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

Abstract

Contact sites in interaction between light-activated rhodopsin and transducin (T) have been investigated by using a chemically preactivated crosslinking reagent, N-succinimidyl 3-(2-pyridyldithio)-propionate. The 3 propionyl-N-succinimidyl group in the reagent was attached by a disulfide exchange reaction to rhodopsin mutants containing single reactive cysteine groups in the cytoplasmic loops. Complex formation between the derivatized rhodopsin mutants and T was carried out by illumination at > 495 nm. Subsequent increase in pH (from 6 to 7.5 or higher) of the complex resulted in crosslinking of rhodopsin to the T␣ subunit. Crosslinking to T ␣ was demonstrated for the rhodopsin mutants K141C, S240C, and K248C, and the crosslinked sites in T␣ were identified for the rhodopsin mutant S240C. The peptides carrying the crosslinking moiety were isolated from the trypsin-digested peptide mixture, and their identification was carried out by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The main site of crosslinking is within the peptide sequence, Leu-19 -Arg-28 at the N-terminal region of T ␣. The total results show that both the N and the C termini of T ␣ are in close vicinity to the third cytoplasmic loop of rhodopsin in the complex between rhodopsin and T.cysteine mutants ͉ disulfide exchange ͉ cytoplasmic domain ͉ matrixassisted laser desorption ionization-time of flight ͉ avidin-biotin affinity chromatography I nteractions of rhodopsin with transducin (T) and rhodopsin kinase are the first steps in the two biochemical cascades that are initiated on light activation in visual signal transduction (1). Because both T ␣␤␥ and rhodopsin kinase are known to bind to the cytoplasmic domain of light-activated rhodopsin, it is important to investigate the actual contact sites in the molecular complexes formed between rhodopsin and the above two proteins. With this long-range aim, we have initiated applications of the broad covalent crosslinking approach. We (2) reported on an initial study in which we used a photoactivatable crosslinking agent (Fig. 1 I). Here we report on a study that uses a crosslinking reagent (Fig. 1 II) carrying an activated carboxyl group. An electrophilic attack by the carbonyl group on unprotonated amino groups in suitable proximity will result in crosslinking by formation of amide linkages. Therefore, only a pH change will be required in the crosslinking strategy to deprotonate the relevant amino groups, usually the -amino groups in lysine residues.As in the accompanying paper (2), rhodopsin mutants containing single reactive cysteines at predetermined positions in the cytoplasmic domain (Fig. 2) form the starting points for study of interaction with T. The crosslinking moiety, R in Fig. 1 II, is transferred to the rhodopsin mutants by a disulfide exchange reaction with the reactive pyridyl thio group in Fig. 2 II (step 1 in Fig. 3). Three mutants of rhodopsin containing single reactive cysteine residues, K141C, S240C, and K248C (Fig. 2), were selected, and all were ...

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Mutational and Computational Analysis of the α1b-Adrenergic Receptor

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Structural Features of Covalently Cross-linked Hydroxylase and Reductase Proteins of Soluble Methane Monooxygenase as Revealed by Mass Spectrometric Analysis

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