Fig. 2: GDID, DAD and RhoA form a negatively cooperative tripartite complex.

A Superimposed ¹H-¹⁵N HSQC spectra of 50 µM [U-¹⁵N]-DAD in the absence (red) and presence (black) of 60 µM GDID (black). The DAD Q1207 cross peak, which is part of the DID-DAD binding interface, undergoes a large change in chemical shift between the bound and free states. B Superimposed ¹H-¹⁵N HSQC spectra of 5 µM [U-¹⁵N]-DAD with 60 µM of RhoA and 50 µM GDID (black) and 5 µM [U-¹⁵N]-DAD with 19 µM of RhoA and 13 µM GDID (red). Free DAD is evident at the lower concentration of the RhoA-GDID complex. C NMR-generated isothermal titration of 5 µM [U-¹⁵N]-DAD binding to the RhoA-GDID complex and fit using a ‘total binding, accounting for ligand depletion’ model to estimate K_d^D/R of 27 ± 5 μM for DAD binding to the RhoA-GDID complex. D ELISA-generated isotherm of RhoA binding to 10 nM GDID fit to a “one site-total and nonspecific binding” model to estimate K_d^R of 8.9 ± 2.4 nM. E ELISA-generated isotherm of RhoA binding to 10 nM GDID-DAD fit to a ‘one site-total and nonspecific binding’ model to estimate K_d^R/D of 840 ± 200 nM. All values for ELISA experiments are represented as mean ± SD and were performed as N = 3 independent experiments. Values from individual experiments are shown as gray dots. Note that the decrease in the plateau values observed in the ELISA titrations are due to the Hook or Prozone effect in which high ligand concentrations result in a decrease in signal strength⁶¹.