Figure 2. Fbx4_G is an Atypical Small GTPase Domain without GTP Binding Activity.

(A) Sequence alignment in the loop regions of Fbx4_G and a group of small GTPases. The conserved residues important for nucleotide binding are highlighted in different colors;the P-loop is in green, Switch I in yellow, Switch II in cyan, G4 in orange and G5 in magenta.

(B) The conformation of Fbx4_G observed in the crystal structure is incompatible with GTP binding. Left: ribbon representation of Fbx4_G with modeled GTP and Mg²⁺. The Ploop, Switch II and the G4-loop of Fbx4_G block the binding of GTP. Right: ribbon representation of the Arf1A-GTP complex (PDB: 2J59). The coloring scheme of the loop regions is the same as in (A). GTP is shown as a stick model and Mg²⁺ a black ball.

(C) Times courses of GTP binding for Fbx4_G and the Fbx4_G-TRF1_TRFH complex. GTP binding was monitored by the increase in fluorescence millipolarization (mP) of a fluorescent GTP analog as it was bound. In this experiment, neither Fbx4_G nor the Fbx4_GTRF1_TRFH complex bound to GTP. RhoA (a small GTPase) and p63RhoGEF (a non GTPase protein) were used as positive and negative controls, respectively.