Bacterial Tubulin Distinct Loop Sequences and Primitive Assembly Properties Support its Origin from a Eukaryotic Tubulin Ancestor

Abstract

The structure of the unique bacterial tubulin BtubA/B from Prosthecobacter is very similar to eukaryotic alphabeta-tubulin, but strikingly, BtubA/B fold without eukaryotic chaperones. Our sequence comparisons indicate that BtubA and BtubB do not really correspond to either alpha or beta-tubulin but have mosaic sequences with intertwining features from both. Their nucleotide binding loops are more conserved and their more divergent sequences correspond to discrete surface zones of tubulin involved in microtubule assembly and binding to eukaryotic chaperonin CCT, which is absent from the P. dejongeii draft genome. BtubA/B cooperatively assembles over a wider range of conditions than alphabeta-tubulin, forming pairs of protofilaments which coalesce into bundles instead of microtubules, and it lacks the abilities to differentially interact with divalent cations and bind typical tubulin drugs. Assembled BtubA/B contain close to one bound GTP and GDP. Both BtubA and BtubB subunits hydrolyze GTP, leading to disassembly. The mutant BtubA/B-S144G in the tubulin signature motif GGG(T/S)G(S/T)G has strongly inhibited GTPase, but BtubA-T147G/B does not, suggesting that BtubB is a more active GTPase, like beta-tubulin. BtubA/B chimera bearing the beta-tubulin loops M, H1-S2 and S9-S10 in BtubB fold, assemble and have reduced GTPase. However, introduction of the alpha-tubulin loop S9-S10 with its unique 8-residue insertion impaired folding. From the sequence analyses, its primitive assembly features and the properties of the chimeras, we propose that BtubA/B were acquired shortly after duplication of a spontaneously folding alpha and beta-tubulin ancestor, possibly by horizontal gene transfer from a primitive eukaryotic cell, followed by divergent evolution