Unveiling the Catalytic Mechanism of GTP Hydrolysis in Microtubules

Abstract

Microtubules (MTs) are large cytoskeletal polymers, composed of {beta}-tubulin heterodimers, capable of stochastically converting from polymerizing to depolymerizing states and vice-versa. Depolymerization is coupled with hydrolysis of GTP within {beta}-tubulin. Hydrolysis is favored in the MT lattice compared to free heterodimer with an experimentally observed rate increase of 500 to 700 fold, corresponding to an energetic barrier lowering of 3.8 to 4.0 kcal/mol. Mutagenesis studies have implicated -tubulin residues, :E254 and :D251, as catalytic residues completing the {beta}-tubulin active site of the lower heterodimer in the MT lattice. The mechanism for GTP hydrolysis in the free heterodimer, however, is not understood. Additionally, there has been debate concerning whether the GTP-state lattice is expanded or compacted relative to the GDP-state and whether a "compacted" GDP-state lattice is required for hydrolysis. In this work, extensive QM/MM simulations with transition-tempered metadynamics free energy sampling of compacted and expanded inter-dimer complexes, as well as free heterodimer, have been carried out to provide clear insight into the GTP hydrolysis mechanism. :E254 was found to be the catalytic residue in a compacted lattice, while in the expanded lattice disruption of a key salt bridge interaction renders :E254 less effective. The simulations reveal a barrier decrease of 3.8 {+/-} 0.5 kcal/mol for the compacted lattice compared to free heterodimer, in good agreement with experimental kinetic measurements. Additionally, the expanded lattice barrier was found to be 6.3 {+/-} 0.5 kcal/mol higher than compacted, demonstrating that GTP hydrolysis is variable with lattice state and slower at the MT tip. Significance StatementMicrotubules (MTs) are large and dynamic components of the eukaryotic cytoskeleton with the ability to stochastically convert from a polymerizing to a depolymerizing state and vice-versa. Depolymerization is coupled to the hydrolysis of guanosine-5-triphosphate (GTP), which is orders of magnitude faster in the MT lattice than in free tubulin heterodimers. Our results computationally ascertain the catalytic residue contacts in the MT lattice that accelerate GTP hydrolysis compared to the free heterodimer as well as confirm that a compacted MT lattice is necessary for hydrolysis while a more expanded lattice is unable to form the necessary contacts and thereby hydrolyze GTP.