Mapping of contact sites in complex formation between transducin and light-activated rhodopsin by covalent crosslinking: Use of a photoactivatable reagent

Abstract

Interaction of light-activated rhodopsin with transducin (T) is the first event in visual signal transduction. We use covalent crosslinking approaches to map the contact sites in interaction between the two proteins. Here we use a photoactivatable reagent, N-[(2-pyridyldithio)-ethyl], 4-azido salicylamide. The reagent is attached to the SH group of cytoplasmic monocysteine rhodopsin mutants by a disulfideexchange reaction with the pyridylthio group, and the derivatized rhodopsin then is complexed with T by illumination at >495 nm. Subsequent irradiation of the complex at 310 nm generates covalent crosslinks between the two proteins. Crosslinking was demonstrated between T and a number of single cysteine rhodopsin mutants. However, sites of crosslinks were investigated in detail only between T and the rhodopsin mutant S240C (cytoplasmic loop V-VI). Crosslinking occurred predominantly with T ␣. For identification of the sites of crosslinks in T ␣, the strategy used involved: (i) derivatization of all of the free cysteines in the crosslinked proteins with N-ethylmaleimide; (ii) reduction of the disulfide bond linking the two proteins and isolation of all of the T ␣ species carrying the crosslinked moiety with a free SH group; (iii) adduct formation of the latter with the Nmaleimide moiety of the reagent, maleimido-butyryl-biocytin, containing a biotinyl group; (iv) trypsin degradation of the resulting T ␣ derivatives and isolation of T␣ peptides carrying maleimido-butyrylbiocytin by avidin-agarose chromatography; and (v) identification of the isolated peptides by matrix-assisted laser desorption͞ionization time-of-flight mass spectrometry. We found that crosslinking occurred mainly to two C-terminal peptides in T ␣ containing the amino acid sequences 310 -313 and 342-345.L ight activation of rhodopsin initiates two biochemical cascades, one leading to sensitization (amplification) and the other to desensitization (quenching). Binding and activation of transducin (T) and rhodopsin kinase are, respectively, the first events in the two cascades (1). Competition between T and rhodopsin kinase for interaction with the light-activated rhodopsin and its phosphorylated form is central to the progression of the two cascades. Therefore, understanding the requirements of the two proteins for binding to rhodopsin and the contact sites in the complexes formed is of much interest. Ideally, three-dimensional structures of the complexes between the proteins are needed, but such structural analysis lies in the future. Alternative approaches have provided some insights into the sites in rhodopsin required for binding of T. Thus, peptides corresponding to the amino acid sequences in the different cytoplasmic loops of rhodopsin inhibit T activation (2), and similarly mutagenic studies (3-5) have indicated that a considerable portion of the cytoplasmic domain of light-activated rhodopsin is involved in binding to T. Some information also has been obtained on the regions of T that contact rhodopsin in the complex. Thus, the effects of a...

Structural and kinetic modeling of an activating helix switch in the rhodopsin-transducin interface

“…3B shows the resulting geometry in the R*⅐G t [empty] complex. The model is in agreement with photo cross-linking experiments in which a photoactivated reagent attached at Ser-240 in loop C3 of R* cross-linked predominantly to G t ␣ sequence 342-345 (38). At the protein concentrations used in the experiment, a small fraction of the R*⅐G t ⅐GDP intermediate complex may have been present in equilibrium, explaining the second weaker cross-link to G t ␣ sequence 310-313 in these experiments.…”

Probing PrPSc Structure Using Chemical Cross-Linking and Mass Spectrometry: Evidence of the Proximity of Gly90 Amino Termini in the PrP 27−30 Aggregate

“…Given the difficulties in applying conventional analytical approaches, such as NMR or X-ray crystallography, as a consequence of the insoluble and polymeric nature of PrP Sc , the design of novel, unconventional analytical approaches appears to be an attractive option. Our study shows that the combination of chemical crosslinking, proteolytic digestion, and mass spectrometry, an approach that has been applied to the structural study of other monomeric and polymeric proteins (15)(16)(17)(18)(19)(20)(21)(22)(23)(24), can be successfully applied to PrP Sc . Using BS 3 , an agent that reacts primarily with amino groups, PrP 27-30 was readily crosslinked with formation of dimers, trimers, and higher-order oligomers.…”

“…Using model proteins of known structure, it has been claimed that the method can be used, in conjunction with threading programs, to identify the fold of a protein (15). The method has been used to obtain valuable information about key features of unknown tertiary and quaternary structures of proteins (15)(16)(17)(18)(19)(20)(21)(22)(23)(24). One particularly relevant example of the use of a variation of this approach was reported by Egnaczyk et al (24), who used it to probe the quaternary structure of -amyloid fibrils.…”

Structural Basis of G Protein-coupled Receptor-Gi Protein Interaction

“…Slessareva et al (71) have shown that the G␣ i1 ␣4 helix-␣4/␤6 loop are involved in 5-HT1a, 5-HT1b, and muscarinic M2 receptor interactions. For the rhodopsin⅐transducin (G␣ t ) complex, residues in the G␣ t ␣ 4 ␤ 6 loop (Arg-310 to Lys-313) were shown to cross-link with residues in the IC-3 loop of rhodopsin using a photoactivatable reagent, N-[(2-pyridyldithio)ethyl],4-azidosalicylamide (72). For the rat M3 muscarinic acetylcholine receptor (M3R)⅐G␣ q complex system, a cross-link has been reported between a D321C mutation on ␣ 4 ␤ 6 loop of G␣ q and a K7.58(548)C mutation on M3R.…”