Portal de Periódicos da CAPES

Mapping Surface Sequences of the Tubulin Dimer and Taxol-Induced Microtubules with Limited Proteolysis

1996; American Chemical Society; Volume: 35; Issue: 45 Linguagem: Inglês

10.1021/bi961356j

1943-295X

José M. de Pereda, José M. Andreu,

14-3-3 protein interactions

Native tubulin αβ dimers and microtubules have been subjected to limited proteolysis with trypsin, chymotrypsin, elastase, clostripain, proteinase lysine-C, thermolysin, protease V8, papain, subtilisin, proteinase K, proteinase aspartic-N, and bromelain. Eighty nicking points have been mapped onto the α- and β-tubulin sequences with the aid of site-directed antibodies, of which 18 sites have been exactly determined by N-terminal sequencing, and the probable position of 6 others deduced from protease specificities. Proteolytic sites cluster into five characteristic zones, including the C termini of both chains. Residues accessible to proteases in the tubulin dimer include α-tubulin Lys40-Thr41-Ile42, Glu168-Phe169-Ser170, Ser178-Thr179-Ala180-Val181, Lys280-Ala281, Glu290-Ile291, Ala294-Cys295, Arg339-Ser340 (plus probably Lys60-His61 and Glu183-Pro184) and β-tubulin Gly93-Gln94, Lys174-Val175, Gly277-Ser278, Tyr281-Arg282-Ala283, Cys354-Asp355 (plus probably Arg121-Lys122, Phe167-Ser168, Tyr183-Asn184, and Glu426-Asp427 or Ala430-Asp431). While the majority of these sites remain accessible at the outer surface of taxol-induced microtubules, α-tubulin Lys280-Ala281, Arg339-Ser340 and β-tubulin Tyr281-Arg282-Ala283 (and probably Arg121-Lys122) become protected from limited proteolysis, suggesting that they are close to or at intermolecular contacts in the assembled structure. The protease nicking points constitute sets of surface constraints for any three-dimensional model structures of tubulin and microtubules. The dimer tryptic site at α-tubulin 339-340 jumps approximately 12−22 residues upstream (probably to Lys326-Asp327 or Lys311-Tyr312) in taxol microtubules, suggesting a tertiary structural change. The cleavage of the approximately 10 C-terminal residues of α-tubulin by protease V8, papain, and subtilisin is inhibited in taxol microtubules compared to tubulin dimers, while the approximately 20 C-terminal residues of β-tubulin are similarly accessible to protease V8, subtilisin, proteinase K, proteinase AspN, and bromelain and show enhanced papain cleavage. This is consistent with models in which the α-tubulin C-terminal zone is near the interdimer contact zone along the protofilaments, whereas the C terminus of β is near the interface between both subunits.