Spatial relationship between heads of dimeric Ncd in the presence of nucleotides and microtubules

Abstract

Kinesins are molecular motors that produce mechanical work at the expense of ATP hydrolysis. Here, we studied Ncd ( non-claret disjunctional), a (−)-end-directed member of this superfamily. To gain insight into the mechanism by which Ncd generates force and movement, we measured distances between the heads in dimeric Ncd-250–700 using fluorescence resonance energy transfer (FRET). About 5% of Ncd heads were labeled with 1,5-IAEDANS (donor), and the remaining thiol groups were modified with QSY35-iodoacetamide (acceptor). Several lines of experimental evidence suggest that the probes were conjugated to Cys-670 in each head of the dimer. The measured donor–acceptor distance was about 35 Å. Nucleotides (ADP, ATP, and AMP-PNP) in the presence and absence of microtubules had only small effects on the interhead distances. Similar results were obtained for bidirectional Ncd mutant in which Asn-340 was replaced by a lysine. The results argue against models of Ncd movement in which the heads undergo large spatial rearrangements during mechanochemical cycle and suggest Gly-347 as a possible pivot point for the head rotation.