Kinetic and Mechanistic Basis of the Nonprocessive Kinesin-3 Motor NcKin3

Abstract

Kinesin-3 motors have been shown to transport cellular cargo along microtubules and to function according to mechanisms that differ from the conventional hand-over-hand mechanism. To find out whether the mechanisms described for Kif1A and CeUnc104 cover the full spectrum of Kinesin-3 motors, we characterize here NcKin3, a novel member of the Kinesin-3 family that localizes to mitochondria of ascomycetes. We show that NcKin3 does not move in a K-loop-dependent way as Kif1A or in a cluster-dependent way as CeUnc104. Its in vitro gliding velocity ranges between 0.30 and 0.64 m/s and correlates positively with motor density. The processivity index (k bi,ratio ) of ϳ3 reveals that not more than three ATP molecules are hydrolyzed per productive microtubule encounter. The NcKin3 duty ratio of 0.03 indicates that the motor spends only a minute fraction of the ATPase cycle attached to the filament. Unlike other Kinesin-3 family members, NcKin3 forms stable dimers, but only one subunit releases ADP in a microtubule-dependent fashion. Together, these data exclude a processive hand-over-hand mechanism of movement and suggest a power-stroke mechanism where nucleotide-dependent structural changes in a single motor domain lead to displacement of the motor along the filament. Thus, NcKin3 is the first plus end-directed kinesin motor that is dimeric but moves in a nonprocessive fashion to its destination.The kinesin superfamily of proteins contains molecular motors that move along microtubules. Members of the founding class, Kinesin-1 (conventional), move according to the socalled hand-over-hand mechanism that leads to processive motility (1). This mechanism involves two motor heads that alternately bind to the microtubule and produce a stepwise progression of the motor. It implies that the catalytic cycles of the motor heads alternate because the nucleotide state of the catalytic domain determines the microtubule affinity (2). This mechanism is referred to as alternating site catalysis, and has been proven by measuring the consecutive release of ADP from head 1 and 2 of the kinesin dimer (3-5). ADP release experiments show that half of the enzyme-bound ADP is liberated upon interaction with the microtubule and the other half upon addition of ATP. Afterward, the motor continues to perform processive catalytic cycles because of the interlaced interaction of both motor heads. In agreement, the rate of the second ADP release is at least as fast as the stepping rate. The kinetic model of alternating site catalysis is consistent with the hand-overhand mechanism that predicts shifted phases of the kinetic cycles of both motor heads. All Kinesin-1 motors investigated so far conform to this model.Conversely, the nonprocessive Kinesin-14 motor Ncd does not show the ADP release pattern typical for alternating site catalysis. Ncd is a homodimeric kinesin from Drosophila melanogaster and is involved in mitosis and meiosis (6 -8). It moves to the microtubule minus end, and it most likely generates motility because of a molecular power stroke...

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

“…Remarkably, the [B, C] type pairing involves a relative rotation of 144°bringing the neck linkers of both molecules in close contact with each other (Figure 6). The neck linkers run almost perpendicular to each other and meet close to their C-terminal ends, so that the main chain oxygen of Q421 in B can form a hydrogen bond with the backbone nitrogen of V419 in C. Since dimerization of full-length NcKin3sas in the case of conventional kinesin (41)sis based on a coiledcoil interaction of the neck helices (30), the neck linkers of the two protomers have to converge to form a dimer. Thus, the asymmetric pairs of molecules in the crystal structure could serve as a plausible model for the contacts that are formed in the true NcKin3 dimer between the two heads.…”

“…The cloning of the plasmid pNcKif434, a pT7-7 derivative, is described elsewhere (30). The coded protein comprises the conserved motor domain of NcKin3, the following 14 amino acids, and a Cys-tag (amino acids PSIVHRKCF (52)).…”

“…In cells defective of Nckin2, another Neurospora Kinesin-3 motor that is essential for the interaction of mitochondria with microtubules, NcKin3 is upregulated, and mitochondria lacking NcKin2 require NcKin3 for binding to microtubules in vitro (29). In contrast to other Kinesin-3 members, NcKin3 forms native dimers; hence, in this case, dimerization is not used for regulation (30). Another surprising result from the analysis of NcKin3's ATPase activity is that only one subunit releases ADP in a microtubule-dependent manner, suggesting a non-processive powerstroke mechanism similar to Ncd.…”

“…Although the header and some loop regions (including the previously mentioned loops L2 and L12) are mostly disordered in the crystal, comparison of the structure with other kinesins, especially with Kif1A in different nucleotide states, revealed unexpected details. These are discussed in the context of the unusual kinetic behavior that NcKin3 exhibits in dimerization and microtubule-stimulated ATPase activity (30).…”

“…The most obvious differences in the primary structure are as follows: (i) Unlike Kif1A and other typical N-type kinesins, the conserved motor domain is preceded by a long extension of about 35 amino acids. The function of the NcKin3-specific header is unclear, and a truncated construct lacking the N-terminal extension has unchanged microtubule-stimulated ATPase activity (30).…”