Abstract
Kinesin-1 is a dimeric motor that transports cargo along microtubules, taking 8.2-nm steps in a hand-over-hand fashion. The ATP hydrolysis cycles of its two heads are maintained out of phase by a series of gating mechanisms, which lead to processive runs averaging ~1 μm. A key structural element for inter-head coordination is the neck linker (NL), which connects the heads to the stalk. To examine the role of the NL in regulating stepping, we investigated NL mutants of various lengths using single-molecule optical trapping and bulk fluorescence approaches in the context of a general framework for gating. Our results show that, although inter-head tension enhances motor velocity, it is crucial neither for inter-head coordination nor for rapid rear-head release. Furthermore, cysteine-light mutants do not produce wild-type motility under load. We conclude that kinesin-1 is primarily front-head gated, and that NL length is tuned to enhance unidirectional processivity and velocity.
Highlights
Kinesin-1, hereafter referred to as kinesin, is an ATP-driven, dimeric motor protein that facilitates the unidirectional transport of intracellular cargo along microtubule (MT) filaments (Vale et al, 1985; Howard et al, 1989; Block et al, 1990; Hackney, 1995)
Our first experiments were designed to probe the influence of neck linker (NL) length on the stepping gate, which might contribute to unidirectional processivity by suppressing the rebinding of the ADPbound tethered head while the motor is in the ATP-waiting state [A] (Figure 1B)
The low mantADP exchange rates observed for our series of constructs are consistent with previous reports (Hackney, 2002; Hackney et al, 2003). These results indicate that kinesin can maintain a stable, 1-HB ATP-waiting state with up to 3 additional amino acids (AA) in its NL, but that extending the NL by 4 AA or more leads to an abrupt increase in rebinding of the rear-head to the MT
Summary
Kinesin-1, hereafter referred to as kinesin, is an ATP-driven, dimeric motor protein that facilitates the unidirectional transport of intracellular cargo along microtubule (MT) filaments (Vale et al, 1985; Howard et al, 1989; Block et al, 1990; Hackney, 1995). Each kinesin head transitions between one or more states that are strongly bound to the MT (the ATP-containing state, and the no-nucleotide state), and one or more states that are weakly bound to the MT (the ADP-containing state) (Block, 2007). A simplified version of this cycle (Figure 1A) may arbitrarily be taken to begin with the one-head-bound (1-HB), ATP-waiting state [α], where the nucleotide-free front head is strongly bound to the MT while the rear, ADP-bound tethered head remains unbound (Hackney, 1994; Asenjo and Sosa, 2009; Guydosh and Block, 2009; Toprak et al, 2009). Following ATP binding to the MT-bound head, the NL of its motor domain undergoes a structural reconfiguration and forms a β-sheet with the head, in a process termed
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