Beata Jastrzebska

Crystal structure of a photoactivated deprotonated intermediate of rhodopsin

The changes that lead to activation of G protein-coupled receptors have not been elucidated at the structural level. In this work we report the crystal structures of both ground state and a photoactivated deprotonated intermediate of bovine rhodopsin at a resolution of 4.15 Å. In the photoactivated state, the Schiff base linking the chromophore and Lys-296 becomes deprotonated, reminiscent of the G protein-activating state, metarhodopsin II. The structures reveal that the changes that accompany photoactivation are smaller than previously predicted for the metarhodopsin II state and include changes on the cytoplasmic surface of rhodopsin that possibly enable the coupling to its cognate G protein, transducin. Furthermore, rhodopsin forms a potentially physiologically relevant dimer interface that involves helices I, II, and 8, and when taken with the prior work that implicates helices IV and V as the physiological dimer interface may account for one of the interfaces of the oligomeric structure of rhodopsin seen in the membrane by atomic force microscopy. The activation and oligomerization models likely extend to the majority of other G protein-coupled receptors.

Efficient coupling of transducin to monomeric rhodopsin in a phospholipid bilayer

G protein-coupled receptors (GPCRs) are seven transmembrane domain proteins that transduce extracellular signals across the plasma membrane and couple to the heterotrimeric family of G proteins. Like most intrinsic membrane proteins, GPCRs are capable of oligomerization, the function of which has only been established for a few different receptor systems. One challenge in understanding the function of oligomers relates to the inability to separate monomeric and oligomeric receptor complexes in membrane environments. Here we report the reconstitution of bovine rhodopsin, a GPCR expressed in the retina, into an apolipoprotein A-I phospholipid particle, derived from high density lipoprotein (HDL). We demonstrate that rhodopsin, when incorporated into these 10 nm reconstituted HDL (rHDL) particles, is monomeric and functional. Rhodopsin·rHDL maintains the appropriate spectral properties with respect to photoactivation and formation of the active form, metarhodopsin II. Additionally, the kinetics of metarhodopsin II decay is similar between rhodopsin in native membranes and rhodopsin in rHDL particles. Photoactivation of monomeric rhodopsin·rHDL also results in the rapid activation of transducin, at a rate that is comparable with that found in native rod outer segments and 20-fold faster than rhodopsin in detergent micelles. These data suggest that monomeric rhodopsin is the minimal functional unit in G protein activation and that oligomerization is not absolutely required for this process.

Structural waters define a functional channel mediating activation of the GPCR, rhodopsin.

Structural water molecules may act as prosthetic groups indispensable for proper protein function. In the case of allosteric activation of G protein-coupled receptors (GPCRs), water likely imparts structural plasticity required for agonist-induced signal transmission. Inspection of structures of GPCR superfamily members reveals the presence of conserved embedded water molecules likely important to GPCR function. Coupling radiolytic hydroxyl radical labeling with rapid H2O18 solvent mixing, we observed no exchange of these structural waters with bulk solvent in either ground state or for the Meta II or opsin states. However, the radiolysis approach permitted labeling of selected side chain residues within the transmembrane helices and revealed activation-induced changes in local structural constraints likely mediated by dynamics of both water and protein. These results suggest both a possible general mechanism for water-dependent communication in family A GPCRs based on structural conservation, and a strategy for probing membrane protein structure.

CacyBP/SIP, a Calcyclin and Siah-1-interacting Protein, Binds EF-hand Proteins of the S100 Family

Recently, a human ortholog of mouse calcyclin (S100A6)-binding protein (CacyBP) called SIP (Siah-1-interacting protein) was shown to be a component of a novel ubiquitinylation pathway regulating β-catenin degradation (Matsuzawa, S., and Reed, J. C. (2001) Mol. Cell 7, 915–926). In murine brain, CacyBP/SIP is expressed at a high level, but S100A6 is expressed at a very low level. Consequently we carried out experiments to determine if CacyBP/SIP binds to other S100 proteins in this tissue. Using CacyBP/SIP affinity chromatography, we found that S100B from the brain extract binds to CacyBP/SIP in a Ca2+-dependent manner. Using a nitrocellulose overlay assay with 125I-CacyBP/SIP and CacyBP/SIP affinity chromatography, we found that this protein binds purified S100A1, S100A6, S100A12, S100B, and S100P but not S100A4, calbindin D9k, parvalbumin, and calmodulin. The interaction of S100 proteins with CacyBP/SIP occurs via its C-terminal fragment (residues 155–229). Co-immunoprecipitation of CacyBP/SIP with S100B from brain and with S100A6 from Ehrlich ascites tumor cells suggests that these interactions are physiologically relevant and that the ubiquitinylation complex involving CacyBP/SIP might be regulated by S100 proteins.

