Supramolecular structure of opsins

Chapter 5

Supramolecular structure of opsins Beata Jastrzebska1, Joseph T. Ortega1 and Paul S.-H. Park2 1

Department of Pharmacology, Case Western Reserve University, Cleveland, OH, United States; 2Department of Ophthalmology and Visual Sciences,

Case Western Reserve University, Cleveland, OH, United States

5.1 Introduction Vision is initiated in the retina by G protein-coupled receptors (GPCRs) called opsins. Two major classes of opsins are found in the vertebrate retina, rhodopsin and cone opsins. All opsins exhibit the characteristic seven a-helical transmembrane architecture of GPCRs and bind the retinoid 11-cis-retinal, which isomerizes to all-trans-retinal upon absorption of a photon of light. The spectral properties of opsins are determined by the protein environment surrounding the bound chromophore. Our understanding about the structure and function of rhodopsin far exceeds that of cone opsins due to the relative abundance of rhodopsin in vertebrate retinas compared to cone opsins. Our molecular and structural concept of rhodopsin has advanced significantly since the early days of research on this receptor, and it is now believed that the receptor is packed into photoreceptor cell membranes as oligomers. In this chapter, we present the progression of the notion of rhodopsin as an oligomer and our current understanding about the structure and function of this supramolecular organization present in photoreceptor cell membranes. Also discussed is our emerging understanding about the oligomerization of cone opsins.

5.2 Rhodopsin oligomerization: a historical perspective Rhodopsin is the light GPCR in rod photoreceptor cells of the retina responsible for scotopic vision. Photoreceptor cells are compartmentalized cells featuring an inner segment and an outer segment (Fig. 5.1A). Rhodopsin is synthesized in the inner segment and must be transported to the outer segment via the connecting cilium (Sung and Chuang, 2010; Insinna and Besharse, 2008; Wang and Deretic, 2014; Nemet et al., 2015; Goldberg et al., 2016). Rhodopsin consists of the apoprotein opsin bound covalently to the inverse agonist 11-cis-retinal. Rhodopsin is incorporated at high concentrations into discs in the outer segment of the photoreceptor cells, which are double lamellar membranes circumscribed by a rim region. It is in these discs that rhodopsin initiates phototransduction, a prototypical G protein-mediated signaling cascade and the initial step of vision (Park, 2014; Jastrzebska, 2013). Structural information about rhodopsin has always been abundant comparatively to other GPCRs. This, in large part, can be attributed to the abundance of rhodopsin in photoreceptor cells and the ability to spectroscopically monitor structural changes in the receptor because of a bound chromophore. Studies on rhodopsin span well over a century (Fig. 5.2). Much of the early studies were conducted prior to a proper understanding about the molecular identity of rhodopsin. Initial studies of rhodopsin were conducted based on its color, which gave rod photoreceptor cells a reddish color and may have first been noted by Heinrich Müller in 1851 (Boll, 1977; Kuhne, 1977). In 1876, Franz Boll noted that the reddish color originating from rhodopsin in photoreceptor cells could be eliminated by light and Friedrich Wilhelm Kühne demonstrated that this effect of light on rhodopsin was photochemical in nature and he was able to extract this pigment with bile salts (Boll, 1977; Kuhne, 1977). By 1920, Selig Hecht suggested that this photochemical reaction initiated by light is a bimolecular process, where rhodopsin is reversibly broken down into two components, and George Wald in 1935 suggested that rhodopsin was a combination of a protein conjugated to a vitamin A derivative (Hecht, 1920a, b; Wald, 1935). Structural information about rhodopsin would only begin to emerge in the early 1980s with the amino acid sequencing of rhodopsin, which would indicate rhodopsin formed seven a-helical transmembrane domains (Hargrave et al., 1983;

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(A)

(B)

FIGURE 5.1 Supramolecular organization of rhodopsin in disc membranes of photoreceptor cells. (A) A cartoon of a rod photoreceptor cell comprising an inner segment and an outer segment. Rhodopsin is packed into disc membranes of the outer segment, where it forms nanodomains of the oligomeric receptor. (B) A model of a rhodopsin oligomer (PDB ID: 1N3M) superimposed on an AFM image of rhodopsin oligomers in native disc membranes of photoreceptor cells. (A). This figure is adapted from Rakshit, T., Senapati, S., Parmar, V.M., Sahu, B., Maeda, A., Park, P.S., 2017. Adaptations in rod outer segment disc membranes in response to environmental lighting conditions. Biochim. Biophys. Acta 1864 1691e1702. (B). This figure is adapted from Park, P.S., Lodowski, D.T., Palczewski, K., 2008b. Activation of G protein-coupled receptors: beyond two-state models and tertiary conformational changes. Annu. Rev. Pharmacol. Toxicol. 48 107e141.

FIGURE 5.2 Evolution of our molecular and structural concept of rhodopsin. A timeline is presented, noting major advancements in our molecular and structural understanding about rhodopsin.

