NMR analysis of rhodopsin-transducin interactions.

Heterotrimeric G-protein activation by an agonist-stimulated G-protein coupled receptor (R*) requires the propagation of structural signals from the receptor interacting surfaces to the guanine nucleotide-binding pocket. Employing high-resolution NMR methods, we are probing heterotrimer-associated and rhodopsin-stimulated changes in an isotope-labeled G-protein alpha-subunit (G(alpha)). A key aspect of the work involves the trapping and interrogation of discrete R*-bound conformations of G(alpha). Our results demonstrate that functionally important changes in G(alpha) structure and dynamics can be detected and characterized by NMR, enabling the generation of robust models for the global and local structural changes accompanying signal transfer from R* to the G-protein.

Evidence for structural changes in carboxyl-terminal peptides of transducin alpha-subunit upon binding a soluble mimic of light-activated rhodopsin.

Although a high-resolution crystal structure for the ground state of rhodopsin is now available, portions of the cytoplasmic surface are not well resolved, and the structural basis for the interaction of the cytoplasmic loops with the retinal G-protein transducin (G(t)) is still unknown. Previous efforts aimed at the design, construction, and functional characterization of soluble mimics for the light-activated state of rhodopsin have shown that grafting defined segments from the cytoplasmic region of bovine opsin onto a surface loop in a mutant form of thioredoxin (HPTRX) is sufficient to confer partial G(t) activating potential [Abdulaev et al. (2000) J. Biol. Chem. 275, 39354-39363]. To assess whether these designed mimics could provide a structural insight into the interaction between light-activated rhodopsin and G(t), the ability of an HPTRX fusion protein comprised of the second (CD) and third (EF) cytoplasmic loops (HPTRX/CDEF) to bind G(t) alpha-subunit (G(t)(alpha)) peptides was examined using nuclear magnetic resonance (NMR) spectroscopy. Transfer NOESY (TrNOESY) experiments show that an 11 amino acid peptide corresponding to the carboxyl terminus of G(t)(alpha) (GtP), as well as a "high-affinity" peptide analogue, HAP1, binds to HPTRX/CDEF in the fast-exchange regime and undergoes similar, subtle structural changes at the extreme carboxyl terminus. Observed TrNOEs suggest that both peptides when bound to HPTRX/CDEF adopt a reverse turn that is consistent with the C-cap structure that has been previously reported for the interaction of GtP with the light-activated signaling state, metarhodopsin II (MII). In contrast, TrNOESY spectra provide no evidence for structuring of the amino terminus of either GtP or HAP1 when bound to HPTRX/CDEF, nor do the spectra show any measurable changes in the CD and EF loop resonances of HPTRX/CDEF, which are conformationally dynamic and significantly exchange broadened. Taken together, the NMR observations indicate that HPTRX/CDEF, previously identified as a functional mimic of MII, is also an approximate structural mimic for this light-activated state of rhodopsin.

Functionally discrete mimics of light-activated rhodopsin identified through expression of soluble cytoplasmic domains.

Numerous studies on the seven-helix receptor rhodopsin have implicated the cytoplasmic loops and carboxyl-terminal region in the binding and activation of proteins involved in visual transduction and desensitization. In our continuing studies on rhodopsin folding, assembly, and structure, we have attempted to reconstruct the interacting surface(s) for these proteins by inserting fragments corresponding to the cytoplasmic loops and/or the carboxyl-terminal tail of bovine opsin either singly, or in combination, onto a surface loop in thioredoxin. The purpose of the thioredoxin fusion is to provide a soluble scaffold for the cytoplasmic fragments thereby allowing them sufficient conformational freedom to fold to a structure that mimics the protein-binding sites on light-activated rhodopsin. All of the fusion proteins are expressed to relatively high levels in Escherichia coli and can be purified using a two- or three-step chromatography procedure. Biochemical studies show that some of the fusion proteins effectively mimic the activated conformation(s) of rhodopsin in stimulating G-protein or competing with the light-activated rhodopsin/G-protein interaction, in supporting phosphorylation of the carboxyl-terminal opsin fragment by rhodopsin kinase, and/or phosphopeptide-stimulated arrestin binding. These results suggest that specific segments of the cytoplasmic surface of rhodopsin can adopt functionally discrete conformations in the absence of the connecting transmembrane helices and retinal chromophore.

[Biochemical characteristics of bovine retina nucleoside diphosphate kinase]. / Biokhimicheskie svoistva nukleoziddifosfatkinazy setchatki glaza byka.

