Molecular Dynamics Study of the Energetic, Mechanistic, and Structural Implications of a Closed Phosphate Tube in ncd

Minehardt, Todd J.; Cooke, Roger; Pate, Edward; Kollman, Peter A.

doi:10.1016/s0006-3495(01)76092-4

Cited by 34 publications

(59 citation statements)

References 61 publications

(83 reference statements)

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“…An atomic model has previously been proposed for the closed switch loop conformation of kinesin (15), based on homology modeling with a crystal structure of kinesin's ancestral relative, myosin. In this switch loop model, closure of the loops around the nucleotide γ-phosphate forms a "phosphate tube," with the triphosphate group from the nucleotide inserted into one end of the tube, while the other end of the tube is open.…”

Section: Resultsmentioning

confidence: 99%

“…The phosphate tube also defines the presumed catalytically active conformation of kinesin's nucleotide pocket (15) by placing residues implicated in catalysis in close proximity with the γ-phosphate. Consequently, our models provide a qualitative explanation for why microtubules are required to fully stimulate catalysis (17): microtubule attachment is required to stabilize the switch II helix extension, so completing the array of stabilizing binding partners for the phosphate tube.…”

Section: How Microtubule Attachment Nucleates the Switch II Helix Extmentioning

confidence: 99%

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An atomic-level mechanism for activation of the kinesin molecular motors

Sindelar

Downing

2010

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

148

223

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Kinesin cytoskeletal motors convert the energy of ATP hydrolysis into stepping movement along microtubules. A partial model of this process has been derived from crystal structures, which show that movement of the motor domain relative to its major microtubule binding element, the switch II helix, is coupled to docking of kinesin's neck linker element along the motor domain. This docking would displace the cargo in the direction of travel and so contribute to a step. However, the crystal structures do not reveal how ATP binding and hydrolysis govern this series of events. We used cryoelectron microscopy to derive 8-9 Å-resolution maps of four nucleotide states encompassing the microtubule-attached kinetic cycle of a kinesin motor. The exceptionally high quality of these maps allowed us to build in crystallographically determined conformations of kinesin's key subcomponents, yielding novel arrangements of kinesin's switch II helix and nucleotide-sensing switch loops. The resulting atomic models reveal a seesaw mechanism in which the switch loops, triggered by ATP binding, propel their side of the motor domain down and thereby elicit docking of the neck linker on the opposite side of the seesaw. Microtubules engage the seesaw mechanism by stabilizing the formation of extra turns at the N terminus of the switch II helix, which then serve as an anchor for the switch loops as they modulate the seesaw angle. These observations explain how microtubules activate kinesin's ATP-sensing machinery to promote cargo displacement and inform the mechanism of kinesin's ancestral relative, myosin.ATPase | cryoelectron microscopy | motility | myosin | structure C ytoskeletal motors are integral to all forms of eukaryotic life, with roles ranging from intracellular transport to cell division. X-ray crystal structures are known for the functional motor domains of kinesin and myosin, the two best-characterized cytoskeletal motors, but both motors substantially change conformations upon binding to their respective filaments (1, 2), and available structural methods have been unable to obtain atomic-resolution descriptions of the motor-filament complexes. Consequently, available structural models do not account for many essential properties of the cytoskeletal motors.In microtubule-attached kinesin, the binding of ATP triggers a conformational change of the neck linker, to which cargo is tethered, leading to cargo displacement along the microtubule (3, 4). This behavior has been linked to a hypothetical mechanism for part of the activity (5), which we will refer to as the switch II helix scheme. In this scheme, inferred through comparisons of crystal structures of kinesin, ATP-triggered movements of the switch II helix would control the conformation of the neck linker through a steric coupling mechanism. CryoEM studies have confirmed key aspects of this scheme, directly resolving ATP analog-induced changes in the neck linker and coincident movement of kinesin's core domain relative to the switch II helix, both consistent with the scheme...

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Section: Resultsmentioning

confidence: 99%

Section: How Microtubule Attachment Nucleates the Switch II Helix Extmentioning

confidence: 99%

An atomic-level mechanism for activation of the kinesin molecular motors

Sindelar

Downing

2010

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

148

223

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“…In addition, development of the Dictyostelium mutant stopped at the mound state and did not proceed through morphogenesis. These studies have been extended by several groups to understand the structural and mechanistic role of the switch I arginine for ATP hydrolysis and mechanochemistry (27)(28)(29)(30)(31)(32)(33)(34).…”

