Structural approaches to understanding retinal proteins needed for vision

Abstract

The past decade has witnessed an impressive expansion of our knowledge of retinal photoreceptor signal transduction and the regulation of the visual cycle required for normal eyesight. Progress in human genetics and next generation sequencing technologies have revealed the complexity behind many inherited retinal diseases. Structural studies have markedly increased our understanding of the visual process. Moreover, technical innovations and improved methodologies in proteomics, macromolecular crystallization and high resolution imaging at different levels set the scene for even greater advances. Pharmacology combined with structural biology of membrane proteins holds great promise for developing innovative accessible therapies for millions robbed of their sight or progressing toward blindness.

Fig. 1. Phylogenetic tree of GPCRs with known X-ray structures

Branches and sub-branches (detailed methods are described elsewhere) of the GPCR phylogenetic tree are shown (adapted from Katritch et al. [25]). Branches of varying thickness represent different subgroups. Known structures from class A (rhodopsin subfamily, light blue branches), class B (secretin subfamily, red branches), and class F (frizzled, green branches) GPCRs are shown, but other groups such as the adhesion and glutamate GPCRs are not included. Structures of known GPCRs obtained in the presence of different ligands are clustered together. Types of ligands are identified by geometrical shapes placed at the top left corner of each individual GPCR structure. Structures with partial agonists are denoted with open triangles, agonists are highlighted with closed triangles, antagonists are identified by open squares, partial inverse agonists are marked by closed squares and inverse agonists are shown with closed circles. GPCRs bound to different inhibitors are identified by their gene names. B-factors for each individual GPCR structure were used to generate the GPCR rainbow color coding (blue through red, for minimum and maximum B-factor values, respectively). The gene names of the clustered structures and their corresponding PDB codes are: RHO (1U19 and 3CAP); ADRB1 (2VT4, 2Y02, and 2Y01); ADRB2 (2R4R and 2RH1); HTR2B (4IB4); HTR1B (4IAQ); CHRM3 (4DAJ); CHRM2 (3UON); DRD3 (3PBL); HRH1 (3RZE); F2R (3VW7); CXCR4 (3ODU); OPRD1 (4EJ4); OPRM1 (4DKL); OPRK1 (4DJH); OPRL1 (4EA3); NTSR1 (4GRV); ADORA2A (3QAK; 3VGA), and 3EML); EDG1 (3V2W); CRF1R (4K5Y); GCGR (4L6R); SMOH (4JKV).

Fig. 2. Structures of rhodopsin, opsin and activated intermediates determined by X-ray crystallography

Color coding of the shown structures was derived from B- factors of the αC atoms as noted in Fig. 1. Ground state rhodopsin (with bound 11-cis-retinal depicted as a red sphere) is activated by a photon of light. Prepared under different experimental conditions, presented GPCR structures were superimposed on ground state rhodopsin and generated using PDB codes: 1U19, 3C9l, 1GZM, 2I35, and 2I36. Photoisomerization of 11-cis-retinal to all-trans-retinal (shown as orange spheres) is accompanied by conformational changes in the transmembrane helices together with a variety of local environmental changes (such as rearrangement of the internal water molecular network). Some of these changes are reflected in B-factor values. The Meta II structure (PDB code 3PXO) contains the all-trans-retinal chromophore. A potential structure of the Meta II-G_t complex is shown in dark grey and light grey shades and indicated by red arrows. The structures of both opsin* and opsin* with bound G_t peptide lack a visual chromophore (PDB codes 3CAP (in gray cartoons because the B-factors for this structure are incorrect) and 3DQB, respectively) and both can be converted to a Meta II state [14]) in the presence of all-trans-retinal (orange sticks) (black arrows, PDB code 3PQR). Opsin structures are not apo-receptors, because they contained bound detergent molecules (depicted in red) [90]. The G_t peptide is represented as grey surface. However, addition of 11-cis-retinal (red sticks) to opsin* crystals cannot successfully regenerate ground state rhodopsin (red arrow). RPE65 (4F3D), a component of the visual cycle, is shown as a dark gray cartoon on a light gray background denoting the retinal pigmented epithelium (RPE). Recycling of all-trans-retinal to 11-cis-retinal through the visual cycle is indicated by gray arrows. Rhodopsin has propensity to oligomerize in both native and expression systems [–93].

Fig. 3. The β₂-adrenergic-G_s complex modeled into a phospholipid membrane bilayer

The β₂-adrenergic receptor in complex with G_s (PDB code: 3SN6) is inserted in a lipid bilayer. The phospholipid units (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) of the lipid bilayer are depicted as gray spheres and the model has been described in detail elsewhere [51]. The position of the β₂-adrenergic transmembrane domain (light blue cartoon) along the z axis of the phospholipid bilayer was chosen by superposition with rhodopsin (not shown). The position of rhodopsin (PDB code: 1U19) in turn is presented such that the palmitoyl fatty acyl chains attached to residues Cys322 and Cys323 are positioned at the same depth as the phospholipid fatty acyl chains of the membrane leaflet. Both the Nb35 nanobody and T4 lysozyme [49] are manifested as light gray structures. The G_s domains are represented as follows: G_sα and G_sγ are shown as cyan cartoons whereas G_sβ is pictured as a blue cartoon. Amino acid residues (Asn121 to Leu153) of the G_sα domain that overlap with the phospholipid head group region of the bilayer model are shown as a red cartoon. The face-on view of the transmembrane region is presented as a z-plane section of the phospholipid bilayer.

Fig. 4. Orientation of GPCR-G protein complexes in a phospholipid membrane

Semi-empirical models of the complex formed between light-activated rhodopsin dimer and a G_t heterotrimer (A and B) [52] and the T4L-β₂AR-G_s-nanobody complex (C) [48] were fitted into a 3D molecular envelope calculated from projections of negatively stained, bis[succinimidyl] 2,2,4,4-glutarate (DSG) crosslinked rhodopsin*-G_t complexes purified in lauryl maltose neopentyl glycol (LMNG). Fitting of the rhodopsin*-G_t model generated with the structure of inactive G_t (PDB code: 1GOT) leaves a significant unoccupied density above one of the rhodopsin molecules (A), which becomes occupied after a 30^o hinge-like motion of the α-helical domain is applied (B). Though fitting the T4L-β₂-adrenergic receptor-G_s-nanobody structure into our EM 3D map leaves sufficient space to accommodate a second molecule of this receptor, conformation of the G_sα helical domain is inconsistent with our EM-density (C). Thus, the favored structure of the rhodopsin-G_t complex appears to be that shown in (B). Photoactivated rhodopsin (Rho*) that binds the C-terminal peptide derived from G_tα and the T4L-β₂-adrenergic receptor-nanobody molecule are both depicted in yellow. The second rhodopsin molecule in (A) and (B) is shown in gray. G_tα, G_tβ, G_tγ are colored pink, dark cyan and dark blue, respectively.