Ovchinnikov Yu, 1982). Low-resolution tertiary structural information revealing the 3D arrangement of the seven a-helical transmembrane domains began emerging in the mid-1990s from projection maps generated by cryo-electron microscopy (cryo-EM) of 2D-crystals of rhodopsin (Unger et al., 1997; Schertler et al., 1993). The first high-resolution tertiary structure of rhodopsin came in 2000 from X-ray crystallography of rhodopsin crystals (Palczewski et al., 2000). This would be the first crystal structure of a GPCR. Crystal structures of rhodopsin in various conformational states would become available several years later (Nakamichi and Okada, 2006a, b; Ruprecht et al., 2004; Salom et al., 2006; Park et al., 2008a; Scheerer et al., 2008; Choe et al., 2011; Kang et al., 2015). The first crystal structure of a GPCR other than rhodopsin would not be solved until 2007, and was that of the b2 adrenergic receptor (Rasmussen et al., 2007; Cherezov et al., 2007). Rhodopsin, like other GPCRs, has been historically considered to exist in the membranes of photoreceptor cells as freely diffusing monomers. This view for rhodopsin resulted, in part, from measurements of rapid lateral and rotational

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diffusion constants for rhodopsin in the membrane that were in apparent disagreement with oligomeric forms of rhodopsin (Liebman and Entine, 1974; Cone, 1972; Poo and Cone, 1974; Wey et al., 1981). Biochemical and biophysical evidence that GPCRs can form dimers or larger oligomers began to accumulate near the turn of the 21st century (Salahpour et al., 2000; Milligan, 2001; Gomes et al., 2001; George et al., 2002; Gazi et al., 2002). Visual evidence that rhodopsin forms oligomers in native photoreceptor cell membranes became possible by atomic force microscopy (AFM), which provided direct evidence that rhodopsin forms oligomers in the form of rows of dimers in the native disc membranes of photoreceptor cells (Fig. 5.1B) (Fotiadis et al., 2003; Liang et al., 2003). This quaternary organization was later confirmed by cryoEM of sectioned rod outer segments of photoreceptor cells and by determination of a low-resolution electron microscopy (EM) structure of a rhodopsin dimer in complex with its G protein transducin (Gt) from single particle reconstructions (Gunkel et al., 2015; Jastrzebska et al., 2011, 2013b). Our molecular and structural understanding of rhodopsin has advanced significantly since the early days of research (Fig. 5.2). The current understanding of the quaternary organization of rhodopsin within disc membranes of photoreceptor cells is discussed here.

5.3 Supramolecular organization of rhodopsin in photoreceptor cells 5.3.1 Visualization of rhodopsin organization in photoreceptor cell membranes The visualization of rhodopsin oligomers within native photoreceptor cell membranes was first made possible by AFM. AFM is uniquely suited to visualize the organization of membrane proteins within biological membranes in physiologically relevant buffer conditions (Whited and Park, 2014; Muller, 2008; Engel and Gaub, 2008). In initial AFM studies, two types of packing arrangements of rhodopsin were observed in the disc membranes of photoreceptor cells (Fotiadis et al., 2003; Liang et al., 2003). The first was a densely packed paracrystalline lattice and the second a less densely packed nanodomain organization. Regardless, in both packing arrangements, rhodopsin formed rows of dimers of variable lengths (Fig. 5.1B). Subsequent AFM studies have revealed the nanodomain organization of rhodopsin to be the likely physiological arrangement. AFM images of rhodopsin arranged as oligomers were in apparent conflict with the previously held belief that rhodopsin exists as monomers in nature and several arguments were raised in opposition to this updated view (Chabre et al., 2003; Chabre and le Maire, 2005). Subsequent AFM and cryo-EM studies have addressed these concerns and ruled out possible artifacts as a cause of the observed supramolecular organization of rhodopsin. The densely packed paracrystalline lattice arrangement of rhodopsin initially observed in AFM images does not appear to be a physiological arrangement as subsequent AFM studies consistently show a nanodomain organization of rhodopsin (Liang et al., 2003, 2004; Fotiadis et al., 2004; Buzhynskyy et al., 2011; Whited and Park, 2015). Discs examined by AFM are isolated from the outer segment of photoreceptor cells, and the structure can be somewhat disrupted, raising a concern that observations by AFM do not reflect what is present in intact photoreceptor cells. Artifacts associated with adsorption of samples on a mica substrate and lateral forces associated with AFM imaging have been ruled out (Rakshit and Park, 2015; Rakshit et al., 2015). Cryo-EM studies on intact or sectioned outer segments of photoreceptor cells have also revealed a nanodomain organization of rhodopsin, where the receptor forms oligomeric complexes arranged as rows of dimers (Nickell et al., 2007; Gunkel et al., 2015). Thus, the nanodomain organization of oligomeric rhodopsin does not appear to be an artifact related to the AFM procedures. A major argument against the oligomeric organization of rhodopsin observed by AFM is that samples are prepared at temperatures below body temperature, which can induce phase separation of lipids in disc membranes and potentially promote the observed supramolecular structure of rhodopsin (Chabre et al., 2003). Phase separation of lipids in disc membranes is only expected to occur in photoreceptor cells of warm-blooded mammalian species and not in cold-blooded vertebrates like frogs (Chabre, 1975). The same nanodomain organization of rhodopsin observed in mammalian species is observed in the cold-blooded X. laevis (Rakshit et al., 2015), which rules out phase separation as a cause of the observed supramolecular organization of rhodopsin. Another major argument against the observed oligomeric organization of rhodopsin is that this type of organization is in apparent disagreement with previously reported lateral diffusion constants of rhodopsin within the membrane of discs (Chabre, 1975). Lateral diffusion constants have been reported within the range of 0.1e0.6 mm2/s (Liebman and Entine, 1974; Poo and Cone, 1973, 1974; Wey et al., 1981; Wang et al., 2008; Gupta and Williams, 1990; Drzymala et al., 1984; Govardovskii et al., 2009; Najafi et al., 2012). The more recent estimates of the lateral diffusion constant are at the lower end of this range (e.g. (Najafi et al., 2012),). The observed size of rhodopsin nanodomains does not necessarily preclude lateral diffusion rates of about 0.1 mm2/s. The diameter of the rhodopsin nanodomains that are most frequently observed is about 20 nm, and the average diameter is about 35 nm (Rakshit and Park, 2015; Rakshit et al., 2017). Lipid rafts of similar