Nucleoside diphosphate kinase (NDP kinase; ATP: NDP phosphotransferase; EC 2.7.4.6) was purified from bovine retina. The molecular mass of the native enzyme was found to be 72 kDa, and those of its subunits were 17.5 and 18.5 kDa. Kinetic characteristics of the enzyme were determined. It was shown that NDP kinase exists in retina in both soluble and membrane-bound forms.

Folding and assembly in rhodopsin. Effect of mutations in the sixth transmembrane helix on the conformation of the third cytoplasmic loop.

Previous studies on bovine opsin folding and assembly have identified an amino-terminal fragment, EF(1-232), which folds and inserts into a membrane only after coexpression with its complementary carboxyl-terminal fragment, EF(233-348). To further characterize this interaction, EF(1-232) production was examined upon coexpression with carboxyl-terminal fragments of varying length and/or amino acid composition. These included fragments with incremental deletions of the third cytoplasmic loop (TH(241-348) and EF(249-348)), a fragment composed of the third cytoplasmic loop and sixth transmembrane helix (HF(233-280)), a fragment composed of the sixth and seventh transmembrane helices (FG(249-312)), and EF(233-348) and TH(241-348) fragments with Pro-267 or Trp-265 mutations. Although EF(1-232) production was independent of the third cytoplasmic loop and carboxyl-terminal tail, both the sixth and seventh transmembrane helices were essential. The effects of mutations in the sixth transmembrane helix on EF(1-232) expression were dependent on the length of the third cytoplasmic loop. Although Pro-267 mutations in EF(233-348) failed to stabilize EF(1-232) expression, their introduction into TH(241-348) was without discernible effects. However, Trp-265 substitutions in the EF(233-348) and TH(241-348) fragments conferred significant EF(1-232) production. Therefore, key residues in the transmembrane helices may exert their effects on opsin folding, assembly, and/or function by influencing the conformation of the connecting loops.

The three-dimensional structures of two isoforms of nucleoside diphosphate kinase from bovine retina.

The crystal structures of two isoforms of nucleoside diphosphate kinase from bovine retina overexpressed in Escherischia coli have been determined to 2.4 A resolution. Both the isoforms, NBR-A and NBR-B, are hexameric and the fold of the monomer is in agreement with NDP-kinase structures from other biological sources. Although the polypeptide chains of the two isoforms differ by only two residues, they crystallize in different space groups. NBR-A crystallizes in space group P212121 with an entire hexamer in the asymmetric unit, while NBR-B crystallizes in space group P43212 with a trimer in the asymmetric unit. The highly conserved nucleotide-binding site observed in other nucleoside diphosphate kinase structures is also observed here. Both NBR-A and NBR-B were crystallized in the presence of cGMP. The nucleotide is bound with the base in the anti conformation. The NBR-A active site contained both cGMP and GDP each bound at half occupancy. Presumably, NBR-A had retained GDP (or GTP) from the purification process. The NBR-B active site contained only cGMP.

Light-induced exposure of the cytoplasmic end of transmembrane helix seven in rhodopsin.

A key step in signal transduction in the visual cell is the light-induced conformational change of rhodopsin that triggers the binding and activation of the guanine nucleotide-binding protein. Site-directed mAbs against bovine rhodopsin were produced and used to detect and characterize these conformational changes upon light activation. Among several antibodies that bound exclusively to the light-activated state, an antibody (IgG subclass) with the highest affinity (Ka approximately 6 x 10(-9) M) was further purified and characterized. The epitope of this antibody was mapped to the amino acid sequence 304-311. This epitope extends from the central region to the cytoplasmic end of the seventh transmembrane helix and incorporates a part of a highly conserved NPXXY motif, a critical region for signaling and agonist-induced internalization of several biogenic amine and peptide receptors. In the dark state, no binding of the antibody to rhodopsin was detected. Accessibility of the epitope to the antibody correlated with formation of the metarhodopsin II photointermediate and was reduced significantly at the metarhodopsin III intermediate. Further, incubation of the antigen-antibody complex with 11-cis-retinal failed to regenerate the native rhodopsin chromophore. These results suggest significant and reversible conformational changes in close proximity to the cytoplasmic end of the seventh transmembrane helix of rhodopsin that might be important for folding and signaling.

Nucleoside diphosphate kinase from bovine retina: purification, subcellular localization, molecular cloning, and three-dimensional structure.

