Molecular dissection of GTP exchange and hydrolysis within the ternary complex of tubulin heterodimers and Op18/stathmin family members.
The ubiquitous Op18 and the neural RB3 and SCG10 proteins are members of the oncoprotein18/stathmin family of microtubule regulators. These proteins bind two tubulin heterodimers via two imperfect helical repeats to form a complex of heterodimers aligned head-to-tail. Here we have analyzed GTP exchange and GTP hydrolysis at the exchangeable GTP-binding site (E-site) of tubulin heterodimers in complex with Op18, RB3, or SCG10. These proteins stimulate a low and indistinguishable rate of GTP hydrolysis, and our results show that GTP exchange is blocked at both E-sites of the ternary complex, whereas GTP hydrolysis only occurs at one of the two E-sites. Results from mutational analysis of clusters of hydrophobic residues within the first helical repeat of Op18 suggest that GTP is hydrolyzed at the E-site that is interfaced between the head-to-tail arranged heterodimers, which is consistent with predicted GTPase productive interactions between the two tubulin heterodimers. Our mutational analysis has also indicated that Op18/stathmin family members actively restrain the otherwise potent GTPase productive interactions that are generated by longitudinal interactions within protofilaments. We conclude that tubulin heterodimers in complex with Op18/stathmin family members are subject to allosteric effects that prevent futile cycles of GTP hydrolysis.
Brännström
Kristoffer
K
Department of Molecular Biology, Umeå University, SE-901 87 Umeå, Sweden.
Segerman
Bo
B
Gullberg
Martin
M
D000494:Allosteric Regulation; D004252:DNA Mutational Analysis; D019281:Dimerization; D004926:Escherichia coli; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D008868:Microtubule Proteins; D009414:Nerve Growth Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D050777:Stathmin; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism
D008868:Microtubule Proteins; D009414:Nerve Growth Factors; D010750:Phosphoproteins; C491795:STMN4 protein, human; D050777:Stathmin; D014404:Tubulin; D006160:Guanosine Triphosphate