G protein binding sites on calnuc (nucleobindin 1) and NUCB2 (nucleobindin 2) define a new class of Gα i-regulatory motifs

Abstract

Heterotrimeric G proteins are molecular switches modulated by families of structurally and functionally related regulators. GIV (Gα-interacting vesicle-associated protein) is the first non-receptor guanine nucleotide exchange factor (GEF) that activates Gαi subunits via a defined, evolutionarily conserved motif. Here we found that Calnuc and NUCB2, two highly homologous calcium-binding proteins, share a common motif with GIV for Gαi binding and activation. Bioinformatics searches and structural analysis revealed that Calnuc and NUCB2 possess an evolutionarily conserved motif with sequence and structural similarity to the GEF sequence of GIV. Using in vitro pulldown and competition assays, we demonstrate that this motif binds preferentially to the inactive conformation of Gαi1 and Gαi3 over other Gα subunits and, like GIV, docks onto the α3/switch II cleft. Calnuc binding was impaired when Lys-248 in the α3 helix of Gαi3 was replaced with M, the corresponding residue in Gαo, which does not bind to Calnuc. Moreover, mutation of hydrophobic residues in the conserved motif predicted to dock on the α3/switch II cleft of Gαi3 impaired the ability of Calnuc and NUCB2 to bind and activate Gαi3 in vitro. We also provide evidence that calcium binding to Calnuc and NUCB2 abolishes their interaction with Gαi3 in vitro and in cells, probably by inducing a conformational change that renders the Gαi-binding residues inaccessible. Taken together, our results identify a new type of Gαi-regulatory motif named the GBA motif (for Gα-binding and -activating motif), which is conserved across different proteins throughout evolution. These findings provide the structural basis for the properties of Calnuc and NUCB2 binding to Gα subunits and its regulation by calcium ions. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.