Obligatory role in GTP hydrolysis for the amide carbonyl oxygen of the Mg2+-coordinating Thr of regulatory GTPases

The crystal structures of transducin α show that in the MgGTP activated conformation (1TND), the amide carbonyl group of the Mg-coordinating Thr177 and the γ-carbonyl group of Gln200 are equidistant from the catalytic H20 located in “front” of the γ-phosphory l of GTP. (A) In the GTPγS-Mg liganded form of the protein, SWI, SWII (to which Gln200 belongs), and Arg174 are oriented in such a way that the catalytic H2O that will capture the γPO3 split off the GTP and leave the active site as part of the newly formed Pi. The Mg-coordinating Ser43 and Thr177 are shown in gray, with the O of the OH groups shown as pink balls, and Mg is shown with four of the six coordinating oxygens: one from the brown γP, one from the brown βP, and one each from the OH groups of Ser and Thr. The coordination shell is completed by the electrons from two additional water oxygens [not shown here, but seen in figure 1 of Zurita and Birnbaumer (17)]. Mg-O distances are shown as green lines with corresponding lengths in A. Distances between the catalytic water oxygen (w) and the Thr177 amide carbonyl O, the γ carbonyl O of Gln200, and the γ-phosphoryl O are also shown as green lines with corresponding lengths in A. The distance between the β-γ ester bond (white) and Arg174, referred to as the “Arg finger” by Wittinghofer and colleagues (24) when GTPase activating G proteins (GAPs) are available to interact with ras-like regulatory GTPases, is also shown as a green line with the corresponding length in A. (B) In the Mg-GDP-Al4F liganded conformation of *Tα stabilized by RGS4 (1FQK)—which is presumed to be equivalent to the transition form of the GTPase—one sees that the backbone carbonyl of Thr177 as well as the γ carbonyl of the Gln have both moved closer to the catalytic H2O oxygen. (C) In the GDP-Mg liganded conformation of Tα (1TAG) none of the critical amino acids are close enough to establish meaningful contacts; especially Thr177 cannot be pulled in to confer an effector regulating conformation to SWII. Crystal structures of Gi1α in the MgGTP conformation [1GIA with GTPγS or 1CIP with GMP-P(NH)P] and GDP conformation (1BOF, 1GP2) and of Gsα in the MgGTPγS conformation (e.g., 1AZT, 1AZS) are in agreement with structural features highlighted in this figure.