Mutational analysis of op18/stathmin-tubulin-interacting surfaces. Binding cooperativity controls tubulin GTP hydrolysis in the ternary complex.
Oncoprotein 18 (Op18) is a microtubule regulator that forms a ternary complex with two tubulin heterodimers. Dispersed regions of Op18 are involved in two-site cooperative binding and subsequent modulation of tubulin GTPase activity. Here we have analyzed specific determinants of Op18 that govern both stoichiometry and positive cooperativity in tubulin binding and consequent stimulatory and inhibitory effects on tubulin GTPase activity. The data revealed that the central and C-terminal regions of Op18 contain overlapping binding-motifs contacting both tubulin heterodimers, suggesting that these regions of Op18 are wedged into the previously noted 1-nm gap between the two longitudinally arranged tubulin heterodimers. Both the N- and C-terminal flanks adjacent to the central region are involved in stabilizing the ternary complex, but only the C-terminal flank does so by imposing positive binding cooperativity. Within the C-terminal flank, deletion of a 7-amino acid region attenuated positive binding cooperativity and resulted in a switch from stimulation to inhibition of tubulin GTP hydrolysis. This switch can be explained by attenuated binding cooperativity, because Op18 under these conditions may block longitudinal contact surfaces of single tubulins with consequent interference of tubulin-tubulin interaction-dependent GTP hydrolysis. Together, our results suggest that Op18 links two tubulin heterodimers via longitudinal contact surfaces to form a ternary GTPase productive complex.
Segerman
B
B
Department of Cell and Molecular Biology, University of Umeå, S-901 87 Umeå, Sweden.
Larsson
N
N
Holmfeldt
P
P
Gullberg
M
M
D000818:Animals; D001665:Binding Sites; D019281:Dimerization; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D046911:Macromolecular Substances; D008868:Microtubule Proteins; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D017384:Sequence Deletion; D050777:Stathmin; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*; D050257:Tubulin Modulators
D046911:Macromolecular Substances; D008868:Microtubule Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D050777:Stathmin; D014404:Tubulin; D050257:Tubulin Modulators; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases