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Oleg G Kisselev

Publications and source records attributed to Oleg G Kisselev.

8 recordsLinked to original sources
PubMed2006

G-protein alpha and beta-gamma subunits interact with conformationally distinct signaling states of rhodopsin.

Light activated rhodopsin interacts with domains on all three subunits of transducin. Two of these domains, the C-terminal regions of the alpha and gamma subunits mimic the ability of transducin to stabilize the active conformation of rhodopsin, metarhodopsin II, but display different roles in transducin activation process. Whether the interactions are with the same or different complimentary sites on Meta II is unknown. We have used chemo-selective thioalkylation of rhodopsin and UV/visible spectroscopy to show that interactions with transducin C-terminal domains can be selectively disrupted. These data provide evidence that formal structural determinants on Meta II for these domains of transducin are different. In a set of complimentary experiments we examined the reactivity of Meta II species produced in the presence of the Gtalpha and Gtgamma subunit peptides to hydroxylamine. Analysis of the rates of Meta II decay confirms that the conformational states of Meta II when bound to Gtalpha and Gtbetagamma represent distinct signaling states of rhodopsin.

Animals↗
PubMed2006

Rhodopsin-interacting surface of the transducin gamma subunit.

The visual signaling pathway is initiated by photoactivation of the GPCR rhodopsin, which activates nucleotide exchange on the heterotrimeric G-protein transducin (Gt). Domains on both Gtalpha and Gtbetagamma subunits participate in coupling to rhodopsin. Previously, we have shown by high-resolution NMR that the farnesylated C-terminal peptide of Gtgamma(60-71), DKNPFKELKGGC, assumes an amphipathic helical conformation during interaction with metarhodopsin II [Kisselev, O. G., and Downs, M. A. (2003) Structure 11, 367-373]. This conformation was docked to the structure of holo-Gt to create a model of rhodopsin-Gt interaction. Here we test this model by mutational analysis of Gt. To evaluate the contribution of specific amino acids of the Gtgamma C-terminal region involved in binding and GTP-dependent release of transducin from native rhodopsin membranes, we have systematically substituted each of the amino acids in the C-terminal region of Gtgamma for alanine. The mutants were co-expressed with six-histidine-tagged Gtbeta subunits in Sf9 insect cells. The Gtbeta-6-His-gamma mutant proteins were purified and assayed in the presence of Gtalpha for the GTP-dependent interactions with light-activated rhodopsin. Several of the alanine mutants, N62A, P63A, and F64A, exhibited significant functional defects at the level of R*-Gt complex formation. These data show that the conserved N-terminal end of the helical domain in the Gtgamma(60-71) region has the most significant effect on rhodopsin-Gt interactions, which places important constraints on the model of the rhodopsin-Gt complex.

Alanine↗
PubMed2006

Relative strength of cation-pi vs salt-bridge interactions: the Gtalpha(340-350) peptide/rhodopsin system.

Interactions between cationic and aromatic side chains of amino acid residues, the so-called cation-pi interaction, are thought to contribute to the overall stability of the folded structure of peptides and proteins. The transferred NOE NMR structure of the G(t)alpha(340-350) peptide bound to photoactivated rhodopsin (R*) geometrically suggests a cation-pi interaction stabilizing the structure between the epsilon-amine of Lys341 and the aromatic ring of the C-terminal residue, Phe350. This interaction has been explored by varying substituents on the phenyl ring to alter the electron density of the aromatic ring of Phe350 and observing the impact on binding of the peptide to R*. The results suggest that while a cation-pi interaction geometrically exists in the G(t)alpha(340-350) peptide when bound to R*, its energetic contribution to the stability of the receptor-bound structure is relatively insignificant, as it was not observed experimentally. The presence of an adjacent and competing salt-bridge interaction between the epsilon-amine of Lys341 and the C-terminal carboxylate of Phe350 effectively shields the charge of the ammonium group. Experimental data supporting a significant cation-pi interaction can be regained through a series of Phe350 analogues where the C-terminal carboxyl has been converted to the neutral carboxamide, thus eliminating the shielding salt-bridge. TrNOE NMR experiments confirmed the existence of the cation-pi interaction in the carboxamide analogues. Various literature estimates of the strength of cation-pi interactions, including some that estimate strengths in excess of salt-bridges, are compromised by omission of the relevant anion in the calculations.

Algorithms↗
PubMed2006

Alternate binding mode of C-terminal phenethylamine analogs of G(t)alpha(340-350) to photoactivated rhodopsin.

