NMR structure of free RGS4 reveals an induced conformational change upon binding G alpha

Heterotrimeric guanine nucleotide-binding proteins (G-proteins) are transdu cers in many cellular transmembrane signaling systems where regulators of C -protein signaling (RGS) act as attenuators of the G-protein signal cascade by binding to the G alpha subunit of G-proteins (G(i alpha l)) and increas ing the rate of GTP hydrolysis, The high-resolution solution structure of f ree RGS4 has been determined using two-dimensional and three-dimensional he teronuclear NMR spectroscopy. A total of 30 structures were calculated by m eans of hybrid distance geometry-simulated annealing using a total of 2871 experimental NMR restraints, The atomic rms distribution about the mean coo rdinate positions for residues 5-134 for the 30 structures is 0.47 +/- 0.05 Angstrom for the backbone atoms, 0.86 +/- 0.05 Angstrom for all atoms, and 0.56 +/- 0.04 Angstrom for all atoms excluding disordered side chains. The NMR solution structure of free RGS4 suggests a significant conformational change upon binding G(i alpha l) as evident by the backbone atomic rms diff erence of 1.94 Angstrom between the free and bound forms of RGS4. The under lying cause of this structural change is a perturbation in the secondary st ructure elements in the vicinity of the Gi alpha l binding site. A kink in the helix between residues K116-Y119 is more pronounced in the RGS4-G(i alp ha l) X-ray structure relative to the free RGS4 NMR structure, resulting in a reorganization of the packing of the N-terminal and C-terminal helices. The presence of the helical disruption in the RGS4-G(i alpha l) X-ray struc ture allows for the formation of a hydrogen-bonding network within the bind ing pocket for G(i alpha l) on RGS4, where RGS4 residues D117, S118, and R1 21 interact with residue T182 from G(i alpha l). The binding pocket for G(i alpha l) on RGS4 is larger and more accessible in the free RGS4 NMR struct ure and does not present the preformed binding site observed in the RGS4-G( i alpha l) X-ray structure. This observation implies that the successful co mplex formation between RGS4 and G(i alpha l) is dependent on both the form ation of the bound RGS4 conformation and the proper orientation of T182 fro m G(i alpha l). The observed changes fur the free RGS4 NMR structure sugges t a mechanism for its selectivity for the G alpha-GTP-Mg2+ complex and a me ans to facilitate the GTPase cycle.