Backsteps induced by nucleotide analogs suggest the front head of kinesin is gated by strain

Guydosh, Nicholas R.; Block, Steven M.

doi:10.1073/pnas.0600931103

Cited by 128 publications

(169 citation statements)

References 54 publications

Supporting

Mentioning

156

Contrasting

Order By: Relevance

“…The dimer then enters the ATP-waiting state with 1 mobile tethered head with ADP still bound to it. In this model, the nucleotidefree head is the leading one according to data indicating that nucleotide dissociation preferentially occurs in the forward-positioned head (29). Two possible configurations for the ATP-waiting state, consistent with our FPM data are shown.…”

Section: Methodssupporting

confidence: 70%

“…Strain between the motor domains or the distinct orientation of the neck linkers on each motor domain provokes an asymmetry (20,21,(29)(30)(31) so that ADP is released from the tethered head when it binds to the microtubule in the forward position but not in the trailing one. An alternative model for the ADP release gate, independent of the microtubule lattice, has been proposed based on the observation that the gate and tubulin binding can operate in the presence of free tubulin (19).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

A mobile kinesin-head intermediate during the ATP-waiting state

Asenjo

Sosa

2009

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

View full text Add to dashboard Cite

Kinesin1 is a motor protein that uses the energy from ATP hydrolysis to move intracellular cargoes along microtubules. It contains 2 identical motor domains, or heads, that coordinate their mechano-chemical cycles to move processively along microtubules. The molecular mechanism of coordination between head domains remains unclear, partly because of the lack of structural information on critical intermediates of the kinesin1 mechano-chemical cycle. A point of controversy has been whether before ATP binding, in the so called ATP-waiting state, 1 or 2 motor domains are bound to the microtubule. To address this issue, here we use ensemble and single molecule fluorescence polarization microscopy (FPM) to determine the mobility and orientation of the kinesin1 heads at different ATP concentrations and in heterodimeric constructs with microtubule binding impaired in 1 head. We found evidence for a mobile head during the ATP-waiting state. We incorporate our results into a model for kinesin translocation that accounts well for many reported experimental results.cytoskeleton ͉ fluorescence ͉ microtubule ͉ single-molecule ͉ polarization K inesin1 is a motor protein that uses the energy of ATP hydrolysis to generate force and movement along microtubules (1). It contains 2 identical motor domains or heads where microtubule binding and ATP hydrolysis occur. It takes 8-nm steps between binding sites along the microtubule (2), hydrolyzing 1 ATP molecule per step (3-5). A stepping event is thought to occur when ATP binds to one of the heads inducing a disorder-to-order transition (docking) of a region called the neck linker, a short segment of Ϸ15 residues located at the C-terminal end of each head (6-8). Kinesin1 is a processive motor that can walk for more than an micrometer, going through hundreds of ATP hydrolysis cycles without dissociating from the microtubule (9). Processivity is achieved by an asymmetric hand-over-hand walking mechanism, in which the 2 heads coordinate their activities to alternate leading and trailing positions at each step (10-13).The molecular mechanism of coordination between kinesin1 motor domains is still unclear. Kinetic studies have identified ADP release and nucleotide binding as one of the coordinating steps. In solution, kinesin1 binds ADP tightly and interaction with the microtubule results in rapid release of 1 ADP molecule per kinesin head dimer. The second ADP molecule is released much more slowly, unless there is ATP present (or a nonhydrolysable ATP analogue) that accelerates the release of the second ADP molecule (14-16). This phenomenon constitutes a coordinating step or gate, as 1 kinesin head has to wait for the partner head to complete a particular step (ATP binding) to proceed (17). Several possible models have been proposed to explain this coordinating gate (18). One model proposes that in the ATPwaiting state the ADP containing head is kept away from the microtubule by interactions with the microtubule-bound head (19). Another model proposes that both motor domains can bind to th...

show abstract

Section: Methodssupporting

confidence: 70%

Section: Discussionmentioning

confidence: 99%

A mobile kinesin-head intermediate during the ATP-waiting state

Asenjo

Sosa

2009

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

View full text Add to dashboard Cite

show abstract

“…Recent singlemolecule (SM) experiments have shown that, with each stepping motion being strongly coupled to the ATP, the kinesin moves toward the (ϩ) ends of MTs by taking discrete 8-nm steps (3-7) in a hand-over-hand fashion (4,7,8). Although the ultimate understanding of kinesin's motility is still far from completion, the SM experiments (3)(4)(5)(6)(7)(8)(9), together with the series of kinetic ensemble measurements (10)(11)(12)(13) and theoretical studies (14)(15)(16)(17)(18)(19), begin providing glimpses to the physical principle of how kinesin walks.…”

