Controlling kinesin by reversible disulfide cross-linking. Identifying the motility-producing conformational change

Area of Science:

• Molecular biology
• Biophysics
• Cellular transport

Background:

• Kinesin is a dimeric molecular motor that moves along microtubules using ATP.
• Two models exist for kinesin's movement: neck linker zippering or neck coiled-coil unwinding.

Purpose of the Study:

• To investigate the structural changes responsible for kinesin's unidirectional movement.
• To differentiate between the neck linker and neck coiled-coil models of kinesin motility.

Main Methods:

• Engineered recombinant kinesin motors with cysteines for disulfide cross-linking.
• Used disulfide cross-linking to prevent specific structural movements.
• Utilized optical traps to measure force generation and processivity.
• Observed kinesin movement on microtubules via microscopy.

Main Results:

• Cross-linking the neck linker abolished unidirectional movement, causing diffusion.
• Partial neck linker restraint allowed directionality but impaired processivity and force generation.
• Cross-linking the neck coiled-coil minimally affected motor activity, reducing run length by 30-50%.

Conclusions:

• Conformational changes in the neck linker, not the neck coiled-coil, are essential for kinesin's processive movement.
• The neck linker plays a critical role in kinesin's directionality, processivity, and force generation.