High resolution crystal structures of human Rab5a and five mutants with substitutions in the catalytically important phosphate-binding loop.
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 A. Of particular interest, the A30P mutant was crystallized in complexes with GDP, GDP+AlF(3), 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 AlF(3) 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 Gln(79) 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.
Zhu
Guangyu
G
Crystallography Research Program of Oklahoma Medical Research Foundation, Oklahoma City 73104, USA.
Liu
Jian
J
Terzyan
Simon
S
Zhai
Peng
P
Li
Guangpu
G
Zhang
Xuejun C
XC
D002384:Catalysis; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D010710:Phosphates / Q000737:chemistry; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics*
D010710:Phosphates; D011994:Recombinant Proteins; D006160:Guanosine Triphosphate; D004247:DNA; D011392:Proline; D020696:rab5 GTP-Binding Proteins