The biochemical and structural properties of bovine retinal nucleoside diphosphate kinase were investigated. The enzyme showed two polypeptides of approximately 17.5 and 18.5 kDa on SDS-PAGE, while isoelectric focusing revealed seven to eight proteins with a pI range of 7.4-8.2. Sedimentation equilibrium yielded a molecular mass of 96 +/- 2 kDa for the enzyme. Carbohydrate analysis revealed that both polypeptides contained Gal, Man, GlcNAc, Fuc, and GalNac saccharides. Like other nucleoside diphosphate kinases, the retinal enzyme showed substantial differences in the Km values for various di- and triphosphate nucleotides. Immunogold labeling of bovine retina revealed that the enzyme is localized on both the membranes and in the cytoplasm. Screening of a retinal cDNA library yielded full-length clones encoding two distinct isoforms (NBR-A and NBR-B). Both isoforms were overexpressed in Escherichia coli and their biochemical properties compared with retinal NDP-kinase. The structures of NBR-A and NBR-B were determined by X-ray crystallography in the presence of guanine nucleotide(s). Both isoforms are hexameric, and the fold of the monomer is similar to other nucleoside diphosphate kinase structures. The NBR-A active site contained both a cGMP and a GDP molecule each bound at half occupancy while the NBR-B active site contained only cGMP.

[Functional expression of bovine retina adenylate cyclase b fragment cDNA in Escherichia Coli cells]. / Funktsional'naia ekspressiia fragmenta kDNK guanilattsiklazy B setchatki byka v kletkakh Escherichia coli.

A 1488-bp fragment of bovine retina guanylate cyclase B gene encoding the catalytic and dimerizing domains as well as part of the protein kinase domain was expressed in Escherichia coli cells. The expression product was obtained as inclusion bodies and solubilized in 6 M guanidine hydrochloride. The fragment of guanylate cyclase B is a dimer close in catalytic activity to the native enzyme.

Functional expression of bovine opsin in the methylotrophic yeast Pichia pastoris.

The methylotrophic yeast Pichia pastoris was examined for functional expression of bovine opsin. An expression plasmid was constructed where the bovine opsin gene was placed downstream from the P. pastoris alcohol oxidase 1 gene promoter and fused at its amino-terminus to the acid phosphatase secretion signal. Quantitative-competitive PCR analysis of a stable yeast transformant showed that one copy of the opsin gene was integrated into the yeast genome. The expression level in this transformant corresponded to approximately 0.3 mg of opsin per liter of cell culture (A600 = 1.0). Sucrose density sedimentation analysis indicated that the opsin was associated exclusively with the membrane fraction. Similar to retinal opsin, P. pastoris-expressed opsin migrated as a single band of approximately 37 kDa on SDS-PAGE and showed high mannose N-glycosylation. A portion of the expressed opsin (approximately 4-15%) reacted with 11-cis-retinal to form the rhodopsin chromophore (lambda max 500 nm), and after purification showed ground and excited state spectral characteristics indistinguishable from those of the native pigment. Further, the metarhodopsin-II-mediated G-protein-activating potential of yeast expressed rhodopsin was similar to that of native rhodopsin. These results show that P. pastoris cells have the capacity to functionally express bovine opsin.

[Nucleoside diphosphate kinase from bovine retina is a glycoprotein]. / Nukleoziddifosfatkinaza setchatki byka iavliaetsia glikoproteinom.

Nucleoside diphosphate (NDP) kinase from bovine retina was found to contain carbohydrates. The subunits of NDP kinase were separated by SDS-PAGE, blotted onto an Immobilon-P membrane, and their carbohydrate content was determined. Both subunits contained equal amounts of Gal, Man, Fuc, Gal-NAc, and Glc-NAc. The total carbohydrate content was 2 to 3% of the protein weight.

Examining rhodopsin folding and assembly through expression of polypeptide fragments.