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size can exhibit lateral diffusion rates similar to those reported for rhodopsin (Cicuta et al., 2007). If rhodopsin nanodomains diffuse similarly as lipid rafts, then the measured diffusion rates may not be contradictory to the supramolecular structure of rhodopsin. Taken together, the supramolecular organization of rhodopsin as oligomers that form nanodomains within the disc membrane of photoreceptor cells appears to represent the physiological organization of the light receptor. This type of supramolecular organization is conserved among vertebrates (Whited and Park, 2015; Rakshit et al., 2015; Buzhynskyy et al., 2011; Liang et al., 2004).

5.3.2 Structure of the rhodopsin-Gt complex Numerous ongoing efforts have been directed at obtaining structural information about the complex between active GPCRs and their respective G proteins to better understand the underlying mechanism of action of these receptors. Major questions in these efforts include: What is the receptor-G protein binding stoichiometry? What is the overall interaction interface between the receptor and G protein? What distinct conformational changes relay information from the receptor to the G protein? Although there is currently no high-resolution structure of the complex between rhodopsin and its cognate G protein Gt, numerous studies have accumulated over the years to provide insights related to questions raised above. Early evidence in support of Gt binding to oligomeric rhodopsin came from binding studies that revealed positive cooperativity in the binding of Gt to rhodopsin in rod outer segment membranes with Hill coefficients of about 2 (Wessling-Resnick and Johnson, 1987; Willardson et al., 1993), which suggested that Gt binds to an oligomer with at least two molecules of rhodopsin. The discovery by AFM that rhodopsin forms rows of dimers in native disc membranes led to the generation of a computational model of the rhodopsin-Gt complex (Filipek et al., 2004). This model indicated that at least two rhodopsin molecules are necessary to bind the alpha subunit of Gt, but the coupling of the heterotrimeric Gt may require a rhodopsin tetramer. A structure of the rhodopsin-Gt complex would be needed to confirm these earlier predictions of the stoichiometry and binding determinants within the signaling complex. The rhodopsin-Gt complex is highly dynamic and short-lived in the native cellular environment. Thus, structure determination has been hampered because of multiple challenges, including obtaining a homogenous and stable preparation. Various strategies for the purification and stabilization of this transient rhodopsin-Gt complex have been developed. For example, in one strategy, complex formation was promoted directly within rod outer segment membranes followed by extensive washing of the membrane to reduce the concentration of released GDP in order to form nucleotide-free rhodopsin-Gt complex (Jastrzebska et al., 2009), which is stable for days (Bornancin et al., 1989). The stability of the nucleotide-free rhodopsin-Gt complex allowed the purification of the complex by sucrose gradient centrifugation of detergent-solubilized membranes. In another strategy, rhodopsin was solubilized from rod outer segment membranes, loaded onto a succinylated concanavalin A affinity resin, photoactivated, and then saturated with purified Gt to form the rhodopsin-Gt complex on the column (Jastrzebska et al., 2011, 2013b). This complex was eluted from the affinity column, chemically cross-linked to stabilize the complex, and further purified by size exclusion chromatography. Although a preparation suitable for X-ray crystallography has yet to be achieved, the strategies employed to purify stable rhodopsin-Gt complexes have led to the determination of low-resolution structures. In 2011, the first low-resolution map of the rhodopsin-Gt complex was obtained based on single particle EM of the signaling complex purified from the native bovine retina (Jastrzebska et al., 2011). The 3D-reconstruction from w12,000 negatively stained particles generated a contour map at 21.6 Å resolution (Fig. 5.3A). Knowledge from biochemical studies and X-ray crystal structures of rhodopsin and Gt were used to achieve the best geometric and biochemical fit of a rhodopsin dimer and heterotrimeric Gt into the EM-derived molecular envelope (Fig. 5.3B). Several lines of evidence from single particle EM studies support the notion that a dimer of rhodopsin forms a complex with a single heterotrimeric Gt. The size of the contour map generated from single particle EM was too large for only a single rhodopsin molecule but was sufficient for a rhodopsin dimer coupled to a single heterotrimeric Gt, suggesting a 2:1 stoichiometry of rhodopsin and Gt (Jastrzebska et al., 2011). Analysis of unstained rhodopsin-Gt complex particles by low-dose dark field scanning transmission EM revealed a particle mass of 221 kDa, which is the expected mass for a 2:1 stoichiometry of rhodopsin and Gt (Jastrzebska et al., 2011). In another single particle EM study where rhodopsin in a rhodopsin-Gt complex was labeled with succinylated concanavalin A, a lectin that binds to mannose in glycosylated rhodopsin, two elongated densities corresponding to succinylated concanavalin A were present in 2D projections (Jastrzebska et al., 2013b), indicating the presence of two rhodopsin molecules within the rhodopsin-Gt complex. In order to more fully understand the mechanism of rhodopsin-Gt signaling, higher resolution structures of the signaling complex are required but currently unavailable. A 4.5 Å resolution cryo-EM structure of a rhodopsin-Gi complex has been determined (Kang et al., 2018). In contrast to previous lower resolution structures, only a single rhodopsin molecule bound to the inhibitory G protein, Gi, was revealed in this structure. This difference likely derives from the fact that the previous

