Unveiling the catalytic mechanism of GTP hydrolysis in microtubules
- Department of Chemistry, Chicago Center for Theoretical Chemistry, Institute for Biophysical Dynamics, and James Franck Institute, The University of Chicago, Chicago, IL 60637
Microtubules (MTs) are large cytoskeletal polymers, composed of αβ-tubulin heterodimers, capable of stochastically converting from polymerizing to depolymerizing states and vice versa. Depolymerization is coupled with hydrolysis of guanosine triphosphate (GTP) within β-tubulin. Hydrolysis is favored in the MT lattice compared to a 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 α-tubulin residues, α:E254 and α:D251, as catalytic residues completing the β-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 quantum mechanics/molecular mechanics simulations with transition-tempered metadynamics free-energy sampling of compacted and expanded interdimer complexes, as well as a free heterodimer, have been carried out to provide clear insight into the GTP hydrolysis mechanism. α: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 α:E254 less effective. The simulations reveal a barrier decrease of 3.8 ± 0.5 kcal/mol for the compacted lattice compared to a 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.
- Research Organization:
- Univ. of Chicago, IL (United States); University of Chicago, IL (United States)
- Sponsoring Organization:
- National Institute of General Medical Sciences (NIGMS); USDOE Office of Science (SC), Basic Energy Sciences (BES)
- Grant/Contract Number:
- SC0023318
- OSTI ID:
- 1986572
- Alternate ID(s):
- OSTI ID: 2368588
OSTI ID: 1994549
- Journal Information:
- Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Issue: 27 Vol. 120; ISSN 0027-8424
- Publisher:
- Proceedings of the National Academy of SciencesCopyright Statement
- Country of Publication:
- United States
- Language:
- English