TUBULIN SECONDARY STRUCTURE-ANALYSIS, LIMITED PROTEOLYSIS SITES, AND HOMOLOGY TO FTSZ

The far-ultraviolet circular dichroism spectrum of the alpha beta-tubu lin dimer analyzed by six different methods indicates an average conte nt of approximately 33% ct helix, 21% beta sheet, and 45% other second ary structure. Deconvolution of Fourier transform infrared spectra ind icates 24% sheet, 37% (maximum) helix, and 38% (minimum) other structu re. Separate alignments of 75 alpha-tubulin, 106 beta-tubulin, and 14 gamma-tubulin sequences and 12 sequences of the bacterial cell divisio n protein FtsZ have been employed to predict their secondary structure s with the multiple-sequence method PHD [Rest, B., & Sander, C. (1993a ) J. Mel. Biol. 232, 584-599]. The predicted secondary structures aver age of 33% a helix, 24% beta sheet, and 43% loop for the alpha beta di mer. The predictions have been compared with sites of limited proteoly sis by 12 proteases at the surfaces of the heterodimer and taxol-induc ed microtubules [de Pereda, J. M., & Andreu, J. M. (1996) Biochemistry , 35, 14184-14202]. From 24 experimentally determined nicking sites, 1 8 are at predicted loops or at the extremes of secondary structure ele ments. Proteolysis zone A (including acetylable Lys40 and probably Lys 60 in alpha-tubulin and Gly93 in beta-tubulin) and proteolysis zone B (extending between residues 167 and 183 in both chains) are accessible in microtubules. Proteolysis zone C, between residues 278 and 295, be comes partially occluded in microtubules. The cl-tubulin nicking site Arg339-Ser340 is at a loop following a predicted or helix in proteolys is zone D. This site is protected in taxol microtubules; however, a ne w tryptic site appears which is probably located at the N-terminal end of the same helix. Zone D also contains beta-tubulin Cys354, which is accessible in microtubules. Proteolysis zone E includes the C-termina l hypervariable loops (10-20 residues) of each tubulin chain. These fo llow the two larger predicted helical zones (residues 372-395 and 405- 432 in beta-tubulin), which also are the longer conserved part of the alpha- and beta-tubulin sequences. Through combination of this with ot her biochemical information, a set of surface and distance constraints is proposed for the folding of P-tubulin. The FtsZ sequences are only 10-18% identical to the tubulin sequences. However, the predicted sec ondary structures show two clearly similar (85-87 and 51-78%) regions, at tubulin positions 95-175 and 305-350, corresponding to FtsZ 65-135 and 255-300, respectively. The first region is flanked by tubulin pro teolysis zones A and B. It consists of a predicted loop1-helix-loop2-s heet-loop3-helix-loop4-sheet fold, which contains the motif (KR)GXXXXG (loop1), and the tubulin-FtsZ signature G-box motif (SAG)GGTG(SAT)G ( loop3). A simple working model envisages loop1 and loop3 together at t he nucleotide binding site, while loops 2 and 4 are at the surface of the protein, in agreement with proteolytic and antigenic accessibility results in tubulin. The model is compatible with studies of tubulin a nd FtsZ mutants. It is proposed that this region constitutes a common structural and evolutionary nucleus of tubulins and FtsZ which is diff erent from typical GTPases.