Volume 3, Issue 5, Pages 649-660 (May 1999) A Molecular Mechanism for the Phosphorylation-Dependent Regulation of Heterotrimeric G Proteins by Phosducin  Rachelle Gaudet, Justin R Savage, Joseph N McLaughlin, Barry M Willardson, Paul B Sigler  Molecular Cell  Volume 3, Issue 5, Pages 649-660 (May 1999) DOI: 10.1016/S1097-2765(00)80358-5

Figure 6 Interactions of Phospho-Phosducin and Gtα with Gtβγ (A) Ribbon diagram of the phosducin/Gtβγ complex where residues 67–86, which become disordered upon Ser-73 phosphorylation, are colored cyan. The N-terminal domain of phosducin is purple, with its 30-residue flexible loop in green. The C-terminal domain is blue. Gtβ is gold, and Gtγ is silver. (B) The transducin heterotrimer (Lambright et al. 1996) with Gtα in red, the GDP in black, and Gtβγ colored as in (A). Residues 67–86 in phosducin overlap the Gtα-Gtβγ interaction surface. (C) Ribbon diagram (stereo pair) of the phosducin/Gtβγ interface near Helix 2. Residues involved in the interactions with phosducin segment from Arg-67 to Asp-86 are shown in ball-and-stick representations. Phosducin residues are labeled with purple or cyan symbols, and Gtβγ residues with black symbols. The complex is viewed in the same “top” orientation in all three panels. Molecular Cell 1999 3, 649-660DOI: (10.1016/S1097-2765(00)80358-5)

Figure 1 Functional Assays of Full-Length Phosducin Variants (A) Comparison of residue structures studied at position 73 of phosducin. (B) Gel-shift assay of the full-length phosducin variants. The S73E variant and phospho-phosducin do not form a gel-shifted complex with Gtβγ. A fraction of phosducin, S73A, S73D, and S73Q form gel-shifted complexes to various degrees. (C) Inhibition of Gt binding to rhodopsin by phosducin. Inhibition of 125I-Gtα binding to light-activated urea-stripped membranes was measured at increasing concentrations of various phosducin variants. Molecular Cell 1999 3, 649-660DOI: (10.1016/S1097-2765(00)80358-5)

Figure 2 Biophysical Analysis of Full-Length Phosducin Variants (A) CD spectra of full-length phosducin variants. (B) Relationship of Trp-29 to the site of phosphorylation, Ser-73. (C) Tryptophan fluorescence spectra of the five full-length phosducin variants. Although error bars are omitted for clarity, both the CD and tryptophan fluorescence spectra are fully overlapping when a 5% error margin in concentration measurements is considered. Molecular Cell 1999 3, 649-660DOI: (10.1016/S1097-2765(00)80358-5)

Figure 3 Analysis of Phosducin’s Isolated Domains (A) Native gel of the unphosphorylated and phospho-phosducin N-terminal domain. The phosphorylated N-terminal domain migrates faster than its unphosphorylated counterpart. Only the unphosphorylated N-terminal domain shows a gel-shifted complex with Gtβγ. (B) CD spectra of phosducin and its domains. (C) Tryptophan fluorescence spectra of phosducin and its domains. Molecular Cell 1999 3, 649-660DOI: (10.1016/S1097-2765(00)80358-5)

Figure 4 One-Dimensional Proton NMR of the N-Terminal Domain (A and C) Amide region. (B and D) Methylene region. The spectrum of the phosphorylated N-terminal domain is shown at the top (A and B) and that of the unphosphorylated N-terminal domain is at the bottom (C and D). Arrows point to spectral features that differ between the two forms of the domain. Molecular Cell 1999 3, 649-660DOI: (10.1016/S1097-2765(00)80358-5)

Figure 5 Electron Density Maps of Phosducin’s Helix 2 after Molecular Replacement Helix 2 residues pointing toward the top of each panel are solvent exposed, whereas those pointing at the bottom of the panel interact with Gtβ and/or phosducin’s Helix 1. (A) and (C) show unbiased Fo–Fc maps calculated for the unphosphorylated phosducin/Gtβγ complex structure and the phospho-phosducin/Gtβγ complex structure, respectively, with rigid body–refined molecular replacement phases where the whole phosducin N-terminal domain, including the region shown in these maps, was omitted from the model. Positive density (ρ > 1.7σ) is cyan, and negative density (ρ < −1.7σ) is magenta. The phospho-phosducin/Gtβγ map shows no density above noise level, whereas the unphosphorylated phosducin/Gtβγ map shows nearly continuous backbone density. (B) and (D) show composite simulated annealing omit maps calculated with the final model of each structure and contoured at 1.3σ. Residues shown in this figure are included in the refined model only for the unphosphorylated phosducin/Gtβγ structure and are not included in the phospho-phosducin/Gtβγ model; the unphosphorylated structure is shown in all panels of this figure for reference. Corresponding maps calculated for the S73E/Gtβγ structure are comparable to the phospho-phosducin/Gtβγ maps. Molecular Cell 1999 3, 649-660DOI: (10.1016/S1097-2765(00)80358-5)

Figure 7 The N-Cap Region of Phosducin’s Helix 2, the Ser-73 Phosphorylation Site (A) The structure of the phosphorylation site in the unphosphorylated state. (B) A phosphoserine was modeled to show the steric clash with Gln-75 and Glu-76. The Ser-73 side chain is fixed in the nonphosphorylated orientation. Molecular Cell 1999 3, 649-660DOI: (10.1016/S1097-2765(00)80358-5)

Figure 8 The Effect of Substitutions of Gln-75 and/or Glu-76 on the Half-Maximal Inhibition of Gt Binding to Rhodopsin The K1/2 values were measured for the unphosphorylated and phosphorylated variants to test the effect of substitutions on phosducin’s regulation by phosphorylation. Molecular Cell 1999 3, 649-660DOI: (10.1016/S1097-2765(00)80358-5)