Evidence for conformational change-induced hydrolysis of β-tubulin-GTP

Paydar, Mohammadjavad; Kwok, Benjamin H.

doi:10.1101/2020.09.08.288019

Cited by 3 publications

(4 citation statements)

References 64 publications

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“…The barrier for the free heterodimer MFEP is 3.8 ± 0.5 kcal/mol higher than compacted, while the barrier for the expanded interdimer complex is 6.3 ± 0.5 kcal/mol higher than compacted. The rate difference between the compacted and free heterodimer–concerted pathways corresponds well to recent experimental measurements of GTP hydrolysis in an MT (0.16 s –1 ) versus free tubulin (0.0003 s –1 ), which translates to an energetic difference of 3.9 kcal/mol ( 17 ). Another recent study comparing wild-type MTs to E254D mutants determined a rate of 0.2 s –1 for GTP hydrolysis in an MT, corresponding to an energy difference with the free tubulin measurement of 4.00 kcal/mol ( 18 ).…”

Section: Resultssupporting

confidence: 85%

“…GTP hydrolysis has been known to be faster in the MT lattice than the free heterodimer, with recent experiments anticipating a rate difference of 500- to 700-fold, corresponding to an energetic barrier difference of 3.9 ± 0.1 kcal/mol ( 17 , 18 ). Cryo-EM measurements and mutagenesis studies have led to the hypothesis that specific α-tubulin residues (α:E254 and α:D251) complete the β-tubulin GTPase site and catalyze hydrolysis ( 18 – 22 ).…”

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confidence: 99%

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Unveiling the catalytic mechanism of GTP hydrolysis in microtubules

Beckett

Voth

2023

Proc. Natl. Acad. Sci. U.S.A.

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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.

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Section: Resultssupporting

confidence: 85%

mentioning

confidence: 99%

Unveiling the catalytic mechanism of GTP hydrolysis in microtubules

Beckett

Voth

2023

Proc. Natl. Acad. Sci. U.S.A.

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“…The rate difference between the compacted and free heterodimer concerted pathways corresponds well to recent experimental measurements of GTP hydrolysis in a MT (0.16 s -1 ) versus free tubulin (0.0003 s -1 ), which translates to an energetic difference of 3.9 kcal/mol. 17 Another recent study comparing wildtype MTs to E254D mutants determined a rate of 0.2 s -1 for GTP hydrolysis in an MT, corresponding to an energy difference with the free tubulin measurement of 4.00 kcal/mol. 18 Comparing these experimental values gives us a kinetic barrer of 3.9 ± 0.1 kcal/mol, which is to within error excellent agreement with our compacted vs. free heterodimer concerted pathway barrier difference of 3.8 ± 0.5 kcal/mol.…”

Section: Resultsmentioning

confidence: 99%

“…15,16 GTP hydrolysis has been known to be faster in the MT lattice than the free heterodimer, with recent experiments anticipating a rate difference of 500 to 700 fold, corresponding to an energetic barrier difference of 3.9 ± 0.1 kcal/mol. 17,18 Cryo-EM measurements and mutagenesis studies on have led to the hypothesis that specific a-tubulin residues (a:E254 and a:D251) complete the β-tubulin GTPase site and catalyze hydrolysis. [18][19][20][21][22] Additionally, when a non-hydrolyzable GTP-mimic, GMPCPP, is used in place of GTP the MT lattice seems to be expanded relative to the GDP lattice, which supports the hypothesis that lattice compaction induced by hydrolysis incurs strain on the MT lattice, which in turn is released by catastrophe.…”

Section: Introductionmentioning

confidence: 99%

Unveiling the Catalytic Mechanism of GTP Hydrolysis in Microtubules

Beckett

Voth

2023

Preprint

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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 GTP within β-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 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 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. 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 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.

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