Section: Discussionmentioning

confidence: 99%

The Role of ATP Hydrolysis for Kinesin Processivity

Farrell¹,

Mackey²,

Klumpp

et al. 2002

Journal of Biological Chemistry

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Conventional kinesin is a highly processive, plus-enddirected microtubule-based motor that drives membranous organelles toward the synapse in neurons. Although recent structural, biochemical, and mechanical measurements are beginning to converge into a common view of how kinesin converts the energy from ATP turnover into motion, it remains difficult to dissect experimentally the intermolecular domain cooperativity required for kinesin processivity. We report here our presteady-state kinetic analysis of a kinesin switch I mutant at Arg 210 (NXXSSRSH, residues 205-212 in Drosophila kinesin). The results show that the R210A substitution results in a dimeric kinesin that is defective for ATP hydrolysis and a motor that cannot detach from the microtubule although ATP binding and microtubule association occur. We propose a mechanistic model in which ATP binding at head 1 leads to the plus-end-directed motion of the neck linker to position head 2 forward at the next microtubule binding site. However, ATP hydrolysis is required at head 1 to lock head 2 onto the microtubule in a tight binding state before head 1 dissociation from the microtubule. This mechanism optimizes forward movement and processivity by ensuring that one motor domain is tightly bound to the microtubule before the second can detach.Kinesin is a highly processive, dimeric mechanoenzyme that travels along microtubules toward their plus-ends in discrete 8-nm steps, each step tightly coupled to a single ATP turnover (1-3). Recent evidence from a variety of experimental approaches has focused our attention to the proposal presented by Rice et al. (4) that ATP binding induces a pronounced conformational change in the neck linker region, which docks the neck linker onto the catalytic core and propels the unattached kinesin head forward to find the next binding site on the microtubule. This model is based on a disorder-to-order transition in the neck linker region for monomeric kinesin constructs. The neck linker of the Mt⅐K 1 complex was shown to be mobile in the presence of ADP, existing in an equilibrium with two predominant conformations trapped by cryo-electron microscopy. However, upon the addition of ATP or nonhydrolyzable ATP analogs to the Mt⅐K complex, the neck mobility ceased with the neck linker element tightly associated with the catalytic core. This ordered state was reversed by the addition of ADP or loss of nucleotide. In addition, the cryo-electron microscopy of this proposed ATP state revealed a single discrete orientation of the neck linker with the carboxyl terminus of the motor domain directed toward the plus-end of the microtubule (4). Xing et al. (5) have reported for a monomeric kinesin motor domain two discrete structural transitions induced by ADP binding and another produced by ATP binding. These three conformations revealed by fluorescence resonance energy transfer were consistent with the results reported by Rice et al. (4). Furthermore, biochemical studies of dimeric kinesin have demonstrated that ATP binding (or nonhydrolyzabl...

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“…Our study does not provide direct evidence that would allow us to identify the nature of these conformational states. However, some insights come from molecular modeling studies of the kinesin-related protein Ncd (25). These propose that ATP binding leads to two structural transitions: movement of switch I to close the phosphate tube and movement of switch II to interact with nucleotide.…”

Section: Discussionmentioning

confidence: 99%

ATP Reorients the Neck Linker of Kinesin in Two Sequential Steps

Rosenfeld

Jefferson

King

2001

Journal of Biological Chemistry

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Recent models of the kinesin mechanochemical cycle provide some conflicting information on how the neck linker contributes to movement. Some spectroscopic approaches suggest a nucleotide-induced order-to-disorder transition in the neck linker. However, cryoelectron microscopic imaging suggests instead that nucleotide alters the orientation of the neck linker when docked on the microtubule surface. Furthermore, since these studies utilized transition state or non-hydrolyzable nucleotide analogs, it is not clear at what point in the ATPase cycle this reorientation of the neck linker occurs. We have addressed this issue by developing a strategy to examine the effect of nucleotide on the orientation of the neck linker based on the technique of fluorescence resonance energy transfer. Transient kinetic studies utilizing this approach support a model in which ATP binding leads to two sequential isomerizations, the second of which reorients the neck linker in relation to the microtubule surface.Models of how molecular motors utilize energy from ATP hydrolysis to generate force or movement are still under active investigation. In the case of myosin, a variety of experimental results suggest that an isomerization associated with or following phosphate release leads to a swinging of a rigid lever arm contained within the regulatory domain (1, 2). However, additional rotation of this lever arm may occur with ADP release in at least some myosin isoforms (3-5). In kinesin motors, the situation is even less clear. In the case of conventional kinesin, a carboxyl-terminal region of the motor domain, referred to as the "neck linker," has been proposed to alter its orientation during the course of ATP binding and/or hydrolysis (6). Studies with spectroscopic and electron-dense labels have led to a model in which the neck linker alternates between an ordered orientation, in which it is docked on the motor domain, and one that is disordered (7). This order-to-disorder transition has been proposed to be dependent on the nature of the nucleotide in the active site. It is the basis for a recently proposed model in which cycles of docking and release of the neck linker cause the unattached kinesin head to find the next binding site on the microtubule (7).However, several lines of evidence suggest that this model may not completely describe the mechanism of processive motility for kinesin. First, cryoelectron microscopy of gold-labeled, kinesin-decorated microtubules showed that in rigor and ADP, the neck linker assumes two distinct orientations, separate from that seen with ATP analogs (7). This suggests the presence of three discrete kinesin states, one induced by ATP and two by ADP or rigor, which is in fact consistent with recent kinetic studies of kinesin motor domain mutants (8). Second, it is not clear at what point in the ATPase cycle the change in state of the neck linker occurs. Both AMP-PNP 1 and ADP ϩ aluminum fluoride immobilize the neck linker and appear to dock it onto the motor domain. However, AMP-PNP is thought to mimic...

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Molecular Dynamics Study of the Energetic, Mechanistic, and Structural Implications of a Closed Phosphate Tube in ncd

Cited by 34 publications

References 61 publications

An atomic-level mechanism for activation of the kinesin molecular motors

An atomic-level mechanism for activation of the kinesin molecular motors

The Role of ATP Hydrolysis for Kinesin Processivity

ATP Reorients the Neck Linker of Kinesin in Two Sequential Steps

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