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FIGURE 5.3 Visualization and 3D reconstruction of the rhodopsin-Gt complex. (A) Negatively stained particles of the rhodopsin-Gt complex (bottom panel) and 2D class averages obtained from manually selected particles. Scale bar, 100Å. (B) 3D map calculated from particle projections and fitted derived model of rhodopsin dimer coupled to the Gt heterotrimer. Scale bar, 20 Å. In the rhodopsin-Gt complex the activated rhodopsin monomer is shown in yellow, and the second monomer is shown in light green. Gt subunits, a, b, and g are colored red, blue, and dark green, respectively. (B). This figure was generated from the data presented in Jastrzebska, B., Ringler, P., Palczewski, K., Engel, A., 2013b. The rhodopsin-transducin complex houses two distinct rhodopsin molecules. J. Struct. Biol. 182 164e172.

complex was isolated from native photoreceptor cells in the retina, whereas this complex was isolated from a heterologous expression system. Moreover, the rhodopsin in the complex is that of a constitutively active mutant, and the G protein is not the native Gt. Similarly, other structures of GPCR-G protein complexes also exhibit a 1:1 stoichiometry between the receptor and G protein. A crystal structure is available for the b2 adrenergic receptor-Gs complex, and several cryo-EM structures are available for various GPCR-G protein complexes (Rasmussen et al., 2011; Zhang et al., 2017; Liang et al., 2017; Garcia-Nafria et al., 2018a, b). In all cases, the structures contain only a single receptor molecule coupled to the G protein. None of these complexes were isolated from natural sources but instead were heterologously expressed, and the receptor and G protein modified to achieve stable and homogeneous preparations. Although structures of a 1:1 complex between receptor and G protein provide some insights about signal transmission, higher resolution structures of the signaling complex from native tissue with a physiologically relevant stoichiometry will be required.

5.3.3 Determinants of rhodopsin supramolecular structure Some of the determinants of the supramolecular structure formed by rhodopsin are beginning to be understood, although much work is still required. Quantitative analysis of AFM images of disc membranes from the photoreceptor cells has revealed that the size of nanodomains formed by rhodopsin, which reflects the size of rhodopsin oligomers, increases with higher concentrations of rhodopsin within the membrane (Rakshit and Park, 2015). A concentration-dependent oligomerization of rhodopsin is also supported by Förster resonance energy transfer (FRET) spectrometry and pulse-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) studies on heterologously expressed rhodopsin tagged with fluorescent proteins (Mishra et al., 2016; Comar et al., 2014). In these studies, an equilibrium of oligomeric forms of rhodopsin was detected, and the size of the oligomers increased with higher concentrations of the receptor. The heterologous expression system produces rhodopsin at lower concentrations compared to native photoreceptor cells, which results in the formation of mainly tetramers in the former and mainly 24-mers in photoreceptor cells (Mishra et al., 2016; Rakshit and Park, 2015). Thus, oligomerization of rhodopsin appears to be a concentration-dependent process where an equilibrium of oligomeric forms exists, and the size of oligomers formed by rhodopsin depends on the concentration of the receptor. Concentration-dependent oligomerization appears to not only underlie the oligomerization of rhodopsin but of other GPCRs as well (Kasai et al., 2011; Hern et al., 2010; Patowary et al., 2013; Calebiro et al., 2013; Ward et al., 2015). Rhodopsin, like other GPCRs, is embedded in the membrane. The lipid composition of membranes can have an impact on the oligomerization of membrane proteins (Gupta et al., 2017). Likewise, the lipids in the membrane appear to play some role in the oligomeric status of rhodopsin. Extraction of rhodopsin from native photoreceptor cell membranes by the detergents n-dodecyl-b-D-maltoside, n-tetradecyl-b-D-maltoside, and n-hexadecyl-b-D-maltoside, results in the preservation of different oligomeric forms of rhodopsin and different levels of coextracted lipids (Jastrzebska et al., 2006). The detergent n-dodecyl-b-D-maltoside extracts the least amount of lipids and results in the disruption of rhodopsin oligomers into dimers and monomers. Similar results are observed in heterologous expression systems where rhodopsin exists as monomers when extracted by this detergent (Ernst et al., 2007; Gragg et al., 2016). The detergent n-tetradecyl-b-Dmaltoside can extract a greater amount of lipids resulting in the extraction of larger oligomers from native photoreceptor

