A P-loop Mutation in Gα Subunits Prevents Transition to the Active State: Implications for G-protein Signaling in Fungal Pathogenesis
Figure 3
Gαi1(G42R) engages inactive conformation-selective binding partners in two nucleotide states.
(A) Wild type Gαi1 binds Gβ1γ1 only in the GDP-bound state, as determined by SPR, while Gαi1(G42R) displayed no nucleotide state-selectivity of Gβ1γ1 binding when liganded with either GDP or GDP·AlF4−. (B) Similarly, fluorescence polarization experiments showed highly nucleotide state-selective binding of the RGS14 GoLoco motif to wild-type Gαi1·GDP (KD = 9.0±1.1 nM (s.e.m.)) compared to the AlF4−-bound form (KD = 8.7±1.0 µM (s.e.m.)), but both nucleotide states of Gαi1(G42R) interacted with the GoLoco motif peptide, with affinity constants of 45±7 nM (s.e.m.) and 168±27 nM (s.e.m.) for GDP and AlF4−, respectively. (C) The activated state-selective peptide KB-1753 preferentially bound the AlF4−-bound form of wild-type Gαi1 (KD = 470±40 nM (s.e.m.)) compared to the GDP-bound form (KD = 6.7±0.4 µM (s.e.m.)), but had low affinity for Gαi1(G42R) in both nucleotide states.