UCSF

The tubulin superfamily is a unique set of GTPases, which, while they have high sequence diversity, have remarkably similar tertiary structures. These proteins share a very conserved longitudinal interface, but are able to form a surprisingly wide variety of super structures within both eukaryotic and prokaryotic cells — from the familiar 13-protofilament microtubule assembled from αβ–tubulin heterodimers to the single protofilaments of the ubiquitous prokaryotic FtsZ seen in vitro. Additionally in eukaryotes, monomeric tubulin family members have the ability to affect the assembly of filaments, such as nucleation of microtubules with γ–tubulin, or involvement in the assembly of B– and C– microtubules in the centriole microtubule triplets (ε– and δ–tubulin respectively). Each tubulin family member, both prokaryotic and eukaryotic is exquisitely tuned to perform its cellular function, with a unique superstructure and dynamics.

The main focus of this manuscript is a structural and biochemical investigation of a recently discovered bacterial tubulin TubZ–Bt. TubZ–Bt is located on the pBtoxis virulence plasmid in Bacillus thuringiensis, and is necessary to maintain stability of the pBtoxis plasmid in conjunction with an upstream DNA–adaptor protein (TubR) and DNAbinding site (tubC). We have determined that TubZ–Bt forms four–stranded filaments in the presence of GTP in vitro, whereas TubZ filaments formed with catalytically compromised protein or non-hydrolysable GTP analogs are predominantly two-stranded. These morphological changes were investigated by high–resolution cryo–electron microscopy of both two– and four–stranded TubZ–Bt filaments, as well as various biochemical approaches, and we propose that TubZ–Bt two–stranded filaments that we observed are an intermediate on pathway to four–stranded filament formation. In addition, we have begun preliminary investigations on the interaction of TubR bind to tubC DNA and the interaction of the TubRC complex with TubZ-Bt filaments.

A minor focus of this manuscript is work on the eukaryotic tubulins, γ–, δ– and ε–tubulin. This work details preliminary attempts at purification of these tubulin family members in order to begin more detailed investigative work on their molecular function. Also included is a published manuscript of work done with Dr. Luke Rice investigating the conformational state of both αβ– and γ–tubulins using solution–state and crystallographic structural studies.