Lethal kinesin mutations reveal amino acids important for ATPase activation and structural coupling

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• Conventional kinesin is a motor protein crucial for intracellular transport.
• Its function relies on the precise interaction between its motor domain, ATP, and microtubules.
• Understanding structure-function relationships is key to deciphering kinesin's role in cellular processes.

Purpose of the Study:

• To investigate the impact of specific mutations in the Drosophila kinesin heavy chain motor domain on its mechanochemical properties.
• To elucidate the role of different structural elements, such as loop 11 and alpha-helix 5, in kinesin function.
• To correlate structural changes with alterations in ATP turnover and microtubule binding.

Main Methods:

• Identification of 13 lethal mutations in the Drosophila kinesin heavy chain motor domain.
• Biochemical and mechanochemical testing of a subset of these mutations.
• Analysis of ATP and microtubule binding affinities and kinetics.
• Characterization of ATP turnover rates in the presence and absence of microtubules.

Main Results:

• The S246F mutation (loop 11) moderately decreased ATP turnover rate, supporting loop 11's role in microtubule-activated ATP hydrolysis.
• The T291M mutation (alpha-helix 5) severely impaired microtubule interaction and increased ATP turnover without microtubules, suggesting a partially activated state.
• The E164K mutation (beta-sheet 5a/loop 8b junction) unexpectedly increased ATP binding affinity and decreased turnover, potentially involving an ionic bridge and alternating site catalysis.

Conclusions:

• Mutations in the kinesin motor domain significantly affect its mechanochemical function.
• Loop 11 and alpha-helix 5 are critical for regulating ATP turnover and microtubule interactions.
• The E164K mutation highlights potential long-range structural communication and coordination in kinesin dimerization.