Kinesin's cover-neck bundle folds forward to generate force

Abstract

Each step of the kinesin motor involves a force-generating molecular rearrangement. Although significant progress has been made in elucidating the broad features of the kinesin mechanochemical cycle, molecular details of the force generation mechanism remain a mystery. Recent molecular dynamics simulations have suggested a mechanism in which the forward drive is produced when the N-terminal cover strand forms a ␤-sheet with the neck linker to yield the cover-neck bundle. We tested this proposal by comparing optical trapping motility measurements of cover strand mutants with the wild-type. Motility data, as well as kinetic analyses, revealed impairment of the force-generating capacity accompanied by a greater load dependence in the mechanochemical cycle. In particular, a mutant with the cover strand deleted functioned only marginally, despite the fact that the cover strand, the Nterminal ''dangling end,'' unlike the neck linker and nucleotidebinding pocket, is not involved with any previously considered energy transduction pathway. Furthermore, a constant assisting load, likely in lieu of a power stroke, was shown to rescue forward motility in the cover strand deletion mutant. Our results support a stepping mechanism driven by dynamic cover-neck bundle formation. They also suggest a strategy to generate motors with altered mechanical characteristics by targeting the force-generating element.biological motor ͉ force generation ͉ optical trap ͉ power stroke ͉ motor protein T ranslocating motors, such as kinesins and certain myosins, form a distinct class of proteins that ''walk'' along biofilament tracks to perform a wide range of vital cellular processes (1). A fundamental, yet poorly understood aspect of these motors is the energy transduction mechanism that converts the chemical energy of ATP binding, hydrolysis, and product release into mechanical work. Kinesins and myosins appear to have a common nucleotide sensor (2, 3) yet have evolved different energy conversion mechanisms to achieve a variety of motile properties. In myosin, a series of structural changes leading to the rotation of its lever arm have been identified (4), but the details of the force generation have yet to be established.Likewise, the force generation mechanism of kinesin (herein, we mainly consider Kinesin-1) is not known. Until recently, the only mechanical element considered was the neck linker (NL), which connects the N-terminal motor head to the ␣-helical stalk. This Ϸ12-residue segment is disordered and flexible in the absence of ATP and ''docks'' when ATP binds to the motor head (5). Mutations in the NL impair the motility while preserving ATPase activity and microtubule (MT) binding (6, 7). However, a mechanism for its contribution to force generation is not available, and, in lieu of this, affinity-driven zippering of the NL to the motor head has been assumed. Unlike the structurally well-defined lever arm of myosin, the NL is short and flexible when detached. Furthermore, it interacts only weakly with the motor head (8), drawing f...

Kinesin processivity is gated by phosphate release

“…The notion that ATP binding by the MT-bound head induces the attachment of its tethered partner to the MT (10, 31, 32), which is a key feature of the prevailing model (2,3,10,14,15,17) (Fig. 3B), is consistent with evidence from biochemical measurements for the release of the fluorescent ADP analog, mantADP, from the tethered head upon nucleotide binding its MT-bound partner (31,(33)(34)(35).…”

“…A key structural element for this coordination is the neck linker (NL), a ∼14-aa segment that connects each catalytic head to a common stalk (9). In the 1-HB state, nucleotide binding is thought to induce a structural reconfiguration of the NL, immobilizing it against the MT-bound catalytic domain (2,3,(10)(11)(12)(13)(14)(15)(16)(17). This transition, called "NL docking," is believed to promote unidirectional motility by biasing the position of the tethered head toward the next MT binding site (2,3,(10)(11)(12)(13)(14)(15)(16)(17).…”

“…This transition, called "NL docking," is believed to promote unidirectional motility by biasing the position of the tethered head toward the next MT binding site (2,3,(10)(11)(12)(13)(14)(15)(16)(17). The completion of an 8.2-nm step (18) entails the binding of this tethered head to the MT, ATP hydrolysis, and detachment of the trailing head, thereby returning the motor to the ATP-waiting state (2,3,(10)(11)(12)(13)(14)(15)(16)(17). Prevailing models of the kinesin mechanochemical cycle (2,3,10,14,15,17), which invoke NL docking upon ATP binding, explain the highly directional nature of kinesin motility and offer a compelling outline of the sequence of events following ATP binding.…”

“…The completion of an 8.2-nm step (18) entails the binding of this tethered head to the MT, ATP hydrolysis, and detachment of the trailing head, thereby returning the motor to the ATP-waiting state (2,3,(10)(11)(12)(13)(14)(15)(16)(17). Prevailing models of the kinesin mechanochemical cycle (2,3,10,14,15,17), which invoke NL docking upon ATP binding, explain the highly directional nature of kinesin motility and offer a compelling outline of the sequence of events following ATP binding. Nevertheless, these abstractions do not speak directly to the branching transitions that determine whether kinesin dissociates from the MT (off-pathway) or continues its processive reaction cycle (onpathway).…”

“…In the 1-HB state, nucleotide binding is thought to induce a structural reconfiguration of the NL, immobilizing it against the MT-bound catalytic domain (2,3,(10)(11)(12)(13)(14)(15)(16)(17). This transition, called "NL docking," is believed to promote unidirectional motility by biasing the position of the tethered head toward the next MT binding site (2,3,(10)(11)(12)(13)(14)(15)(16)(17). The completion of an 8.2-nm step (18) entails the binding of this tethered head to the MT, ATP hydrolysis, and detachment of the trailing head, thereby returning the motor to the ATP-waiting state (2,3,(10)(11)(12)(13)(14)(15)(16)(17).…”