The Complex Interplay between the Neck and Hinge Domains in Kinesin-1 Dimerization and Motor Activity

Bathe, Friederike; Hahlen, Katrin; Dombi, Renate; Driller, Lucia; Schliwa, Manfred; Woehlke, Günther

doi:10.1091/mbc.e04-11-0957

Cited by 27 publications

(31 citation statements)

References 44 publications

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“…Conversely, organisms that express KHCs without associated KLCs exhibit a different motif even though the nearby regulatory region has high sequence identity across all species. These KLC-less KHCs may have a specific mechanism of inhibition based on their distinct sequence (48,49).…”

Section: Resultsmentioning

confidence: 99%

Kinesin’s light chains inhibit the head- and microtubule-binding activity of its tail

Wong

Rice

2010

Proc. Natl. Acad. Sci. U.S.A.

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Kinesin-1 is a microtubule-based motor comprising two heavy chains (KHCs) and two light chains (KLCs). Motor activity is precisely regulated to avoid futile ATP consumption and to ensure proper intracellular localization of kinesin-1 and its cargoes. The KHC tail inhibits ATPase activity by interacting with the enzymatic KHC heads, and the tail also binds microtubules. Here, we present a role for the KLCs in regulating both the head- and microtubule-binding activities of the kinesin-1 tail. We show that KLCs reduce the affinity of the head-tail interaction over tenfold and concomitantly repress the tail’s regulatory activity. We also show that KLCs inhibit tail-microtubule binding by a separate mechanism. Inhibition of head-tail binding requires steric and electrostatic factors. Inhibition of tail-microtubule binding is largely electrostatic, pH dependent, and mediated partly by a highly negatively charged linker region between the KHC-interacting and cargo-binding domains of the KLCs. Our data support a model wherein KLCs promote activation of kinesin-1 for cargo transport by simultaneously suppressing tail-head and tail-microtubule interactions. KLC-mediated inhibition of tail-microtubule binding may also influence diffusional movement of kinesin-1 on microtubules, and kinesin-1’s role in microtubule transport/sliding.

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Section: Resultsmentioning

confidence: 99%

Kinesin’s light chains inhibit the head- and microtubule-binding activity of its tail

Wong

Rice

2010

Proc. Natl. Acad. Sci. U.S.A.

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“…1B). The longest truncation, NcKin3-558 was used in most experiments described here, and a reactive cysteine was added at the C terminus, allowing modifications with maleimide labels (29,30). The truncated, biotin-tagged NcKin3-558 kinesin was equally fast in gliding assays as the recombinant fulllength NcKin3 protein.…”

Section: Resultsmentioning

confidence: 99%

“…At the C terminus of NcKin3-558, an NgoMIV restriction site was inserted for the addition of amino acids 432-546 of human kinesin tail (hTail). 3 The hTail DNA was transferred from an NcKin hTail pT7-based plasmid (30) and inserted into pT7.7-NcKin3-558 via the NgoMIV and HindIII restriction sites.…”

Section: Methodsmentioning

confidence: 99%

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

Adio

Bloemink

Hartel

et al. 2006

Journal of Biological Chemistry

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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...

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“…Understanding this active system, which is constantly driven away from the thermodynamics equilibrium, has been a challenge for physicists, biologists, and chemists. Nowadays, the genetic approach, together with an increasing amount of structural information (1,2) and single-molecule studies (3)(4)(5)(6)(7)(8)(9)(10), has supplied a detailed description of molecular motor dynamics. To describe how those machines move and develop force, several hypothesis have been proposed.…”

mentioning

confidence: 99%

Myosin V stepping mechanism

Cappello

Pierobon

Symonds

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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We observe the myosin V stepping mechanism by traveling wave tracking. This technique, associated with optical tweezers, allows one to follow a scattering particle in a two-dimensional plane, with nanometer accuracy and a temporal resolution in the microsecond range. We have observed that, at the millisecond time scale, the myosin V combines longitudinal and vertical motions during the step. Because at this time scale the steps appear heterogeneous, we deduce their general features by aligning and averaging a large number of them. Our data show that the 36-nm step occurs in three main stages. First, the myosin center of mass moves forward 5 nm; the duration of this short prestep depends on the ATP concentration. Second, the motor performs a fast motion over 23 nm; this motion is associated to a vertical movement of the myosin center of mass, whose distance from the actin filament increases by 6 nm. Third, the myosin head freely diffuses toward the next binding site and the vertical position is recovered. We propose a simple model to describe the step mechanism of the dimeric myosin V. molecular motor ͉ single molecule ͉ traveling wave tracking ͉ total internal reflection ͉ interference M olecular motors convert the chemical energy, obtained from ATP hydrolysis, into mechanical work in a very efficient way. Understanding this active system, which is constantly driven away from the thermodynamics equilibrium, has been a challenge for physicists, biologists, and chemists. Nowadays, the genetic approach, together with an increasing amount of structural information (1, 2) and single-molecule studies (3-10), has supplied a detailed description of molecular motor dynamics. To describe how those machines move and develop force, several hypothesis have been proposed. They range from purely mechanical models, in which a conformational change takes place during the chemical cycle and drives the motor to the final state (11-13), to stochastic descriptions based on thermal ratchets. In the latter models, the molecular motor thermally explores the energy landscape and the arrival to the final state triggers the chemical cycle and prevents the motor from stepping back (14-21).A detailed description of the mechanical cycle (existence of internal subcycles and their dynamics) is essential to discriminate among the various theoretical models. The myosin V-actin complex is naturally a good candidate for this study: at each chemical cycle the myosin V center of mass moves toward the plus-end of the actin filaments, by steps of 36 nm (5). This movement is among the widest steps observed in molecular motors, and it facilitates the observation and characterization of the mechanical substeps. In addition, myosin V, like many dimeric machines, moves along the filament in a hand-over-hand manner (9), which seems to be a very general feature of processive motors (22,23).The single-molecule approach has been indispensable to understand the myosin V dynamics: this motor steps back only occasionally at zero load, whereas the backward/forward...

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The Complex Interplay between the Neck and Hinge Domains in Kinesin-1 Dimerization and Motor Activity

Cited by 27 publications

References 44 publications

Kinesin’s light chains inhibit the head- and microtubule-binding activity of its tail

Kinesin’s light chains inhibit the head- and microtubule-binding activity of its tail

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

Myosin V stepping mechanism

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