Section: F Irst Recognized By Means Of Their Close Relationship Betweenmentioning

confidence: 99%

Mechanical control of the directional stepping dynamics of the kinesin motor

Hyeon

Onuchic²

2007

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

129

157

View full text Add to dashboard Cite

Among the multiple steps constituting the kinesin mechanochemical cycle, one of the most interesting events is observed when kinesins move an 8-nm step from one microtubule (MT)-binding site to another. The stepping motion that occurs within a relatively short time scale (Ϸ100 s) is, however, beyond the resolution of current experiments. Therefore, a basic understanding to the real-time dynamics within the 8-nm step is still lacking. For instance, the rate of power stroke (or conformational change) that leads to the undocked-to-docked transition of neck-linker is not known, and the existence of a substep during the 8-nm step still remains a controversial issue in the kinesin community. By using explicit structures of the kinesin dimer and the MT consisting of 13 protofilaments, we study the stepping dynamics with varying rates of power stroke (k p ATPase activity and organelle transport along microtubules (MTs) (1, 2), kinesins have received broad attention as a prototype of molecular motors for the past two decades. Recent singlemolecule (SM) experiments have shown that, with each stepping motion being strongly coupled to the ATP, the kinesin moves toward the (ϩ) ends of MTs by taking discrete 8-nm steps (3-7) in a hand-over-hand fashion (4,7,8). Although the ultimate understanding of kinesin's motility is still far from completion, the SM experiments (3-9), together with the series of kinetic ensemble measurements (10-13) and theoretical studies (14-19), begin providing glimpses to the physical principle of how kinesin walks.Along the kinesin mechanochemical cycle (supporting information (SI) Fig. 7), one of the main observations of the SM experiments is the stepping dynamics that enables the kinesin to move forward. The actual time spent for the stepping motion itself (Շ100 s), compared with the ATP binding and hydrolysis (տ10 ms), is too short, however, to detect the details of the dynamics with the spatial and temporal resolution of current instruments. Thus, it is still difficult to answer many basic questions (20) related to the stepping dynamics. Some of those questions are: (i) During the 8-nm step, how does the swiveling motion of the tethered head occur? Does any detectable substep exist that reflects a transient intermediate (5,21,22)? (ii) What fraction of the time and length scales is contributed from the power stroke and the diffusional search? (iii) Does the kinesin walk parallel to the single protofilament (PF) or walk astride by using two parallel PFs (23, 24)? To shed light on these questions, we propose to take advantage of the native topology of kinesins and MTs. For instance, our earlier study clarified the regulation mechanism between the two heads (6, 25) by using the native topology-based, two-head bound model of kinesin on the MT (19). Following the same line of thought, we show that even the dynamical pathways of kinesins reflect ''the topological constraints emanating from the molecular architecture.'' In the present work, we have adapted our previous two-head bound model (19) to stud...

show abstract

“…For kinesin, the T and the E state of each motor head are strongly bound whereas the D state is only weakly bound to the filament [13][14][15]. In order to make a forward mechanical step, the trailing head must detach from the filament whereas the leading head must be firmly attached to it.…”

Section: Network Representationsmentioning

confidence: 99%

“…Thus, these chemical transitions take much longer than the mechanical steps. When the catalytic domain of one motor head is occupied by ADP, this head is only loosely bound to the microtubule [13][14][15] and most likely to unbind from it.…”

Section: Introductionmentioning

confidence: 99%

Energy Conversion by Molecular Motors Coupled to Nucleotide Hydrolysis

2009

View full text Add to dashboard Cite

Recent theoretical work on the energy conversion by molecular motors coupled to nucleotide hydrolysis is reviewed. The most abundant nucleotide is provided by adenosine triphosphate (ATP) which is cleaved into adenosine diphosphate (ADP) and inorganic phosphate. The motors have several catalytic domains (or active sites), each of which can be empty or occupied by ATP or ADP. The chemical composition of all catalytic domains defines distinct nucleotide states of the motor which form a discrete state space. Each of these motor states is connected to several other states via chemical transitions. For stepping motors such as kinesin, which walk along cytoskeletal filaments, some motor states are also connected by mechanical transitions, during which the motor is displaced along the filament and able to perform mechanical work. The different motor states together with the possible chemical and mechanical transitions provide a network representation for the chemomechanical coupling of the motor molecule. The stochastic motor dynamics on these networks exhibits several distinct motor cycles, which represent the dominant pathways for different regimes of nucleotide concentrations and load force. For the kinesin motor, the competition of two such cycles determines the stall force, at which the motor velocity vanishes and the motor reverses its direction of motion. In general, kinesin is found to be governed by the competition of three distinct chemomechanical cycles. The corresponding network representation provides a unified description for all motor properties that have been determined by single molecule experiments.

show abstract

Backsteps induced by nucleotide analogs suggest the front head of kinesin is gated by strain

Cited by 128 publications

References 54 publications

A mobile kinesin-head intermediate during the ATP-waiting state

A mobile kinesin-head intermediate during the ATP-waiting state

Mechanical control of the directional stepping dynamics of the kinesin motor

Energy Conversion by Molecular Motors Coupled to Nucleotide Hydrolysis

Contact Info

Product

Resources

About