The C-terminus of the Galpha-subunit of transducin plays an important role in receptor recognition. Synthetic peptides corresponding to the last 11 residues of the subunit have been shown to stabilize the photoactivated form of rhodopsin, Rh*. The Rh*-bound structure of the G(t)alpha(340-350) peptide has been determined using transferred nuclear overhauser effect NMR. In that structure, we observed two interactions between Lys341 and Phe350, a cation-pi interaction between the epsilon-amine and the aromatic ring of Phe350 and a salt-bridge between the epsilon-amine and the C-terminal carboxylate. A series of C-terminal phenethylamine analogs of the G(t)alpha(340-350) peptide were synthesized, lacking the C-terminal carboxylate group, to investigate the forces that contribute to the stability of the Rh*-bound conformation of the peptide. Rh*-stabilization assay data suggest that the C-terminal carboxylate is not necessary to maintain binding affinity. Transferred nuclear overhauser effect NMR experiments reveal that these C-terminal phenethylamine peptides adopt an Rh*-bound structure that is similar overall, but lacking some of the intramolecular interactions observed in the native Rh*-bound G(t)alpha(340-350) structure. These studies suggest that the binding site for G(t)alpha(340-350) on Rh* is adaptable, and we propose that the charged carboxylate of Phe350 does not play a significant role in the interaction with Rh*, but helps stabilize the Rh*-bound confirmation of the native peptide.

Amino Acid Sequence↗
PubMed2004

Conformational changes in the phosphorylated C-terminal domain of rhodopsin during rhodopsin arrestin interactions.

Phosphorylation of activated G-protein-coupled receptors and the subsequent binding of arrestin mark major molecular events of homologous desensitization. In the visual system, interactions between arrestin and the phosphorylated rhodopsin are pivotal for proper termination of visual signals. By using high resolution proton nuclear magnetic resonance spectroscopy of the phosphorylated C terminus of rhodopsin, represented by a synthetic 7-phosphopolypeptide, we show that the arrestin-bound conformation is a well ordered helix-loop structure connected to rhodopsin via a flexible linker. In a model of the rhodopsin-arrestin complex, the phosphates point in the direction of arrestin and form a continuous negatively charged surface, which is stabilized by a number of positively charged lysine and arginine residues of arrestin. Opposite to the mostly extended structure of the unphosphorylated C-terminal domain of rhodopsin, the arrestin-bound C-terminal helix is a compact domain that occupies a central position between the cytoplasmic loops and occludes the key binding sites of transducin. In conjunction with other binding sites, the helix-loop structure provides a mechanism of shielding phosphates in the center of the rhodopsin-arrestin complex and appears critical in guiding arrestin for high affinity binding with rhodopsin.

Animals↗
PubMed2004

The arrestin-bound conformation and dynamics of the phosphorylated carboxy-terminal region of rhodopsin.

Visual arrestin binds to the phosphorylated carboxy-terminal region of rhodopsin to block interactions with transducin and terminate signaling in the rod photoreceptor cells. A synthetic seven-phospho-peptide from the C-terminal region of rhodopsin, Rh(330-348), has been shown to bind arrestin and mimic inhibition of signal transduction. In this study, we examine conformational changes in this synthetic peptide upon binding to arrestin by high-resolution proton nuclear magnetic resonance (NMR). We show that the peptide is completely disordered in solution, but becomes structured upon binding to arrestin. A control, unphosphorylated peptide that fails to bind to arrestin remains highly disordered. Specific NMR distance constraints are used to model the arrestin-bound conformation. The models suggest that the phosphorylated carboxy-terminal region of rhodopsin, Rh(330-348), undergoes significant conformational changes and becomes structured upon binding to arrestin.

Animals↗
PubMed2003

Rhodopsin controls a conformational switch on the transducin gamma subunit.

Rhodopsin, a prototypical G protein-coupled receptor, catalyzes the activation of a heterotrimeric G protein, transducin, to initiate a visual signaling cascade in photoreceptor cells. The betagamma subunit complex, especially the C-terminal domain of the transducin gamma subunit, Gtgamma(60-71)farnesyl, plays a pivotal role in allosteric regulation of nucleotide exchange on the transducin alpha subunit by light-activated rhodopsin. We report that this domain is unstructured in the presence of an inactive receptor but forms an amphipathic helix upon rhodopsin activation. A K65E/E66K charge reversal mutant of the gamma subunit has diminished interactions with the receptor and fails to adopt the helical conformation. The identification of this conformational switch provides a mechanism for active GPCR utilization of the betagamma complex in signal transfer to G proteins.

Amino Acid Sequence↗