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J. Biol. Chem., Vol. 283, Issue 34, 23104-23112, August 22, 2008

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On the Significance of Toc-GTPase Homodimers^*

Patrick Koenig¹, Mislav Oreb, Karsten Rippe^¶, Claudia Muhle-Goll^||, Irmgard Sinning, Enrico Schleiff, and Ivo Tews²

From the Heidelberg University Biochemistry Center, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany, JWGU Frankfurt am Main, Cluster of Excellence Macromolecular Complexes, Department of Biosciences, Max-von-Laue Strasse 9, 60439 Frankfurt, Germany, ^¶Deutsches Krebsforschungszentrum and BIOQUANT, Research Group Genome Organization and Function, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany, and ^||Karlsruhe Institute of Technology, Institut für Biologische Grenzflächen, POB 3640, 76021 Karlsruhe, Germany

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 2.0Å. 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.

Received for publication, December 31, 2007 , and in revised form, April 15, 2008.

The atomic coordinates and structure factors (code 3DEF) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* The work was supported by Deutsche Forschungsgemeinschaft Grant SFB594-B11 (to E. S.) and by a grant from the Volkswagenstiftung (to E. S. and K. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by a fellowship of the interdisciplinary Ph.D. program "Molecular machines: mechanisms and functional interconnections" of the Land Baden-Württemberg (to I. S.).

² To whom correspondence should be addressed. Tel.: 49-6221-5447-85; Fax: 49-6221-5447-90; E-mail: ivo.tews{at}bzh.uni-heidelberg.de.

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