PT  - JOURNAL ARTICLE
AU  - Neil A. Venables
AU  - Till Bretschneider
AU  - Robert A. Cross
TI  - TOG domain MT polymerases accelerate MT plus end growth via electrostatically-steered diffusion-to-capture and electrostatic templating of GTP-tubulin
AID  - 10.1101/282897
DP  - 2018 Jan 01
TA  - bioRxiv
PG  - 282897
4099  - http://biorxiv.org/content/early/2018/03/15/282897.short
4100  - http://biorxiv.org/content/early/2018/03/15/282897.full
AB  - TOG domain microtubule polymerases track microtubule plus ends, bind GTP-tubulin and catalyse microtubule growth, by mechanisms that are not yet understood. In this work, we use computational analysis and simulation to probe the detailed mechanism of tubulin capture and exchange by TOG domains. TOG domains display a ridge of 5 surface loops that form the core of the TOG-tubulin interface. Using computational mutagenesis, we confirm that this row of loops, which is positively charged, plays a dominant role in setting the overall electrostatic field on the TOG domain. Brownian dynamics simulations establish that diffusion-to-capture of TOGs by tubulin is very strongly electrostatically steered. Under a range of conditions and in all trajectories examined, TOGs are initially captured and oriented by tubulin at high radius so that their basic loops faced inwards towards the tubulin. Thereafter, the loops continue to face inwards towards the tubulin and to scan its surface until stereospecific docking the crystallographic binding site occurs. We find that the acidic C-terminal tails of tubulin are not required for electrostatic steering, but instead serve to widen the acceptance angles for electrostatically steered diffusion-to-capture. All-atom normal mode analysis indicates that TOGs are remarkably stiff, enabling them to drive free GTP-tubulin into a partially-curved state by conformational selection. Electrostatic free energy calculations show that the complex that each TOG makes with its cognate tubulin is stable. Our work argues that TOGs accelerate microtubule plus end growth by two complementary electrostatic mechanisms, first by electrostatically steered diffusion-to-capture, and second by electrostatic stabilisation of a partially bent conformation of GTP-tubulin that exchanges rapidly into the tip-lattice. To explain this rapid exchange, we propose a model in which simultaneous binding of the GTP-tubulin to the TOG and the microtubule tip-lattice can occur and is required to de-stabilise the crystallographic complex and release and recycle the TOG.Author Summary TOG domain microtubule polymerases are protein machines that accelerate the growth of microtubule plus ends by capturing tubulin building blocks from solution and feeding them into the growing microtubule tip. Exactly how TOGs manage to do this remains unclear. Several lines of evidence suggest that electrostatic interactions play a key role, but the detailed role of electrostatics in the polymerase mechanism of TOGs is so far little explored. Here using linked computational approaches we analyse the electrostatic fields of TOGs from the TOG polymerase superfamily and simulate their tubulin binding trajectories. We find that each TOG domain has a shaped electrostatic field that is precisely matched to its tubulin binding partner, such that each TOG is electrostatically orientated at high radius and thereafter electrostatically guided to its capture site. Our work shows that electrostatic steering dramatically accelerates the diffusion-to-capture of tubulin by TOGs. The resulting TOG-tubulin complexes are electrostatically stabilized and we suggest that release of the TOG from this complex requires that tubulin first be incorporated into the growing microtubule, thereby being driven into a TOG-incompatible conformation.