Previous work on the expression of bovine opsin fragments separated in the cytoplasmic region has allowed the identification of specific polypeptide segments that contain sufficient information to fold independently, insert into a membrane, and assemble to form a functional photoreceptor. To further examine the contributions of these and other polypeptide segments to the mechanism of opsin folding and assembly, we have constructed 20 additional opsin gene fragments where the points of separation occur in the intradiscal, transmembrane, and cytoplasmic regions. Nineteen of the fragments were stably expressed in COS-1 cells. A five-helix fragment was stably produced only after coexpression with its complementary two-helix fragment. Two fragments composed of the amino-terminal region and the first transmembrane helix were not N-glycosylated and were only partially membrane integrated. One of the singly expressed fragments, which is truncated after the retinal attachment site, bound 11-cis-retinal. Of the coexpressed complementary fragments, only those separated in the second intradiscal and third cytoplasmic regions formed noncovalently linked rhodopsin. Both of the pigments showed reduced transducin activation. Therefore, while many opsin fragments contain enough information to fold and insert into a membrane, only those separated at specific locations assemble to a retinal-binding opsin.

Conformation of surface exposed N-terminus part of bacteriorhodopsin studied by transferred NOE technique.

Interaction of the monoclonal antibody A5 raised against native bacteriorhodopsin (BR) with the synthetic peptide pGlu1-Ala-Gln-Ile-Thr-Gly-Arg7-NH2, corresponding to the amino acid sequence 1-7 was studied by transferred nuclear Overhauser effect (TRNOE) spectroscopy. The denaturing reagents and the specially designed pulse sequences which eliminate broad signals from the TRNOE spectra were used to favour evaluation of the TRNOE peaks. On the basis of the data obtained, the conformation of peptide bound with A5 was calculated. A model of the mutual arrangement of bacteriorhodopsin N-terminus and the first transmembrane alpha-helical segment 8-32 was proposed.

[Guanylate kinase from bovine retina: isolation, primary structure, and expression in E. coli]. / Guanilatkinaza iz setchatki byka: vydelenie, pervichnaia struktura i ékspressiia v E. coli.

Guanylate kinase (EC 2.7.4.8), catalysing the reaction GMP+ATP = GDP+ADP, was purified to homogeneity from bovine retina. Primary structure of the enzyme was determined by parallel analyses of amino acid sequences of its peptides and nucleotide sequence of the corresponding cDNA. It is shown that the bovine retinal guanylate kinase like the analogous enzyme from yeast Saccharomyces cerevisiae contains a characteristic glycine-rich motif, involved in ATP binding. All of the amino acids, involved in GMP binding in the yeast enzyme, are conserved or conservatively substituted in the bovine retinal guanylate kinase. The bovine retinal enzyme was expressed in E. coli as a fusion protein. Data are presented on the purification of the fusion protein, its digestion by enteropeptidase, purification of the recombinant enzyme and its functional characteristics.

Enzymes of the cyclic GMP metabolism in bovine retina. I. Cloning and expression of the gene for guanylate kinase.

Guanylate kinase (EC 2.7.4.8) catalyzing the reaction GMP + ATP = GDP + ADP, was purified to homogeneity from bovine retina. Using oligonucleotides based on the amino acid sequence of this enzyme, the cDNA encoding guanylate kinase (GK) was isolated and its nucleotide sequence was determined. Expression of the GK cDNA in E. coli, and the purification and functional characterization of the expressed enzyme are presented. It is shown that bovine retinal GK, like its yeast counterpart, contains the characteristic glycine-rich motif and all the amino acids involved in GMP binding. Bovine retinal enzyme is extended for several amino acid residues both at the N- and C-termini, compared to the yeast enzyme.

[Detection of expression of a membrane form of the guanylate cyclase type of GC-B in cattle retina]. / Obnaruzhenie ékspressii membrannoi formy guanilattsiklazy tipa GC-B v setchatke byka.

cDNA clones encoding the central and C-terminal parts of a membrane-bound guanylate cyclase (GC) were isolated from the lambda ZAP bovine retinal library. All of the analysed recombinants appeared to carry inserts encoding the guanylate cyclase GC-B. Analysis of the determined nucleotide and deduced amino acid sequences showed extremely high level of homology to the sequences of known GC-B. The results indicate that a mRNA for GC-B is expressed in the bovine retina.

[p26--a calcium binding protein from photoreceptor cells in the bovine retina: primary structure and expression in E. coli]. / p26--kal'tsiisviazyvaiushchii belok fotoretseptornykh kletok setchatki byka: pervichnaia struktura i ékspressiia v E. coli.

The primary structure of the bovine retinal calcium binding protein P26 has been determined by the parallel analysis of the protein and the corresponding cDNA. This protein is identical to recovering and shares 59% homology with visinin, a cone specific calcium binding protein from chicken retina. P26 was expressed in E. coli as a fusion protein and, after purification by affinity chromatography on IgG-Sepharose 6, cleaved off with enteropeptidase.