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cell membranes and the detergent n-hexadecyl-b-D-maltoside extracts the greatest amount of lipids resulting in the extraction of even larger oligomers of rhodopsin in the form of the rows of dimers observed in AFM images (Jastrzebska et al., 2006). Detergent-solubilized and purified monomeric rhodopsin can self-associate when reconstituted into lipid vesicles (Mansoor et al., 2006; Ploier et al., 2016). Thus, lipids and the membrane appear to be required to stabilize and promote the oligomerization of rhodopsin. It is unclear whether or not specific lipids modulate the oligomerization of rhodopsin. Disc membranes of photoreceptor cells have a unique lipid composition that is high in docosahexaenoic acid (DHA) (Boesze-Battaglia and Albert, 1989). DHA in the membrane is predicted to impact the quaternary structure of rhodopsin-based on in vitro studies in artificial lipid vesicles (Periole et al., 2007; Botelho et al., 2006). However, AFM studies on disc membranes and FRET studies on HEK293 cells heterologously expressing tagged rhodopsin demonstrated that the oligomeric status of rhodopsin in native photoreceptor cells and HEK293 cells is unaltered by changes in the level of DHA in the membrane (Senapati et al., 2018). While the oligomeric status of rhodopsin may be independent of DHA in the membrane, the kinetics of oligomerization may be altered, an observation made with other GPCRs (Guixa-Gonzalez et al., 2016). A high-resolution structure of oligomeric rhodopsin is currently unavailable. Thus, the assignment of rhodopsinrhodopsin association interfaces is based on indirect observations. The current working model of a rhodopsin oligomer is that derived computationally based on the geometric constraints observed in AFM images of rod outer segment disc membranes (Liang et al., 2003; Fotiadis et al., 2004). This model highlights two possible dimer interfaces: one involving transmembrane helices TM4 and TM5 in an intradimeric interface and another involving transmembrane helices TM1 and TM2 and the amphipathic helix H8 in an interdimeric interface between adjacent rows of dimers (Fig. 5.4). Although this model should be updated in the future with higher-resolution structural information, current studies are in general agreement with this model. Earlier structural studies are consistent with the two dimeric interfaces observed in the computational working model. The dimeric organization of dark state rhodopsin for the first time was observed in 2D crystals of the receptor isolated from bovine and frog retinas (Schertler et al., 1993; Schertler and Hargrave, 1995). The dimeric interface in the crystals involved

FIGURE 5.4 Rhodopsin dimer interacting interfaces. Arrangement of rhodopsin dimers in a model derived from geometric constraints in AFM images of rhodopsin in rod outer segment disc membranes (PDB ID: 1N3M). Two possible dimerization interfaces are highlighted. A dimer interface involving TM4/TM5 is shown in blue, and the dimer interface involving TM1/TM2/H8 is shown in green.

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TM1 and H8 of each molecule. Similar dimers were also observed in 2D crystals of photoactivated rhodopsin in the metarhodopsin I conformation (Ruprecht et al., 2004). The first 3D crystal structure of dark state rhodopsin revealed a dimeric association involving TM1; however, unlike in 2D crystals, the dimer was in a nonphysiological antiparallel orientation (Palczewski et al., 2000). Parallel dimers with similar contacts between TM1, TM2, and H8, as in the initial 2D crystals, was found later in the 3D crystal structures of photoactivated bovine rhodopsin (Salom et al., 2006; Park et al., 2006). The 3D crystal structure solved for squid rhodopsin revealed another dimer interface between TM4 and TM5 (Enami et al., 2006). Although rhodopsin packed into crystals do not necessarily represent a physiological scenario, cumulatively, the solved structures agree with the possibility of rhodopsin associating via at least two interfaces. In addition to structural studies, the working model of oligomeric rhodopsin has been validated by a variety of biochemical, biophysical, and computational studies. Chemical cross-linking studies indicate both TM4/TM5 and TM1/H8 dimer interfaces. Heterologously expressed cysteine-modified rhodopsin revealed cross-linking at Trp175Cys present in the extracellular loop connecting TM4 and TM5 and Tyr206Cys located on the extracellular part of TM5 (Kota et al., 2006). Cross-linking of endogenous cysteine residues in rhodopsin from native rod outer segments followed by partial proteolysis and mass-spectrometry indicated the presence of a TM1/H8 dimer interface (Knepp et al., 2012). These two dimer interfaces also emerge in self-assembly coarse-grained molecular dynamic simulations (Periole et al., 2012). Disruption of rhodopsin oligomers has been demonstrated by in vitro competition experiments using peptides corresponding to the transmembrane helices in rhodopsin. Synthetic peptides corresponding to TM1, TM2, TM4, and TM5 were able to disrupt rhodopsin oligomerization using several experimental approaches including size exclusion chromatography, rhodopsin cross-linking within rod outer segment disc membranes, and bioluminescence energy resonance transfer in HEK293 cells stably expressing rod opsin (Jastrzebska et al., 2015). Consistent with in vitro studies, peptide competition studies conducted in vivo in mice also demonstrated the ability to disrupt rhodopsin oligomerization. Peptides corresponding to TM1 and H8 appeared to disrupt rhodopsin oligomerization; however, peptides corresponding to TM4 and TM5 appeared to have no effect (Zhang et al., 2016). In contrast to in vitro studies, disruption of rhodopsin oligomerization in vivo was only assessed indirectly by examining the ability of peptides to cause rhodopsin to mislocalize. Thus, dimerization via the TM1/TM2/H8 dimer interface may be a requirement for proper trafficking, whereas dimerization via the TM4/TM5 interface is not.

