Coupling of Kinesin-1 Neck Linker Docking to the Nucleotide Binding Site

Abstract

Coupling of nucleotide binding to docking of the neck linker of kinsin-1 is important for generation of directional motility. One approach towards determining the magnitude of this coupling is to use isotopic exchange reactions to evaluate the free energy differences between states. Kinesin-1 monomer head domains catalyze the slow MT-dependent synthesis of bound ATP from bound ADP and free Pi ( MT•E•ADP + Pi → MT•E•ATP + HOH) that results in oxygen isotopic exchange of 18O/16O between water and Pi. The tethered head domain of a kinesin dimer bound to MTs, however, catalyzes ATP synthesis at a 20-fold faster rate (Proc.Natl.Acad.Sci.USA 102, 18228 (2005)). This more rapid rate of ATP synthesis with a dimer suggests that the tethered head can bind to the microtubule behind the strongly attached head, because this positions the neck linker of the tethered head toward the plus end of the microtubule and would facilitate its docking on synthesis of ATP.Isotopic exchange analysis of other constructs with alterations in the neck linker is in progress. One approach is to delete part of the neck linker and therefore prevent reversible docking. DKH335 has lost the C-terminal part of the neck linker that makes extensive contacts with the core. During net ATP hydrolysis, the full length head domain DKH346 resynthesizes ATP on average once in 40 turnovers. In contrast, DKH335 is reported here to hydrolyze ATP with no detectable ATP resynthesis (ATP resynthesis occurs only once in >500 turnovers). This is consistent with more rapid Pi release in the absence of a requirement for coupled neck linker undocking or with destabilization of bound ATP in the absence of neck linker docking.Supported by NSF grant MCB-0615549.