Single-Molecule Studies on the Motion and Force Generation of the Kinesin-3 Motor KIF1A

Rao, Lu; Gennerich, Arne

doi:10.1007/978-1-0716-2229-2_21

Cited by 5 publications

(2 citation statements)

References 81 publications

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“…MTBR fractions were used for the single-molecule TIRF assay and the dilutions were adjusted to an appropriate density of motors on MTs. The assay was performed as follows 106 . A flow chamber was assembled with a glass slide (Fisher #12-550-123), an ethanol-cleaned coverslip (Zeiss #474030-9000-000), and two stripes of parafilm.…”

Section: Methodsmentioning

confidence: 99%

Cryo-EM unveils kinesin KIF1A’s processivity mechanism and the impact of its pathogenic variant P305L

Benoit,

Rao,

Asenjo

et al. 2024

Nat Commun

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Mutations in the microtubule-associated motor protein KIF1A lead to severe neurological conditions known as KIF1A-associated neurological disorders (KAND). Despite insights into its molecular mechanism, high-resolution structures of KIF1A-microtubule complexes remain undefined. Here, we present 2.7-3.5 Å resolution structures of dimeric microtubule-bound KIF1A, including the pathogenic P305L mutant, across various nucleotide states. Our structures reveal that KIF1A binds microtubules in one- and two-heads-bound configurations, with both heads exhibiting distinct conformations with tight inter-head connection. Notably, KIF1A’s class-specific loop 12 (K-loop) forms electrostatic interactions with the C-terminal tails of both α- and β-tubulin. The P305L mutation does not disrupt these interactions but alters loop-12’s conformation, impairing strong microtubule-binding. Structure-function analysis reveals the K-loop and head-head coordination as major determinants of KIF1A’s superprocessive motility. Our findings advance the understanding of KIF1A’s molecular mechanism and provide a basis for developing structure-guided therapeutics against KAND.

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Section: Methodsmentioning

confidence: 99%

Cryo-EM unveils kinesin KIF1A’s processivity mechanism and the impact of its pathogenic variant P305L

Benoit,

Rao,

Asenjo

et al. 2024

Nat Commun

Self Cite

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show abstract

“…MTBR fractions were used for the single-molecule TIRF assay and the dilutions were adjusted to an appropriate density of motors on MTs. The assay was performed as described before 103 except the motility buffer was BRB80 (80 mM PIPES, 2 mM MgCl 2 , 1 mM EGTA, 1 mM DTT, 10 µM taxol, 0.5% Pluronic F-127 (w/v), 2 mM ATP, 5 mg/mL BSA, 1 mg/mL α-casein, gloxy oxygen scavenging system, and 10% glycerol, pH 6.8). Images were acquired with 200 ms per frame (total 600 frames per movie), and then analyzed via a custom-written MATLAB software.…”

Section: Methodsmentioning

confidence: 99%

Cryo-EM Unveils the Processivity Mechanism of Kinesin KIF1A and the Impact of its Pathogenic Variant P305L

Benoit

Rao

Asenjo

et al. 2023

Preprint

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Mutations in the Kinesin-3 motor KIF1A, a microtubule (MT)-associated motor protein, cause devastating neurodevelopmental and neurodegenerative diseases termed KIF1A-associated neurological disorders (KAND). While the mechanism of KIF1A is increasingly understood, high resolution (<4 Å) structural information of KIF1A-MT complexes is lacking. Here, we present 2.7-3.4 Å resolution structures of dimeric MT-bound wild-type (WT) KIF1A and the pathogenic P305L mutant as a function of the nucleotide state. Our structures reveal that 1) the KIF1A dimer binds MTs in one- and two-heads-bound states, 2) that both MT-bound heads assume distinct conformations with tight inter-head connection, 3) the position and conformation of the class-specific loop 12 (the K-loop), and 4) that the P305L mutation causes structural changes in the K-loop that result in a weakly MT-bound state. Motivated by our structural insights, we performed structure-function studies that reveal that both the K-loop and head-head coordination are major determinants of KIF1A's superprocessive motility. Our work provides key insights into the mechanism of KIF1A and provides near-atomic structures of WT and mutant KIF1A for future structure-guided drug-design approaches to treat KAND.

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Insect Cell-Based Expression of Cytoskeletal Motor Proteins for Single-Molecule Studies

Liu,

Gennerich

2023

Methods in Molecular Biology

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Single-Molecule Studies on the Motion and Force Generation of the Kinesin-3 Motor KIF1A

Cited by 5 publications

References 81 publications

Cryo-EM unveils kinesin KIF1A’s processivity mechanism and the impact of its pathogenic variant P305L

Cryo-EM unveils kinesin KIF1A’s processivity mechanism and the impact of its pathogenic variant P305L

Cryo-EM Unveils the Processivity Mechanism of Kinesin KIF1A and the Impact of its Pathogenic Variant P305L

Insect Cell-Based Expression of Cytoskeletal Motor Proteins for Single-Molecule Studies

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