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

Pashkov, V. S.; Balashova, T. A.; Zhemaeva, Ljubov V.; Sikilinda, Nina N.; Kutuzov, Mikhail A.; Abdulaev, N.G.; Arseniev, Alexander S.

doi:10.1016/0014-5793(96)00094-4

Cited by 6 publications

(3 citation statements)

References 15 publications

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“…Information on the relative arrangement of the seven transmembrane helices in G protein-coupled receptors (GPCRs) is based on previous detailed structural studies of rhodopsin (43), but little is known about the structures of the intra-and extracellular termini and loops of GPCRs. The determination of these structural elements is essential for understanding the biomolecular interactions between agonists and heterotrimeric G proteins with their specific receptors (32,34,(44)(45)(46)(47).…”

Section: Discussionmentioning

confidence: 99%

Photoaffinity Labeling of Mutant Neurokinin-1 Receptors Reveals Additional Structural Features of the Substance P/NK-1 Receptor Complex

Macdonald

Mierke

et al. 2001

Biochemistry

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Photoaffinity labeling, receptor site-directed mutagenesis, and high-resolution NMR spectroscopy have been combined to further define the molecular details of the binding of substance P (SP) to the rat neurokinin-1 (NK-1) receptor. Mutant NK-1 receptors were constructed by substituting Ala for Met174 and/or Met181: residues previously identified as the sites of covalent attachment of radioiodinated, photoreactive derivatives of SP containing p-benzoyl-L-phenylalanine (Bpa) in positions 4 and 8, respectively. Photoaffinity labeling of the M181A mutant using radioiodinated Bpa8-SP resulted in a marked reduction in photoincorporation efficiency compared to the wild-type receptor. In contrast, photoaffinity labeling of the M174A mutant using radioiodinated Bpa4-SP gave the unexpected result of an increase in the efficiency of photoincorporation compared to the wild-type receptor. Enzymatic and chemical fragmentation analysis of the photolabeled receptor mutants established that the sites of covalent attachment were not the substituted alanine, but rather the other methionine on the second extracellular (E2) loop sequence, that is not the primary site of attachment in the wild-type receptor. The results thus suggest a close spatial relationship between Met174 and Met181 on the NK-1 receptor. To evaluate this structural disposition, NMR analyses were performed on a synthetic peptide with a sequence corresponding to the entire E2 loop and segments of the adjoining transmembrane helices to anchor the peptide in the lipids used to mimic a membrane. The structural features of the E2 loop include a centrally located alpha-helix, extending from Pro175 to Glu183, as well as smaller alpha-helices at the termini, corresponding to the transmembrane regions. The two methionine residues are located on the same face of the central alpha-helix, approximately 11 A apart from each other, and are therefore consistent with the conclusions of the photoaffinity labeling results.

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Section: Discussionmentioning

confidence: 99%

Photoaffinity Labeling of Mutant Neurokinin-1 Receptors Reveals Additional Structural Features of the Substance P/NK-1 Receptor Complex

Macdonald

Mierke

et al. 2001

Biochemistry

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“…While the placement and relative arrangement of the seven TM helices in G-protein coupled receptors is relatively well understood from the studies of rhodopsin and bacteriorhodopsin (11) and other membrane embedded proteins (chiefly the bacterial reaction center), little is known about the structure of the intra-and extracellular termini and loops. The determination of the structural elements of the loops and termini would afford important insight into the biomolecular interactions of the receptor with the G-protein (intracellular domain) (40)(41)(42) and with the hormone-ligand (extracellular domain) (43,44). These studies would reveal the determinants which confer the specificity of ligand- recognition and signal transduction.…”