Functional and structural characterization of rhodopsin oligomers

A major question in G protein-coupled receptor signaling concerns the quaternary structure required for signal transduction. Do these transmembrane receptors function as monomers, dimers, or larger oligomers? We have investigated the oligomeric state of the model G protein-coupled receptor rhodopsin (Rho), which absorbs light and initiates a phototransduction-signaling cascade that forms the basis of vision. In this study, different forms of Rho were isolated using gel filtration techniques in mild detergents, including n-dodecyl-β-d-maltoside, n-tetradecyl-β-d-maltoside, and n-hexadecyl-β-d-maltoside. The quaternary structure of isolated Rho was determined by transmission electron microscopy, demonstrating that in micelles containing n-dodecyl-β-d-maltoside, Rho exists as a mixture of monomers and dimers whereas in n-tetradecyl-β-d-maltoside and n-hexadecyl-β-d-maltoside Rho forms higher ordered structures. Especially in n-hexadecyl-β-d-maltoside, most of the particles are present in tightly packed rows of dimers. The oligomerization of Rho seems to be important for interaction with its cognate G protein, transducin. Although the activated Rho (Meta II) monomer or dimers are capable of activating the G protein, transducin, the activation process is much faster when Rho exists as organized dimers. Our studies provide direct comparisons between signaling properties of Meta II in different quaternary complexes.

Retinol Dehydrogenase (RDH12) Protects Photoreceptors from Light-induced Degeneration in Mice

RDH12 has been suggested to be one of the retinol dehydrogenases (RDH) involved in the vitamin A recycling system (visual cycle) in the eye. Loss of function mutations in the RDH12 gene were recently reported to be associated with autosomal recessive childhood-onset severe retinal dystrophy. Here we show that RDH12 localizes to the photoreceptor inner segments and that deletion of this gene in mice slows the kinetics of all-trans-retinal reduction, delaying dark adaptation. However, accelerated 11-cis-retinal production and increased susceptibility to light-induced photoreceptor apoptosis were also observed in Rdh12-/- mice, suggesting that RDH12 plays a unique, nonredundant role in the photoreceptor inner segments to regulate the flow of retinoids in the eye. Thus, severe visual impairments of individuals with null mutations in RDH12 may likely be caused by light damage1.

A Naturally Occurring Mutation of the Opsin Gene (T4R) in Dogs Affects Glycosylation and Stability of the G Protein-coupled Receptor

Rho (rhodopsin; opsin plus 11-cis-retinal) is a prototypical G protein-coupled receptor responsible for the capture of a photon in retinal photoreceptor cells. A large number of mutations in the opsin gene associated with autosomal dominant retinitis pigmentosa have been identified. The naturally occurring T4R opsin mutation in the English mastiff dog leads to a progressive retinal degeneration that closely resembles human retinitis pigmentosa caused by the T4K mutation in the opsin gene. Using genetic approaches and biochemical assays, we explored the properties of the T4R mutant protein. Employing immunoaffinity-purified Rho from affected RHOT4R/T4R dog retina, we found that the mutation abolished glycosylation at Asn2, whereas glycosylation at Asn15 was unaffected, and the mutant opsin localized normally to the rod outer segments. Moreover, we found that T4R Rho* lost its chromophore faster as measured by the decay of meta-rhodopsin II and that it was less resistant to heat denaturation. Detergent-solubilized T4R opsin regenerated poorly and interacted abnormally with the G protein transducin (Gt). Structurally, the mutation affected mainly the “plug” at the intradiscal (extracellular) side of Rho, which is possibly responsible for protecting the chromophore from the access of bulk water. The T4R mutation may represent a novel molecular mechanism of degeneration where the unliganded form of the mutant opsin exerts a detrimental effect by losing its structural integrity.

