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The Ultrastructure and Nucleotide-regulated Assembly Mechanism of the Bacteriophage-encoded Tubulin Homologue, PhuZ

  • Author(s): Zehr, Elena A.
  • Advisor(s): Agard, David A
  • et al.
Abstract

Tubulin is a universally conserved molecule, found in all three domains of life. Tubulin filaments use energy derived from GTP binding and hydrolysis to organize cytoplasm. Although tubulin homologues share the same fold to their monomers and similar protofilament architecture, they assemble filaments with unique geometric designs. Moreover, despite sharing the mechanism of self-assembly, wherein tubulin subunits require GTP binding to polymerize, and their polymerization stimulates GTP hydrolysis, tubulins exhibit distinctive dynamic properties. Together, unique filament architectures and dynamic properties determine a specific set of biological functions each tubulin family performs. The origins of tubulin filament architectural diversity and distinctive dynamic behavior are not well understood.

We have been studying PhuZ tubulins, which are encoded by a few very large Pseudomonas ϕKZ-like bacteriophages. Our studies have shown that PhuZ assembles dynamically unstable spindle-like arrays that organize bacteriophage DNA at the cell midpoint, which somehow facilitates phage infectivity. Moreover, PhuZ monomer structure has the canonical tubulin fold, with a unique, highly conserved and extended C-terminus. The C-terminus mediates protofilament contacts and is critical for polymerization both in vitro and in vivo.

The main focus of this manuscript is the structural investigation of the molecular mechanisms underlying PhuZ dynamic behavior. In an attempt to explain how GTP binding and hydrolysis drives PhuZ filament turnover, we have conducted a number of electron cryomicroscopy studies (cryo-EM) on PhuZ filaments in both pre- and post- hydrolysis states. PhuZ forms a polar three-stranded polymer with an unusual subunit orientation. Its C-terminus guides a cooperative assembly of the three-stranded filament by mediating both longitudinal and lateral interactions. Upon assembly, the longitudinal interface, C-terminus and tubulin fold undergo polymerization-competent rearrangements. We propose that the energy of GTP binding is stored in the displacement of these structural elements. Also, our structural studies have revealed how the energy of GTP hydrolysis is stored in the PhuZ filament lattice. In particular, GTP hydrolysis, initially sensed by the T3 loop and C-terminus, is accompanied by unwinding and supercoiling of the filament lattice. Based on these observations, we propose an assembly/disassembly pathway of PhuZ filament.

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