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. 2001 Apr 24;98(9):4877-82.
doi: 10.1073/pnas.051632898.

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

K Cai  1 , Y Itoh, H G Khorana
Affiliations

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

K Cai et al. Proc Natl Acad Sci U S A. .

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 disulfide-exchange reaction with the pyridylthio group, and the derivatized rhodopsin then is complexed with T by illumination at lambda >495 nm. Subsequent irradiation of the complex at lambda310 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(alpha). For identification of the sites of crosslinks in T(alpha), 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(alpha) species carrying the crosslinked moiety with a free SH group; (iii) adduct formation of the latter with the N-maleimide moiety of the reagent, maleimido-butyryl-biocytin, containing a biotinyl group; (iv) trypsin degradation of the resulting T(alpha) derivatives and isolation of T(alpha) peptides carrying maleimido-butyryl-biocytin by avidin-agarose chromatography; and (v) identification of the isolated peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We found that crosslinking occurred mainly to two C-terminal peptides in T(alpha) containing the amino acid sequences 310-313 and 342-345.

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Figures

Figure 1
Figure 1
A secondary structure model of rhodopsin showing positions of the single cysteine-containing mutants that were tested for attachment of R (I) and showed photocrosslinking with heterotrimeric Tαβγ (circled in blue). Mutant S240C (circled in red) was taken through all steps in the strategy described in Fig. 3.
Figure 2
Figure 2
The photoactivatable reagent N-[(2-pyridyldithio)ethyl]-4-azido-salicylamide (I). The group R is transferred to the SH group of cysteine residue in a rhodopsin mutant by a disulfide-exchange reaction with the reactive pyridylthio group in I.
Figure 3
Figure 3
Steps in strategy for photocrosslinking (A) and subsequent analysis of crosslinked sites in Tαβγ (B). See text for details.
Figure 4
Figure 4
Structure of MBB, an SH-specific reagent containing a biotinyl group.
Figure 5
Figure 5
Light-dependent binding of GDP/Tαβγ to wild-type rhodopsin bound to 1D4-Sepharose. Analysis was performed by SDS/PAGE (reducing). Tαβγ was visualized by immunoblotting using Ab Mab4A against Tα. Samples were treated as described in Materials and Methods. After extensive washing of 1D4 Sepharose beads both in the dark (D) and after illumination (L), Tα and rhodopsin were eluted together from the matrix with the epitope peptide (lane 2, dark; lane 3, after illumination). Tα was released also alone from the matrix by the addition of GTP (lane 4, dark; lane 5, after illumination). Controls are: lane 1, Tαβγ; lanes 6 and 7, controls of lanes 3 and 5 without 1D4-Sepharose-bound rhodopsin.
Figure 6
Figure 6
SDS/PAGE analysis of products formed in crosslinking of rhodopsin derivative S240C-R and Tαβγ. The photocrosslinking procedure was performed as described in Fig. 3. All samples were treated with 10 mM DTT immediately after the addition of Laemmli SDS sample buffer. The gel was visualized by Coomassie blue staining. (A) After GTP washing (Step 4) to remove uncrosslinked Tαβγ (lane 2), sample V was reduced by DTT and the eluted protein is shown in lane 1. Lane 3 contains 25 pmol of purified Tαβγ as a control. (B) Sample V in Fig. 3 was treated with NEM and eluted with DTT, and the eluate was then derivatized with MBB (Steps 5–7) in Fig. 3B. Sample VIII (Fig. 3B) then was analyzed by Western blotting using antibodies against Tα (I), Tβ (II), and biotinyl moiety (III). Lane 1 in all three cases contained purified Tαβγ as a control.
Figure 7
Figure 7
MALDI analysis of the MBB-labeled tryptic fragments of T2 obtained after avidin affinity purification (Fig. 3B, ). Peaks labeled in red are the identified fragments. Amino acid sequences and the predicted masses of these fragments are indicated in the insets. Unidentified peaks labeled in blue also appeared in the two control samples (trypsin alone and Tα-MBB alone, data not shown).
Figure 8
Figure 8
Amino acid sequence of Tα. Shaded sequences indicate the secondary structural elements in the C-terminal region (26). The two regions identified from photocrosslinking are highlighted in red. Sequence 310–313 (R DVK), is located between the α4 and β6 regions of Tα, and the sequence 342–345 is near the C terminus.
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Comment in

  • How activated receptors couple to G proteins.
    Hamm HE. Hamm HE. Proc Natl Acad Sci U S A. 2001 Apr 24;98(9):4819-21. doi: 10.1073/pnas.011099798. Proc Natl Acad Sci U S A. 2001. PMID: 11320227 Free PMC article. No abstract available.

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