On the significance of Toc-GTPase homodimers.
Precursor protein translocation across the outer chloroplast membrane depends on the action of the Toc complex, containing GTPases as recognizing receptor components. The G domains of the GTPases are known to dimerize. In the dimeric conformation an arginine contacts the phosphate moieties of bound nucleotide in trans. Kinetic studies suggested that the arginine in itself does not act as an arginine finger of a reciprocal GTPase-activating protein (GAP). Here we investigate the specific function of the residue in two GTPase homologues. Arginine to alanine replacement variants have significantly reduced affinities for dimerization compared with wild-type GTPases. The amino acid exchange does not impact on the overall fold and nucleotide binding, as seen in the monomeric x-ray crystallographic structure of the Arabidopsis Toc33 arginine-alanine replacement variant at 2.0A. We probed the catalytic center with the transition state analogue GDP/AlF(x) using NMR and analytical ultracentrifugation. AlF(x) binding depends on the arginine, suggesting the residue can play a role in catalysis despite the non-GAP nature of the homodimer. Two non-exclusive functional models are discussed: 1) the coGAP hypothesis, in which an additional factor activates the GTPase in homodimeric form; and 2) the switch hypothesis, in which a protein, presumably the large Toc159 GTPase, exchanges with one of the homodimeric subunits, leading to activation.
Heidelberg University Biochemistry Center, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany.
Patrick
P
Koenig
Mislav
M
Oreb
Karsten
K
Rippe
Claudia
C
Muhle-Goll
Irmgard
I
Sinning
Enrico
E
Schleiff
Ivo
I
Tews
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000378:metabolism; D001703:Biophysics / Q000379:methods; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray / Q000379:methods; D019281:Dimerization; D020558:GTP Phosphohydrolases / Q000737:chemistry*; D008565:Membrane Proteins / Q000378:metabolism*; D008954:Models, Biological; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009154:Mutation; D010940:Plant Proteins / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D008565:Membrane Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; C112475:Toc34 protein, plant; D020558:GTP Phosphohydrolases