Kinesin tail domains and Mg2+ directly inhibit release of ADP from head domains in the absence of microtubules.
Kinesin-1 is a vesicle motor that can fold into a compact inhibited conformation that is produced by interaction of the heavy chain C-terminal tail region with the N-terminal motor domains (heads). Binding of the tail domains to the heads inhibits net microtubule-stimulated ATPase activity by blocking the ability of the heads to bind to microtubules with coupled acceleration of ADP release. We now show that folding of kinesin-1 also directly inhibits ADP release even in the absence of microtubules. With long heavy chain constructs such as DKH960 that exhibit a high degree of regulation by folding, the basal rate of ADP release is inhibited up to 30-fold compared to that of truncated DKH894 which has lost the inhibitory tail domains and does not fold. Inhibition of ADP release is also observed when separate head and tail domain constructs are mixed at low salt concentrations. This inhibition of ADP release by tail domains is formally analogous to the action of nucleotide dissociation inhibitors (NDI or GDI) for regulatory GTPases. In contrast to their inhibition of ADP release, tail domains accelerate the rate of ADP binding to nucleotide-free kinesin-1. Inhibition of release of ADP by tail domains is reversed by Unc-76 (FEZ1) which is a potential regulator of kinesin-1. Tail domains only weakly inhibit the initial slow release of Mg (2+) from the kinesin-MgADP complex but strongly inhibit the fast release of Mg (2+)-free ADP.
Hackney
David D
DD
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA. ddh@andrew.cmu.edu
Stock
Maryanne F
MF
D000244:Adenosine Diphosphate / Q000378:metabolism*; D016547:Kinesins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D017510:Protein Folding; D017434:Protein Structure, Tertiary
D000244:Adenosine Diphosphate; D011189:Potassium Chloride; D016547:Kinesins; D008274:Magnesium