Modulation of the Kinesin ATPase Cycle by Neck Linker Docking and Microtubule Binding
Kinesin motor proteins use an ATP hydrolysis cycle to perform various functions in eukaryotic cells. Many questions remain about how the kinesin mechanochemical ATPase cycle is fine-tuned for specific work outputs. In this study, we use isothermal titration calorimetry and stopped-flow fluorometry to determine and analyze the thermodynamics of the human kinesin-5 (Eg5/KSP) ATPase cycle. In the absence of microtubules, the binding interactions of kinesin-5 with both ADP product and ATP substrate involve significant enthalpic gains coupled to smaller entropic penalties. However, when the wild-type enzyme is titrated with a non-hydrolyzable ATP analog or the enzyme is mutated such that it is able to bind but not hydrolyze ATP, substrate binding is 10-fold weaker than ADP binding because of a greater entropic penalty due to the structural rearrangements of switch 1, switch 2, and loop L5 on ATP binding. We propose that these rearrangements are reversed upon ATP hydrolysis and phosphate release. In addition, experiments on a truncated kinesin-5 construct reveal that upon nucleotide binding, both the N-terminal cover strand and the neck linker interact to modulate kinesin-5 nucleotide affinity. Moreover, interactions with microtubules significantly weaken the affinity of kinesin-5 for ADP without altering the affinity of the enzyme for ATP in the absence of ATP hydrolysis. Together, these results define the energy landscape of a kinesin ATPase cycle in the absence and presence of microtubules and shed light on the role of molecular motor mechanochemistry in cellular microtubule dynamics
- Research Organization:
- Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source
- Sponsoring Organization:
- DOE - OFFICE OF SCIENCE
- DOE Contract Number:
- DE-AC02-98CH10886
- OSTI ID:
- 1019967
- Report Number(s):
- BNL-95508-2011-JA; JBCHA3; TRN: US201115%%603
- Journal Information:
- Journal of Biological Chemistry, Vol. 285, Issue 33; ISSN 0021-9258
- Country of Publication:
- United States
- Language:
- English
Similar Records
High-resolution structures of kinesin on microtubules provide a basis for nucleotide-gated force-generation
A Kinesin Motor In A Force-producing Conformation
Related Subjects
99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE
AFFINITY
CALORIMETRY
ENZYMES
HYDROLYSIS
KINETICS
MICROTUBULES
MODULATION
MOTORS
MUTAGENESIS
NUCLEOTIDES
PHOSPHATES
PROTEINS
SUBSTRATES
THERMODYNAMICS
TITRATION
national synchrotron light source