Decoration of microtubules in solution by the kinesin-14, Ncd.
The kinesin-14, Ncd, is a cellular motor involved in microtubule spindle assembly and contraction during mitosis and meiosis. Like other members of the kinesin superfamily, Ncd consists of two motor heads connected by a linker and a long cargo-carrying stalk. The motor heads hydrolyze ATP to ADP to provide the power stroke that moves them and the cargo along the microtubule. Whereas conventional kinesins move processively along the sense of the microtubule right-handed helix, Ncd moves in the opposite direction, apparently using a different motive mechanism. According to the current model, the microtubule-binding state of Ncd is bound by one head and then released during the motive cycle. This is distinguished from the binding states of conventional kinesins, in which the motor heads are always bound in the motive cycle with alternating one-head and two-head binding. The objective was to determine the extent of binding, the binding states of Ncd in the presence of an ATP analogue, AMPPNP, and whether the binding is cooperative. Small-angle neutron scattering (SANS) of microtubules decorated with a deuterated Ncd construct, Ncd281, in solution containing 42% D(2)O was used. These conditions render the microtubule `invisible' to SANS, while amplifying the SANS from the Ncd constructs. In the presence of AMPPNP, 75% of Ncd281 was not bound. The remainder was bound cooperatively by one of its motor heads to the microtubule.
Los Alamos Neutron Science Center, Los Alamos National Laboratory, Los Alamos, NM 87545, USA. hjelm@lanl.gov
Rex P
RP
Hjelm
Deborah Bennett
DB
Stone
Robert J
RJ
Fletterick
Robert A
RA
Mendelson
D000266:Adenylyl Imidodiphosphate / Q000378:metabolism*; D000818:Animals; D029721:Drosophila Proteins / Q000737:chemistry* / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000378:metabolism; D016547:Kinesins / Q000737:chemistry* / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D033363:Neutron Diffraction; D053838:Scattering, Small Angle; D012996:Solutions
D029721:Drosophila Proteins; D012996:Solutions; C066696:ncd protein, Drosophila; D000266:Adenylyl Imidodiphosphate; D016547:Kinesins