5.3.4 Modulation of rhodopsin packing in photoreceptor cells The outer segment of photoreceptor cells is a dynamic structure. The outer segment contains up to 500e2000 discs stacked on top of each other that are continually renewed (Daemen, 1973) (Fig. 5.1A). Rhodopsin is incorporated into the discs that are continuously formed at the base of the outer segment whereas old discs at the distal end of the outer segment are phagocytosed daily by retinal pigment epithelial cells (Young, 1967; Ding et al., 2015; Volland et al., 2015). While this type of dynamic behavior has been appreciated for some time, less is known about the plasticity present in the packing of rhodopsin within the disc membranes. Rod photoreceptor cells are exquisitely sensitive and capable of generating a response to a single photon of light (Baylor et al., 1979). As the initiator of phototransduction, rhodopsin is an important contributor to the sensitivity displayed by rod photoreceptor cells. It has been proposed previously based on computational studies that rhodopsin is packed into disc membranes at a density that is optimal for signaling (Saxton and Owicki, 1989). The notion of an optimal packing density of rhodopsin is supported experimentally by quantitative AFM studies. A correlation is observed between the size of the discs and the number of rhodopsin molecules packed into the disc, yet no correlation was observed between the size of the disc and the density of rhodopsin within the membrane (Whited and Park, 2015). These observations suggested that rod photoreceptor cells adjust the size of discs to accommodate different numbers of rhodopsin being packed into the membrane to maintain an optimal packing density. Studies on heterozygous rhodopsin knockout mice, which express half the amount of rhodopsin as wild-type mice, confirmed this idea, as the reduction in rhodopsin expression resulted in smaller discs but maintained the same rhodopsin density within the membrane (Rakshit and Park, 2015). The adaptations of rod photoreceptor cells to maintain an optimal packing density of rhodopsin appears to be related to the signaling capacity of the photoreceptor cells. This concept is related to the proposed photostasis phenomenon, which posits that photoreceptor cells adapt to maintain an optimal photon catch capability (Penn and Williams, 1986). Although earlier studies focused on the dimension of the outer segment of rod photoreceptor cells, which appeared to change size depending on the lighting environment (Penn and Williams, 1986; Cunea et al., 2013), more recent studies have indicated that the packing density of rhodopsin in the disc membranes also changes in response to different lighting environments (Rakshit et al., 2017). When photons are scarce, the density of rhodopsin increases and improves visual function as assessed by electroretinography. Thus, the optimal density of rhodopsin for signaling may not occur under normal cyclic

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lighting conditions. The signal for this adaptation requires phototransduction and inhibition of phototransduction by either genetic or environmental means can cause the same adaptations (Rakshit et al., 2017; Senapati et al., 2018). The adaptations of photoreceptor cells that change the density of rhodopsin within the membrane of discs do not alter the most frequently observed size of oligomers (i.e., 24-mer), but rather, can change the level of a minor population of larger oligomers (Rakshit et al., 2017).

5.3.5 Pathogenic quaternary structure of rhodopsin While properly folded rhodopsin form oligomers natively, another type of quaternary structure exists for misfolded rhodopsin molecules. When rhodopsin is misfolded, the receptor is retained in the endoplasmic reticulum and forms toxic aggregates (Illing et al., 2002; Saliba et al., 2002). A majority of the mutations in rhodopsin that cause retinitis pigmentosa, a progressive retinal degenerative disease, have been shown to result in misfolding and aggregation of the receptor (Athanasiou et al., 2018). The toxic rhodopsin aggregates are thought to play a role in causing photoreceptor cell death (Athanasiou et al., 2012; Vasireddy et al., 2011; Gorbatyuk et al., 2010). The interactions in rhodopsin oligomers and those in rhodopsin aggregates are fundamentally different. Misfolding mutants have an altered secondary structure that results in a decrease in a-helical structure and an increase in b-sheet structure (Miller et al., 2015; Liu et al., 1996). There is specificity in the aggregation of misfolded rhodopsin molecules as misfolded mutants do not aggregate or oligomerize with properly folded rhodopsin (Gragg et al., 2016; Gragg and Park, 2018). Relatively little is known about the nature of aggregates formed by misfolded rhodopsin molecules in contrast to rhodopsin oligomerization, and therefore, more structural interrogation of rhodopsin aggregates is necessary.