Section: Discussionmentioning

confidence: 99%

Binding Domain of Human Parathyroid Hormone Receptor: From Conformation to Function^,

et al. 1998

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A 31 amino acid fragment of the extracellular N-terminus of the human G-protein coupled receptor for parathyroid hormone (PTH1R) has been structurally characterized by NMR and molecular dynamics simulations. The fragment PTH1R[168-198] includes residues 173-189, shown by photoaffinity cross-linking to be a contact domain with position 13 of parathyroid hormone (PTH). The structure of PTH1R[168-198], determined in a micellar solution of dodecylphosphocholine to mimic the membrane environment, consists of three alpha-helices, separated by a well-defined turn and a flexible region. The topological orientation of PTH1R[168-198] was determined from nitroxide-radical induced relaxation of NMR signals utilizing 5- and 16-doxylstearic acid. The C-terminal helix (residues 190-196), consisting of seven amino acids of the first transmembrane domain, is very hydrophobic and embedded in the lipid core. This helix is preceded by a well-defined turn, forming an approximate 90 degrees bend, placing the other helices (residues 169-176 and 180-189), both of which are amphipathic, on the surface of the micelle. In this orientation, many hydrophilic residues of the receptor, including Glu177, Arg179, Arg181, Glu182, Asp185, and Arg186, are projecting toward the solvent available to form complementary Coulombic interactions with the polar residues of the principal binding domain of the ligand (e.g., Arg25, Lys26, Lys27, Asp30, and His32). Given that the binding domain of PTH adopts an amphipathic alpha-helix which lies on the membrane, we visualize ligand binding as a two stage process involving a nonspecific hydrophobic interaction of amphipathic helices with the membrane, followed by two-dimensional diffusion leading to highly specific, ligand-receptor interaction.

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“…Bovine retinae were from W. L. Lawson (Lincoln, NE), and 11-cisretinal was a gift of R. Crouch (Medical University of South Carolina and the National Eye Institute). The A5 mAb, which is specific for the amino-terminal sequence of bacteriorhodopsin, has been described (27). Other materials used in this investigation have been described (28).…”

Section: Methodsmentioning

confidence: 99%

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

Abdulaev

Ridge

1998

Proc. Natl. Acad. Sci. U.S.A.

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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 (K a Ϸ 6 ؋ 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.Rhodopsin, a member of a large family of seven-transmembrane (TM) helix receptors ( Fig. 1), triggers a light-dependent, phosphodiesterase-mediated, cyclic guanosine monophosphate-regulated, and G protein-coupled signal transduction cascade (1-7). Rhodopsin contains 11-cis-retinal covalently bound via a protonated Schiff base (PSB) to the -amino group of Lys-296 on TM7. The carboxyl group of Glu-113 on TM3, which resides well within the membrane, neutralizes the positive charge on the retinyl-PSB in the dark state (8-10). This dominant electrostatic interaction and the steric constraint imposed by the very nature of the chromophore in its immediate protein environment supports the inactive ground-state conformation. Upon light absorption, retinal isomerizes to the all-trans configuration that drives the protein through a series of transient and thermally stable intermediates (11). Charge separation between the retinyl-PSB and Glu-113 with subsequent protonation of the latter culminates in formation of metarhodopsin II (Meta II), an active intermediate that accommodates the high-affinity sites for interaction with several regulatory proteins of the visual transduction cascade.Recently, there has been considerable progress in understanding retinal-protein interactions and the structural rearrangements that the protein undergoes upon cis 3 trans isomerization of the chromophore (12, 13). Despite these advances, a clear understanding of how changes deep in the membrane are relayed to the surface of the protein, thereby allowing an opening of its conformation...

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

Cited by 6 publications

References 15 publications

Photoaffinity Labeling of Mutant Neurokinin-1 Receptors Reveals Additional Structural Features of the Substance P/NK-1 Receptor Complex

Photoaffinity Labeling of Mutant Neurokinin-1 Receptors Reveals Additional Structural Features of the Substance P/NK-1 Receptor Complex

Binding Domain of Human Parathyroid Hormone Receptor: From Conformation to Function^,

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

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

Cited by 6 publications

References 15 publications

Photoaffinity Labeling of Mutant Neurokinin-1 Receptors Reveals Additional Structural Features of the Substance P/NK-1 Receptor Complex

Photoaffinity Labeling of Mutant Neurokinin-1 Receptors Reveals Additional Structural Features of the Substance P/NK-1 Receptor Complex

Binding Domain of Human Parathyroid Hormone Receptor: From Conformation to Function,

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

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Binding Domain of Human Parathyroid Hormone Receptor: From Conformation to Function^,