Context

Microtubules are dynamic filaments that can polymerise and depolymerise. Because of their instability, they need to be regulated by a number of proteins. Such proteins include polymerases that can increase their polymerisation rate. Alp14, Saccharomyces pombe microtubule polymerase has two Tumor Overexpressed Gene (TOG) domains that can bind tubulin dimers at its N terminus and a dimerisation interface at its C terminus. How polymerases increase the polymerisation rate of microtubules is not clearly understood and different models have been proposed. In these papers, the authors used X-ray crystallography, biochemistry, in vitro reconstitution and in vivo studies to propose and test a new model explaining how the different TOG domains of polymerases can come together to accelerate the polymerisation rate of microtubules.

Key findings – Structural Basis of Tubulin Recruitment and Assembly by Tumor Overexpressed Gene (TOG) domain array Microtubule Polymerases

After showing that the affinity for tubulin of TOG1 was stronger than TOG2, the authors obtained a crystal structure of four TOG domains (Saccharomyces kluyveri ortholog of Alp14) forming a homodimer containing two TOG1 and two TOG2 domains each bound to a tubulin dimer. This structure formed a square with the TOG domains orientated on the inside and the tubulin dimers on the outside. Using crosslinking-mass spectrometry and EM they showed this structure was likely adopted by the protein in solution.

The authors then obtained a second crystal structure containing only two TOG domains; TOG1 and TOG2 bound to two polymerised tubulin dimer. This second crystal structure allowed them to have a better insight of what might be happening during polymerisation. Their model explains that one polymerase can bind to four tubulin dimers using its four TOG domains and hold them in the square conformation. Using its SK rich region, the polymerase can diffuse to the plus end of the microtubule where the first tubulin dimer is incorporated, which disrupts the square conformation due to steric clashes and allows the second tubulin dimer held by TOG2 to be incorporated into the microtubule.

Key findings – Roles for tubulin recruitment and self-organization by TOG domain arrays in Microtubule plus-end tracking and polymerase

Cook et al. further explore the “polarized unfurling” model, designing Alp14 mutants from the predicted functions of the TOG domains to test in vitro and in vivo.  Using TIRF microscopy and in vivo assays they show that the functions of the TOG1 and TOG2 domains of Alp14-GFP are distinct, with the TOG1 domain being crucial for plus-end tracking and the TOG2 domain important for polymerase activity. Both mutants significantly decreased microtubule growth rates. Cook et al. next created mutants targeting the interfaces for square assembly (Nithianantham et al.), both inter- and intra-dimer mutations reduce polymerase activity and plus tip residency.  This was also shown to be true in vivo, showing that both plus end tracking and polymerase activity are reduced with disruption of the square assembly.

Cook et al. conclude that the TOG1 domain anchors the complex to the microtubule end using is high tubulin affinity whilst TOG2 with a faster tubulin exchange drives the addition of tubulin to the microtubule end and a faster polymerisation process.  Defects in the formation of the square complex affect the recruitment of tubulin prior to polymerisation.

Why we chose these preprints

I saw this data being presented at the Microtubule Symposium in Heidelberg two years ago and I enjoyed the structural biology and biochemistry techniques used. This model is very interesting as it explains using two different structures how polymerases work. It also shows that slight differences in affinity can drive important conformational changes. The fact that the authors also tested their model in vitro and in vivo makes it more convincing.

Open questions

When the polymerase arrives at the plus end and the square conformation starts to unroll, what happens to the other TOG1-TOG2 domain?

How might this model translate to other TOG proteins eg.ch-TOG and XMAP215 with more TOG domains?

Is diffusion the only mechanism for the polymerase to get to the plus end?

Tags: alp14, microtubule, polymerase, tog

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