5.4 Functional role of rhodopsin supramolecular structure 5.4.1 Signaling efficiency within a crowded membrane environment Rhodopsin is densely packed in the disc membranes of photoreceptor cells. The light receptor represents greater than 90% of all proteins in the disc membranes (Papermaster and Dreyer, 1974). It is packed at a density of about 20,000 mm 2 and occupies about a quarter of the surface area of the disc membrane (Rakshit and Park, 2015; Whited and Park, 2015; Gunkel et al., 2015). The density of rhodopsin present in native photoreceptor cell membranes is orders of magnitude higher than that of typical GPCRs in their native membranes, with receptor density estimates of 5e30 mm 2 (Herrick-Davis et al., 2015; Hegener et al., 2004). Signaling mediated by rhodopsin must be efficient to facilitate the exquisite sensitivity of photoreceptor cells. The high concentrations of rhodopsin in the disc membrane may be beneficial for photoreceptor cell function by maximizing the probability of photon capture; however, they may be detrimental for diffusion-mediated signaling events that occur in phototransduction. The supramolecular organization of rhodopsin within the disc membranes may provide the necessary order and structure to counteract the impediments of a crowded environment and facilitate the efficiency and sensitivity required for the photoreceptor cell response (Cangiano and Dell’Orco, 2013; Dell’Orco, 2013). Computational studies demonstrate that the supramolecular organization of rhodopsin is necessary to facilitate efficient signaling that would otherwise impede diffusion-mediated signaling events (Gunkel et al., 2015; Dell’Orco and Schmidt, 2008; Schoneberg et al., 2014; Ramirez and Leidy, 2018). The supramolecular organization of rhodopsin can provide a platform for transient precoupling of rhodopsin with Gt prior to activation and provide the platform for positive cooperativity observed in the binding of Gt to activated rhodopsin (Dell’Orco and Koch, 2011; Park et al., 2004; Wessling-Resnick and Johnson, 1987; Willardson et al., 1993). Although monomeric rhodopsin can itself signal to Gt (Bayburt et al., 2007; Ernst et al., 2007; Whorton et al., 2008; Banerjee et al., 2008), oligomeric rhodopsin in native disc membranes appears to provide an appropriate scaffold for efficient and sensitive signaling in the midst of a crowded membrane environment.

5.4.2 Asymmetric function e rhodopsin-Gt and rhodopsin-arrestin complexes The oligomerization of rhodopsin provides the possibility of allosteric communication between rhodopsin protomers, which can facilitate signaling efficiency and increase the range of available responses by the photoreceptor cell. Molecular dynamics simulations demonstrate asymmetry between rhodopsin molecules in a dimeric complex with the potential for allosteric communication (Filizola et al., 2006). Moreover, molecular dynamics simulations also indicate that the activation of a rhodopsin dimer can proceed in an asymmetric manner where photoactivation of one rhodopsin protomer in the dimer can promote the activation of a second rhodopsin protomer (Neri et al., 2010). This cross-talk allows for a potential tandem

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activation mechanism where one rhodopsin molecule detects light and the other rhodopsin molecule signals to Gt, which can enhance signaling efficiency. Asymmetric activation of rhodopsin within an oligomeric complex is supported by in vivo experiments in mice, which demonstrate cross-phosphorylation between activated and nonactivated rhodopsin in rod outer segment disc membranes (Shi et al., 2005). The EM-derived structure of the rhodopsin-Gt complex shows that each rhodopsin molecule binds asymmetrically to Gt, with only one rhodopsin molecule of the dimer binding to the C-terminus of the alpha subunit of Gt (Fig. 5.3B). This structural asymmetry points to a possible functional asymmetry in the rhodopsin dimer. Functional characterization of the rhodopsin molecules within the rhodopsin-Gt complex have revealed that the functional properties of each rhodopsin protomer in the complex is not equivalent but can adopt distinct active states (Jastrzebska et al., 2013a, b). In the canonical view of rhodopsin signaling, rhodopsin adopts the active metarhodopsin II (MII) state when light causes the isomerization of bound 11-cis-retinal to all-trans-retinal, which is later released from the binding pocket leaving the empty apoprotein opsin (Park, 2014). In the rhodopsin-Gt complex, photoactivation does not lead to these expected events in one of the rhodopsin molecules in the dimer. Upon light activation of the rhodopsin-Gt complex, only one of the rhodopsin protomers in the dimer appears to have gone through the canonical activation cycle, resulting in the formation of the chromophorefree opsin form that could be regenerated with the chromophore. In contrast, the other rhodopsin protomer was still conjugated to all-trans-retinal, presumably stabilized by the direct contact made by this protomer with Gt as this effect required an intact rhodopsin-Gt complex. Thus, the two rhodopsin molecules within the dimer in the signaling complex exhibited different functional properties. Interestingly, when all-trans-retinal was depleted from the binding pocket of the activated signaling complex and regenerated with 11-cis-retinal, the bound chromophore immediately converted to alltrans-retinal in the absence of light activation. This unexpected behavior suggests that the rhodopsin molecule stabilized by Gt increases the rigidity of the receptor, thereby promoting light-independent isomerization. This increase in rigidity promoted by Gt is supported by hydrogen-deuterium exchange studies (Orban et al., 2012). The rhodopsin dimer appears to be functionally asymmetric with one rhodopsin protomer within the dimer remaining active while engaged with Gt and the other rhodopsin protomer being available for regeneration. This asymmetry may allow for signaling efficiency that may not be possible within a signaling complex involving monomeric rhodopsin (Jastrzebska et al., 2013a). Asymmetry is also detected within a rhodopsin dimer when it is bound to arrestin. Photoactivated rhodopsin is desensitized by phosphorylation of its C-terminal tail by rhodopsin kinase and coupling to arrestin, via the same binding interface as Gt, before it decays to opsin and free all-trans-retinal (Szczepek et al., 2014). Binding of arrestin prevents coupling of rhodopsin with Gt and promotes the termination of signaling. The bimodal structure of arrestin presents two potential receptor binding interfaces that can each accommodate a single rhodopsin molecule, which suggests that a rhodopsin dimer can bind a single arrestin molecule (Modzelewska et al., 2006). A high-resolution crystal structure of the rhodopsin-arrestin complex revealed only a 1:1 stoichiometry between rhodopsin and arrestin (Kang et al., 2015). However, this complex was formed with modified rhodopsin heterologously expressed and may not represent the physiological form of the receptor. Indeed, in Fourier transform infrared spectroscopy and pull-down studies involving rhodopsin obtained from native rod outer segment disc membranes, rhodopsin-arrestin complex stoichiometries of 1:1 were observed at low light intensities whereas stoichiometries of 2:1 were observed for rhodopsin and arrestin at higher light intensities (Beyriere et al., 2015; Sommer et al., 2011, 2012). Similarly to the case of the rhodopsin-Gt complex, asymmetry is observed within the rhodopsin dimer in the rhodopsin-arrestin complex. The N-domain lobe of arrestin engages the MII activated form of rhodopsin and can facilitate the binding of all-trans-retinal whereas the C-domain lobe of arrestin binds to the apoprotein opsin that is in an inactive form incapable of binding all-trans-retinal (Sommer et al., 2012; Beyriere et al., 2015). The asymmetry observed within the rhodopsin dimer in the rhodopsin-arrestin complex has been proposed to be a protective mechanism for photoreceptor cells under bright light conditions. In such a scenario, a large flux of all-trans-retinal is expected since all rhodopsin molecules within the photoreceptor cell become activated. The ability to retain some of the isomerized all-trans-retinal within the rhodopsin binding pocket when bound to arrestin would reduce the accumulation of all-trans-retinal and the buildup in the retina of toxic condensation products such as di-retinoidpyridinium-ethanolamine, which can cause retinal degenerative diseases (Maeda et al., 2008; Kim et al., 2007).

