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 Goalpha mutant protein (D273N) did not bind either xanthine nucleotides or guanine nucleotides. Interestingly, the introduction of a second mutation to generate the Goalpha subunit D273N/Q205L switched nucleotide binding specificity to xanthine nucleotide. The double mutant protein GoalphaD273N/Q205L (GoalphaX) bound xanthine triphosphate, but not guanine triphosphate. Recombinant GoalphaX (GoalphaD273N/Q205L) formed heterotrimers with betagamma complexes only in the presence of xanthine diphosphate (XDP), and the binding to betagamma was inhibited by xanthine triphosphate (XTP). Furthermore, as a result of binding to XTP, the GoalphaX protein underwent a conformational change similar to that of the activated wild-type Goalpha. In transfected COS-7 cells, we demonstrate that the interaction between GoalphaX and betagamma 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 alpha subunit.
Highlights
§ To whom correspondence should be addressed: Division of Biology, California Institute of Technology, Pasadena, CA 91125
To change the binding specificity of Goa from guanine nucleotides to xanthine nucleotides, we replaced Asp-273 by an asparagine residue, which was expected on the basis of structural analysis to coordinate with xanthine instead of guanine (Fig. 1b)
We engineered a mutant of Goa that switched nucleotide binding activity from guanine nucleotides to xanthine nucleotides
Summary
Crystal structures of the a subunits of transducin and Gi have been recently solved (8 –11) Both Ga structures had nearly identical binding pockets for the guanine nucleotide, which was similar to the guanine nucleotide binding pocket revealed in the crystal structures of Ras [12] and EF-Tu [13, 14]. It has been shown that the characteristic hydrogen bond formed with the aspartic acid residue determines the specificity of guanine nucleotide binding in other GTP-binding proteins, such as EF-Tu and Ras [15, 16]. A mutation of aspartate to asparagine at this position in several GTP binding proteins, including EF-Tu [17, 18], Ypt1 [19], rab-5 [20, 21], and FtsY [22] and adenylosuccinate synthetase [23], leads to active proteins regulated by xanthine nucleotides instead of guanine nucleotides. We studied the effect of the similar D273N mutation on nucleotide binding specificity of Goa
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