Control of the structural stability of the tubulin dimer by one high affinity bound magnesium ion at nucleotide N-site.
Tubulin liganded with GTP at the N-site in the alpha-subunit and with GDP at the E-site in the beta-subunit (GDP-tubulin) reversibly binds one high affinity Mg2+ cation (Kb = 1.1 x 10(7) M-1), whereas tubulin liganded with GTP at both subunits (GTP-tubulin) binds one more high affinity Mg2+. The two cation binding loci are identified as nucleotide sites N and E, respectively. Mg2+ at the N-site controls the stability and structure of the alphabeta-tubulin dimer. Mg2+ dissociation is followed by the slow release of bound nucleotide and functional inactivation. Mg2+ bound to the N-site significantly increases the thermal stability of the GDP-tubulin dimer (by 10 degrees C and approximately 50 kcal mol-1 of experimental enthalpy change). However, the thermal stability of Mg2+-liganded GDP- and GTP-tubulin is the same. Mg2+ binding to the N-site is linked to the alphabeta-dimer formation. The binding of Mg2+ to the alpha-subunit communicates a marked enhancement of fluorescence to a colchicine analogue bound to the beta-subunit. Colchicine, in turn, thermally stabilizes Mg2+-depleted tubulin. The tubulin properties described would be simply explained if the N-site and the colchicine site are at the alpha-beta dimerization interface. It follows that the E-site would be at the beta-end of the tubulin dimer, consistent with the known functional role of the E nucleotide gamma-phosphate and coordinated cation controlling microtubule stability.
Menéndez
M
M
Instituto de Química Física, Consejo Superior de Investigaciones Científicas, 28006 Madrid, Spain.
Rivas
G
G
Díaz
J F
JF
Andreu
J M
JM
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D019281:Dimerization; D005453:Fluorescence; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D013816:Thermodynamics; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism; D050257:Tubulin Modulators
D014404:Tubulin; D050257:Tubulin Modulators; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D008274:Magnesium