X-ray Structure and Microtubule Interaction of the Motor Domain of Neurospora crassa NcKin3, a Kinesin with Unusual Processivity^,

Abstract

Neurospora crassa kinesin NcKin3 belongs to a unique fungal-specific subgroup of small Kinesin-3-related motor proteins. One of its functions appears to be the transport of mitochondria along microtubules. Here, we present the X-ray structure of a C-terminally truncated monomeric construct of NcKin3 comprising the motor domain and the neck linker, and a 3-D image reconstruction of this motor domain bound to microtubules, by cryoelectron microscopy. The protein contains Mg‚ADP bound to the active site, yet the structure resembles an ATP-bound state. By comparison with structures of the Kinesin-3 motor Kif1A in different nucleotide states (Kikkawa, M. et al. (2001) Nature (London, U.K.) 411, 439-445), the NcKin3 structure corresponds to the AMPPCP complex of Kif1A rather than the AMPPNP complex. NcKin3-specific differences in the coordination of the nucleotide and asymmetric interactions between adjacent molecules in the crystal are discussed in the context of the unusual kinetics of the dimeric wild-type motor and the monomeric construct used for crystal structure analysis. The NcKin3 motor decorates microtubules at a stoichiometry of one head per R -tubulin heterodimer, thereby forming an axial periodicity of 8 nm. In spite of unusual extensions at the N-terminus and within flexible loops L2, L8a, and L12 (corresponding to the K-loop of monomeric kinesins), the microtubule binding geometry is similar to that of other members of the kinesin family.Kinesins form a large superfamily that comprises molecules of different architecture and function, which have in common a high-homology catalytic domain or head, which is able to bind and hydrolyze ATP 1 and to interact with microtubules in a nucleotide-dependent manner (1, 2). Most of the kinesins are motor proteins that use the energy from ATP hydrolysis for directed transport of various cellular cargoes. Conventional kinesin, the founding member of the Kinesin-1 family, is a dimeric motor protein, which moves processively along microtubules (3-5). While doing so, it hydrolyses one molecule of ATP per step in a strictly alternating way (6-10), suggesting that at any time, at least one head or motor domain is firmly attached to the microtubule. The hand-over-hand mechanism as a model of conventional kinesins processive movement is now wellsupported by single molecule microscopy techniques with nanometer resolution (11-15) and high-resolution EM of microtubule-bound motor constructs (16), although it is still a matter of debate as to how the two heads are coordinated in detail (17).Hand-over-hand walking is not the only mechanism used by kinesin motors to produce directed movement. The minus end directed C-type motor Ncd (Kinesin-14 family) seems to use a powerstroke mechanism that involves repetitive attachment and detachment of the motor (18)(19)(20). Keeping on track is not a problem for Ncd since this motor normally works in concert with many other Ncd molecules. Members of the Unc104/Kif1A family (Kinesin-3 family) are mostly monomeric, plus end directed ...

Interactions between Subunits in Heterodimeric Ncd Molecules

“…The amino acid sequences of the heads are identical, but only one of the motor domains participates in hydrolyzing ATP and MT binding (7,44). The x-ray structure of the NcKin3 motor domain did not reveal any unusual features that could be linked to this unusual behavior of the motor domains (8). Comparison of the kinetic properties of mono-meric and dimeric NcKin3 constructs have led Adio and Woehlke (44) to propose that such an asymmetric domain structure is important for triggering the detachment of the dimeric motor after one kinetic cycle.…”

“…In kinesin NcKin3, only one of the heads interacts with the microtubule and generates force (7). The x-ray derived structure of the NcKin3 motor domain did not provide a clue as to the origin of the head asymmetry (8). Generally speaking, the reason for the occurrence of two heads in nonprocessive motors is unclear.…”

Protein complexes at the microtubule organizing center regulate bipolar spindle assembly

“…Co-crystalization of monomeric and dimeric Klps with microtubules has provided increasingly higher resolution to define the tubulin surface [61][62][63][64][65][66][67][68][69] as well as the interacting Klp domains in contact with the protofilament. [70][71][72][73][74][75][76] Our work with γ-tubulin helix 11 indicates that Klp/tubulin binding is unlikely to be an exclusive feature of microtubule α/β-tubulins.…”

“…Members of both families of Klps cross-link microtubules and move with opposite polarity that contributes to a counterbalance of forces on microtubules. [67][68][69] In the Drosophila syncitia Kinesin-5 KLP61F and Kinesin-14 Ncd have been shown to act after bipolar spindle assembly to maintain interpolar spacing using a sliding filament mechanism. 41 In fungi, Kinesin-14 Klps have been proposed to contribute to the establishment of bipolarity in addition to regulating anaphase.…”

Multiple Conformations of the Nucleotide Site of Kinesin Family Motors in the Triphosphate State

“…The sharp spikes in spectrum E, of particular note on the low-field side of component P2, are termed satellite peaks. They arise from interaction of the unpaired electron in the spin moiety with 13 C (methyl groups) isotopes 64 and not from motor-bound analog. Additional conformation of this identification comes from the fact that the spectral shift between the satellite component and the P1 peak arising from 13 C is 0.47 mT, the same as observed for 2′,3′-SLADP in solution in the absence of protein.…”