsin or a conformationally selective nanobody in the case of the β(2)AR) or not (A(2A)AR). The agonist-induced conformational changes in the ligand-binding pocket are largely receptor specific due to the different chemical nature of the agonists. However, several similarities can be observed, including a relocation of conserved residues W6.48 and F6.44 towards L5.51 and P5.50, and of I/L3.40 away from P5.50. This transmission switch links agonist binding to the movement of TM5 and TM6 through the rearrangement of the TM3-TM5-TM6 interface, and possibly constitutes a common theme of GPCR activation.

Serine and Threonine Residues Bend α-Helices in the χ1 = g− Conformation

The relationship between the Ser, Thr, and Cys side-chain conformation (chi(1) = g(-), t, g(+)) and the main-chain conformation (phi and psi angles) has been studied in a selection of protein structures that contain alpha-helices. The statistical results show that the g(-) conformation of both Ser and Thr residues decreases their phi angles and increases their psi angles relative to Ala, used as a control. The additional hydrogen bond formed between the O(gamma) atom of Ser and Thr and the i-3 or i-4 peptide carbonyl oxygen induces or stabilizes a bending angle in the helix 3-4 degrees larger than for Ala. This is of particular significance for membrane proteins. Incorporation of this small bending angle in the transmembrane alpha-helix at one side of the cell membrane results in a significant displacement of the residues located at the other side of the membrane. We hypothesize that local alterations of the rotamer configurations of these Ser and Thr residues may result in significant conformational changes across transmembrane helices, and thus participate in the molecular mechanisms underlying transmembrane signaling. This finding has provided the structural basis to understand the experimentally observed influence of Ser residues on the conformational equilibrium between inactive and active states of the receptor, in the neurotransmitter subfamily of G protein-coupled receptors.

An activation switch in the rhodopsin family of G protein coupled receptors: The thyrotropin receptor

We aimed at understanding molecular events involved in the activation of a member of the G protein-coupled receptor family, the thyrotropin receptor. We have focused on the transmembrane region and in particular on a network of polar interactions between highly conserved residues. Using molecular dynamics simulations and site-directed mutagenesis techniques we have identified residue Asn-7.49, of the NPxxY motif of TM 7, as a molecular switch in the mechanism of thyrotropin receptor (TSHr) activation. Asn-7.49 appears to adopt two different conformations in the inactive and active states. These two states are characterized by specific interactions between this Asn and polar residues in the transmembrane domain. The inactive gauche+ conformation is maintained by interactions with residues Thr-6.43 and Asp-6.44. Mutation of these residues into Ala increases the constitutive activity of the receptor by factors of ∼14 and ∼10 relative to wild type TSHr, respectively. Upon receptor activation Asn-7.49 adopts the trans conformation to interact with Asp-2.50 and a putatively charged residue that remains to be identified. In addition, the conserved Leu-2.46 of the (N/S)LxxxD motif also plays a significant role in restraining the receptor in the inactive state because the L2.46A mutation increases constitutive activity by a factor of ∼13 relative to wild type TSHr. As residues Leu-2.46, Asp-2.50, and Asn-7.49 are strongly conserved, this molecular mechanism of TSHr activation can be extended to other members of the rhodopsin-like family of G protein-coupled receptors.

The Role of Internal Water Molecules in the Structure and Function of the Rhodopsin Family of G Protein‐Coupled Receptors

Membrane receptors coupled to guanine nucleotide-binding regulatory proteins (commonly known as G protein-coupled ACHTUNGTRENNUNGreceptors, GPCRs) constitute one of the most attractive pharmaceutical targets, as around 40% of clinically prescribed drugs and 25% of the top-selling drugs act at these receptors. GPCRs are receptors for sensory signals of external origin such as odors, pheromones, or tastes; and for endogenous signals such as neurotransmitters, (neuro)peptides, divalent cations, proteases, glycoprotein hormones, and purine ligands. Phylogenetic analyses of the human genome have permitted GPCR sequences to be classified into five main families: rhodopsin (class A or family 1), secretin (class B or family 2), glutamate (class C or family 3), adhesion, and frizzled/ taste2. Specialized databases of GPCRs can be found at http://www.gpcr.org/7tm, http://gris.ulb.ac.be/, and http:// www.iuphar-db.org. Due to the low natural abundance of GPCRs and the difficulty in producing and purifying recombinant protein, only one member of this family, rhodopsin, the photoreceptor protein of rod cells, has been crystallized so far. Five structural models of inactive rhodopsin are available at the Protein Data Bank, at resolutions of 2.8 A (PDB IDs: 1F88 and 1HZX), 2.65 A (1GZM), 2.6 A (1L9H), and 2.2 A (1U19). Structural models of rhodopsin photointermediates such as bathorhodopsin (2G87), lumirhodopsin (2HPY), metarhodopsin I, and a photoactivated deprotonated intermediate reminiscent of metarhodopsin II (2I37) are also available. Rhodopsin is formed by an extracellular N terminus of four b-strands, seven transmembrane helices (TM1 to TM7) connected by alternating intracellular (I1 to I3) and extracellular (E1 to E3) hydrophilic loops, a disulfide bridge between E2 and TM3, and a cytoplasmic C terminus containing an a-helix (Hx8) parallel to the cell membrane. Statistical analysis of the residues forming the TM helices of the rhodopsin family of GPCRs shows a large number of conserved sequence patterns; this suggests a common TM structure. Thus, the availability of the rhodopsin structure allows the use of homology modeling techniques to build three-dimensional models of other homologous GPCRs. The putative structural homology between rhodopsin and other GPCRs probably does not extend to the extracellular domain, since the extracellular N terminus and loop fragments are highly variable in length and amino acid content. The class A family of GPCRs contains highly conserved Pro residues in the middle of TMs 5 (P5.50, conserved in 77% of the sequences), 6 (P6.50, 100%), and 7 (P7.50, 96%; residues are identified by the generic numbering scheme of Ballesteros and Weinstein, which allows easy comparison among residues in the 7TM segments of different receptors). Pro residues are normally observed in the TM helices of membrane proteins where they usually induce a significant distortion named a “Pro-kink”. This break arises in order to avoid a steric clash between the pyrrolidine ring of the Pro side chain (at position i) and the carbonyl oxygen of the residue in the preceding turn (position i 4) and leads to a distortion of the helical structure. However, TM segments of rhodopsin, either with or without Pro residues in their sequence are far from being standard Pro-kinked or ideal helices, respectively. Their distortions are energetically stabilized through complementary intraand interhelical interactions involving polar side chains, backbone carbonyls, and, in some cases, specific structural and functional water molecules embedded in the TM bundle. Here we review the role of these water molecules in the structure and function of GPCRs and in building computergenerated homology models of class A GPCRs. We propose that water molecules present in the vicinity of highly conserved motifs are most likely present in the rhodopsin family of GPCRs, being another conserved structural element in the family.