Abstract
Kinesin-1 is an ATP-dependent motor protein that moves towards microtubules (+)-ends. Whereas structures of isolated ADP-kinesin and of complexes with tubulin of apo-kinesin and of ATP-like-kinesin are available, structural data on apo-kinesin-1 in the absence of tubulin are still missing, leaving the role of nucleotide release in the structural cycle unsettled. Here, we identified mutations in the kinesin nucleotide-binding P-loop motif that interfere with ADP binding. These mutations destabilize the P-loop (T87A mutant) or magnesium binding (T92V), highlighting a dual mechanism for nucleotide release. The structures of these mutants in their apo form are either isomorphous to ADP-kinesin-1 or to tubulin-bound apo-kinesin-1. Remarkably, both structures are also obtained from the nucleotide-depleted wild-type protein. Our results lead to a model in which, when detached from microtubules, apo-kinesin possibly occupies the two conformations we characterized, whereas, upon microtubule binding, ADP-kinesin converts to the tubulin-bound apo-kinesin conformation and releases ADP. This conformation is primed to bind ATP and, therefore, to run through the natural nucleotide cycle of kinesin-1.
Highlights
Structures are mostly similar to those of ADP-kinesin or of tubulin-bound apo-kinesin; these conformations are adopted by the parental, nucleotide-depleted, wild-type protein
Either a threonine or a serine, this residue is conserved in all nucleotide-binding proteins’ P-loop motif (GxxxxGK(S/T)), which is known as the Walker A motif and required for coordinating α and β phosphates[22]
In kinesins, when a serine is found at the last P-loop position, a higher ADP release rate is observed
Summary
Structures are mostly similar to those of ADP-kinesin or of tubulin-bound apo-kinesin; these conformations are adopted by the parental, nucleotide-depleted, wild-type protein. An initial indication that residues on the other side of ADP as compared to Mg2+ are important for nucleotide binding came from the study of the kinesin E236A mutant, which releases ADP about 100-fold faster than wild-type kinesin[19], a result we were able to confirm (Fig. 1c, Table 1).
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