5.5 Cone opsin oligomerization In contrast to the oligomerization of rhodopsin, little is known about the oligomeric status of cone opsins. There are three human cone opsins: blue, green, and red cone opsins that correspond to OPN1SW, OPN1MW, and OPN1LW genes, respectively. The cone opsins share about 40% sequence homology with rhodopsin (Stenkamp et al., 2002). Cone opsins are expressed in cone photoreceptor cells and are required for color vision and visual acuity and function under photopic

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FIGURE 5.5 Oligomeric status of cone opsins in the membrane. (A) Based on PIE-FCCS experiments (Jastrzebska et al., 2017), only red cone opsin diffuses in the cell membrane as dimers, whereas blue and green cone opsins diffuse as monomers. (B) Amino acid residues at positions 230, 233, and 236 located in TM5 are involved in the self-association of red cone opsin. Changing these residues in the green cone opsin to the respective residues in the red cone opsin results in the ability to form dimers. Changing these residues in the red cone opsin to the respective residues present in the green cone opsin disrupts dimerization.

conditions. Compared to rod photoreceptor cells, cone photoreceptor cells are much less abundant in the retina, precluding the isolation of high levels of native protein for structural and molecular characterizations and determining the organization of the receptor within the native membrane. Recently, the propensity of heterologously expressed human cone opsins to oligomerize in the membrane of live cells was tested by fluorescence correlation spectroscopy and PIE-FCCS (Jastrzebska et al., 2017). The receptors were fused to either green or red fluorescent protein and heterologously expressed in Cos-7 cells to detect the oligomers of human cone opsins. The measured molecular brightness of fluorescent proteins fused to the cone opsins and computed diffusion coefficients were consistent with the notion that red cone opsin was larger in size compared to either the green or blue cone opsins. Computed cross-correlation functions determined for each receptor by PIE-FCCS indicated that red cone opsin forms dimers, whereas the other two cone opsins were monomeric (Fig. 5.5A). Although blue cone opsin does not appear to dimerize, an in vivo study in a transgenic mouse indirectly indicates that blue cone opsin can associate with rhodopsin (Zhang et al., 2016). Blue cone opsin shares only about 40% sequence similarity with the green or red cone opsins, and the green and red cone opsins differ by only 15 amino acid residues (Nathans et al., 1986). Based on sequence similarity, the green and red cone opsins would be predicted to behave similarly; however, this is not the case. Analysis of the sequence difference between the green and red cone opsins revealed differences in TM5 at residues in positions 230, 233, and 236 (IAM in the red cone opsin and TSV in the green cone opsin) (Fig. 5.5B), indicating that residues at these positions may determine the propensity for dimer formation. The amino acid residues at these three positions in green cone opsin were swapped for those in the red cone opsin and vice versa, which resulted in a reversal in dimeric properties, supporting the idea that these residues in TM5 are a determinant for dimer formation (Jastrzebska et al., 2017). In addition, swapping the amino acid residues at these positions changed the spectral properties of the respective mutants. Although a direct connection between cone opsin dimerization and color tuning has yet to be established, these in vitro studies suggest a potential physiological role of cone opsin dimerization in modulating spectral sensitivity. Many more studies will be required to better understand the oligomeric properties and function of cone opsins.

Acknowledgments This work was supported by grants from the National Institutes of Health (EY025214 to B.J. and EY021731 to P.S.-H.P.).

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