Characterization of a Goα Mutant That Binds Xanthine Nucleotides

Abstract

Several GTP binding proteins, including EF-Tu, Ypt1, rab-5, and FtsY, and adenylosuccinate synthetase have been reported to bind xanthine nucleotides when the conserved aspartate residue in the NKXD motif was changed to asparagine. However, the corresponding single Goα mutant protein (D273N) did not bind either xanthine nucleotides or guanine nucleotides. Interestingly, the introduction of a second mutation to generate the Goα subunit D273N/Q205L switched nucleotide binding specificity to xanthine nucleotide. The double mutant protein GoαD273N/Q205L (GoαX) bound xanthine triphosphate, but not guanine triphosphate. Recombinant GoαX (GoαD273N/Q205L) formed heterotrimers with βγ complexes only in the presence of xanthine diphosphate (XDP), and the binding to βγ was inhibited by xanthine triphosphate (XTP). Furthermore, as a result of binding to XTP, the GoαX protein underwent a conformational change similar to that of the activated wild-type Goα. In transfected COS-7 cells, we demonstrate that the interaction between GoαX and βγ occurred only when cell membranes were permeabilized to allow the uptake of xanthine diphosphate. This is the first example of a switch in nucleotide binding specificity from guanine to xanthine nucleotides in a heterotrimeric G protein α subunit.