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J. Biol. Chem., Vol. 278, Issue 4, 2452-2460, January 24, 2003

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High Resolution Crystal Structures of Human Rab5a and Five Mutants with Substitutions in the Catalytically Important Phosphate-binding Loop^*

Guangyu Zhu, Jian Liu^§, Simon Terzyan, Peng Zhai, Guangpu Li^§, and Xuejun C. Zhang^¶

From the  Crystallography Research Program of Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104 and the ^§ Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104

GTPase domain crystal structures of Rab5a wild type and five variants with mutations in the phosphate-binding loop are reported here at resolutions up to 1.5 Å. Of particular interest, the A30P mutant was crystallized in complexes with GDP, GDP+AlF₃, and authentic GTP, respectively. The other variant crystals were obtained in complexes with a non-hydrolyzable GTP analog, GppNHp. All structures were solved in the same crystal form, providing an unusual opportunity to compare structures of small GTPases with different catalytic rates. The A30P mutant exhibits dramatically reduced GTPase activity and forms a GTP-bound complex stable enough for crystallographic analysis. Importantly, the A30P structure with bound GDP plus AlF₃ has been solved in the absence of a GTPase-activating protein, and it may resemble that of a transition state intermediate. Conformational changes are observed between the GTP-bound form and the transition state intermediate, mainly in the switch II region containing the catalytic Gln⁷⁹ residue and independent of A30P mutation-induced local alterations in the P-loop. The structures suggest an important catalytic role for a P-loop backbone amide group, which is eliminated in the A30P mutant, and support the notion that the transition state of GTPase-mediated GTP hydrolysis is of considerable dissociative character.

The atomic coordinates and the structure factors (code 1N6H, 1N6I, 1N6K, 1N6L, 1N6N, 1N6O, 1N6P, and 1N6R) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

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