Functional role of internal water molecules in rhodopsin revealed by x-ray crystallography

Abstract

Activation of G protein-coupled receptors (GPCRs) is triggered and regulated by structural rearrangement of the transmembrane heptahelical bundle containing a number of highly conserved residues. In rhodopsin, a prototypical GPCR, the helical bundle accommodates an intrinsic inverse-agonist 11-cis-retinal, which undergoes photo-isomerization to the all-trans form upon light absorption. Such a trigger by the chromophore corresponds to binding of a diffusible ligand to other GPCRs. Here we have explored the functional role of water molecules in the transmembrane region of bovine rhodopsin by using x-ray diffraction to 2.6 Å. The structural model suggests that water molecules, which were observed in the vicinity of highly conserved residues and in the retinal pocket, regulate the activity of rhodopsin-like GPCRs and spectral tuning in visual pigments, respectively. To confirm the physiological relevance of the structural findings, we conducted single-crystal microspectrophotometry on rhodopsin packed in our three-dimensional crystals and show that its spectroscopic properties are similar to those previously found by using bovine rhodopsin in suspension or membrane environment. C ell surface membrane receptors mediate a variety of biological signaling processes that are triggered by a multitude of diffusible molecules or light in the case of visual pigments. G protein-coupled receptors (GPCRs), including the rhodopsinlike family as a dominant subgroup, are the largest group of membrane receptors. Many of the members are considered as primary drug targets for various medical and pharmacological interventions. These receptors appear to be activated by a mechanism involving common heptahelical transmembrane architecture that undergoes major rearrangement upon signal reception, resulting in activation of the heterotrimeric G protein molecules.Rhodopsin from retinal rod cells mediates scotopic vision. It is a unique member among GPCRs in that it contains an intrinsic inverse-agonist, the 11-cis-retinal. Photon absorption results in retinal isomerization to the all-trans configuration, which drives the protein to the active metarhodopsin form (M II) (1). Visual pigments mediating color vision in cone cells share a common mechanism to evoke a cellular signaling cascade (2) through interaction between the M II state and the G protein. A view of the seven helices of bovine rhodopsin was provided by electron crystallography in 1993 (3), and an x-ray crystallographic study recently determined its structure at 2.8-Å resolution (4). This structure provided the template model at high resolution for the rhodopsin-like GPCRs and has been further refined at the same resolution (5).The 11-cis-retinal chromophore is covalently bound to Lys-296 in transmembrane helix VII by a protonated Schiff base linkage, which is stabilized by the negatively charged counterion Glu-113 in helix III. Disruption of this salt bridge (6) upon proton transfer is thought to trigger conformational changes in rhodopsin (7), which are necessary for G protein ...

Diversity of actions of GnRHs mediated by ligand-induced selective signaling

“…This restoration of ligand binding by reciprocal mutation demonstrates that the side-chains of the two residues in TMs 2 and 7 have complementary roles in maintaining the structure of the receptor and occupy the same microenvironment within the receptor helical bundle. Subsequently, the crystal structure of bovine rhodopsin revealed that these residues are capable of interacting through a water molecule [110] thus validating the GnRH receptor models. Interestingly, this exchange prevents the human GnRH receptor coupling to phospholipase D by the small G-proteins ARF and RhoA, implying receptor conformationdependent signaling selectivity [98].…”

Generation of a homology model of the human histamine H3 receptor for ligand docking and pharmacophore-based screening

“…One internal water molecule was included in the receptor model, which was located in proximity to D2.50 and linked helices 2, 3 and 7 at a comparable position as water molecule 1b in the structure of bovine rhodopsin [26].…”

“…Different to the model of Sippl et al which suggested an indirect influence of amino-acids varying between species, in the model of Yao antagonists made a direct contact to those residues, resulting in a ligand placement extending from D3.32 orthogonal to the membrane plane down to residue D2.50 [25,26]. In 2005, a model of the rat H 3 R was published by Lorenzi et al which was used to guide the successful design of further imidazole-containing H 3 R compounds [27].…”

“…After truncating the 3rd intracellular loop to a comparable length as present in the template structure bovine rhodopsin by excising the stretch A240-Q346 from the hH 3 R sequence, amino acid side chain conformations were added using program SCWRL3.0 [30]. One internal water molecule was included in the hH 3 R receptor model, which was located in proximity to D2.50 and linked helices 2, 3 and 7 at a comparable position as water molecule 1b in the structure of bovine rhodopsin [26].…”

The Seventh Transmembrane Domains of the δ and κ Opioid Receptors Have Different Accessibility Patterns and Interhelical Interactions

“…structures have been elucidated (1)(2)(3). Rhodopsin has an extracellular N-terminal domain, seven transmembrane helices (TMs) connected by alternating intracellular and extracellular hydrophilic loops and an intracellular C-terminal domain.…”