Asymmetry of the rhodopsin dimer in complex with transducin

A large body of evidence for G‐proteincoupled receptor (GPCR) oligomerization has accumulated over the past 2 decades. The smallest of these oligomers in vivo most likely is a dimer that buries 1000‐Å2 intramolecular surfaces and on stimulation forms a complex with heterotrimeric G protein in 2:1 stoichiometry. However, it is unclear whether each of the monomers adopts the same or a different conformation and function after activation of this dimer. With bovine rhodopsin (Rho) and its cognate bovine G‐protein transducin (Gt) as a model system, we used the retinoid chromophores 11‐cis‐retinal and 9‐cis‐retinal to monitor each monomer of the dimeric GPCR within a stable complex with nucleotide‐free Gt. We found that only 50% of Rho* in the Rho*‐Gt complex is trapped in a Meta II conformation, while 50% evolves toward an opsin conformation and can be regenerated with 9‐cis‐retinal. We also found that all‐trans‐retinal can regenerate chromophore‐depleted Rho*e complexed with Gt and FAK*TSA peptide containing Lys296 with the attached all‐trans retinoid (m/ z of 934.5[MH]+) was identified by mass spectrometry. Thus, our study shows that each of the monomers contributes unequally to the pentameric (2:1:1:1) complex of Rho dimer and Gt heterotrimer, validating the oligomeric structure of the complex and the asymmetry of the GPCR dimer, and revealing its structural/functional signature. This study provides a clear functional distinction between monomers of family A GPCRs in their oligomeric form.—Jastrzebska, B., Orban, T., Golczak, M., Engel, A., Palczewski, K. Asymmetry of the rhodopsin dimer in complex with transducin. FASEB J. 27, 1572–1584 (2013). www.fasebj.org

Ca2+-dependent Translocation of the Calcyclin-binding Protein in Neurons and Neuroblastoma NB-2a Cells

The calcyclin-binding protein (CacyBP) binds calcyclin (S100A6) at physiological levels of [Ca2+] and is highly expressed in brain neurons. Subcellular localization of CacyBP was examined in neurons and neuroblastoma NB-2a cells at different [Ca2+] i . Immunostaining indicates that CacyBP is present in the cytoplasm of unstimulated cultured neurons in which resting [Ca2+] i is known to be ∼50 nm. When [Ca2+] i was increased to above 300 nm by KCl treatment, the immunostaining was mainly apparent as a ring around the nucleus. Such perinuclear localization of CacyBP was observed in untreated neuroblastoma NB-2a cells in which [Ca2+] i is ∼120 nm. An additional increase in [Ca2+] i to above 300 nm by thapsigargin treatment did not change CacyBP localization. However, when [Ca2+] i in NB-2a cells dropped to 70 nm, because of BAPTA/AM treatment, perinuclear localization was diminished. Ca2+-induced translocation of CacyBP was confirmed by immunogold electron microscopy and by fluorescence of NB-2a cells transfected with an EGFP-CacyBP vector. Recombinant CacyBP can be phosphorylated by protein kinase C in vitro. In untreated neuroblastoma NB-2a cells, CacyBP is phosphorylated on a serine residue(s), but exists in the dephosphorylated form in BAPTA/AM-treated cells. Thus, phosphorylation of CacyBP occurs in the same [Ca2+] i range that leads to its perinuclear translocation.

Conformational Dynamics of Activation for the Pentameric Complex of Dimeric G Protein-Coupled Receptor and Heterotrimeric G Protein

Photoactivation of rhodopsin (Rho), a G protein-coupled receptor, causes conformational changes that provide a specific binding site for the rod G protein, G(t). In this work we employed structural mass spectrometry techniques to elucidate the structural changes accompanying transition of ground state Rho to photoactivated Rho (Rho(∗)) and in the pentameric complex between dimeric Rho(∗) and heterotrimeric G(t). Observed differences in hydroxyl radical labeling and deuterium uptake between Rho(∗) and the (Rho(∗))(2)-G(t) complex suggest that photoactivation causes structural relaxation of Rho following its initial tightening upon G(t) coupling. In contrast, nucleotide-free G(t) in the complex is significantly more accessible to deuterium uptake allowing it to accept GTP and mediating complex dissociation. Thus, we provide direct evidence that in the critical step of signal amplification, Rho(∗) and G(t) exhibit dissimilar conformational changes when they are coupled in the (Rho(∗))(2)-G(t) complex.