Premium
Gαi binding sites on Calnuc and NUCB2 define a new family of structurally‐related Gαi‐regulatory motifs
Author(s) -
GarciaMarcos Mikel,
Kietrsunthorn Patrick,
Wang HongHui,
Ghosh Pradipta,
Farquhar Marilyn G
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.1010.4
Subject(s) - heterotrimeric g protein , binding site , sequence motif , biology , conserved sequence , plasma protein binding , structural motif , genetics , biochemistry , g protein , chemistry , peptide sequence , receptor , gene
Heterotrimeric G proteins are molecular switches modulated by families of structurally and functionally related regulators. GIV is the first non‐receptor GEF that activates Gαi‐subunits via a defined, evolutionarily conserved motif. Here we found that Calnuc and its closely related homolog NUCB2, two calcium‐binding proteins, share a common motif with GIV for Gαi binding and activation. Bioinformatic searches and structural analysis revealed that Calnuc and NUCB2 possess an evolutionarily conserved motif with sequence and structural similarity to GIV's GEF sequence. 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 docks onto the same site as GIV, i.e., the α3/Switch II cleft. Calnuc binding was impaired when K248 in the α3 helix of Gαi3 was replaced by the corresponding M residue in Gαo, which does not bind to Calnuc. Moreover, mutation of hydrophobic residues from the conserved motif predicted to dock on the α3/Switch II cleft of Gαi3 abolished Calnuc's binding to Gαi3 and impaired its GEF activity in vitro . We also demonstrate that calcium binding to Calnuc and NUCB2 abolishes their interaction with Gαi3, probably via a conformational change that renders their Gαi‐binding residues unexposed. Taken together our results define a new class of structurally‐related Gαi regulatory motifs and provide the structural basis for the properties of Calnuc and NUCB2 binding to Gα‐subunits and its regulation by calcium ions. Support: NIH grants DKI7780 and CA100768 to M.G.F. Susan G. Komen fellowship KG080079 to M.G‐M CAMS (BWF) and RSA (AGA) to P.G.