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A seesaw model for intermolecular gating in the kinesin motor protein.

Sindelar CV - Biophys Rev (2011)

Bottom Line: Recent structural observations of kinesin-1, the founding member of the kinesin group of motor proteins, have led to substantial gains in our understanding of this molecular machine.The new structural information revises or replaces key details of earlier models of kinesin's ATPase cycle that were based principally on crystal structures of free kinesin, and demonstrates that high-resolution characterization of the kinesin-microtubule complex is essential for understanding the structural basis of the cycle.I discuss the broader implications of the seesaw mechanism within the cycle of a fully functional kinesin dimer and show how the seesaw can account for two types of "gating" that keep the ATPase cycles of the two heads out of sync during processive movement.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, SHMC-E25, 333 Cedar Street, New Haven, CT 06520-8024 USA.

ABSTRACT
Recent structural observations of kinesin-1, the founding member of the kinesin group of motor proteins, have led to substantial gains in our understanding of this molecular machine. Kinesin-1, similar to many kinesin family members, assembles to form homodimers that use alternating ATPase cycles of the catalytic motor domains, or "heads", to proceed unidirectionally along its partner filament (the microtubule) via a hand-over-hand mechanism. Cryo-electron microscopy has now revealed 8-Å resolution, 3D reconstructions of kinesin-1•microtubule complexes for all three of this motor's principal nucleotide-state intermediates (ADP-bound, no-nucleotide, and ATP analog), the first time filament co-complexes of any cytoskeletal motor have been visualized at this level of detail. These reconstructions comprehensively describe nucleotide-dependent changes in a monomeric head domain at the secondary structure level, and this information has been combined with atomic-resolution crystallography data to synthesize an atomic-level "seesaw" mechanism describing how microtubules activate kinesin's ATP-sensing machinery. The new structural information revises or replaces key details of earlier models of kinesin's ATPase cycle that were based principally on crystal structures of free kinesin, and demonstrates that high-resolution characterization of the kinesin-microtubule complex is essential for understanding the structural basis of the cycle. I discuss the broader implications of the seesaw mechanism within the cycle of a fully functional kinesin dimer and show how the seesaw can account for two types of "gating" that keep the ATPase cycles of the two heads out of sync during processive movement.

No MeSH data available.


Experimental observation of elongation by helix α6 following ATP analog binding, in 8 Å cryo-EM maps. a Cryo-EM density map of microtubule-attached, no-nucleotide kinesin, showing a shortened “sausage” of density for α6 that corresponds to a non-helical conformation of the C terminus as seen in crystal structures of "ADP-like" kinesin. b Density map of microtubule-attached, ADP•Al•Fx kinesin showing elongation of α6, corresponding to occupation of the docking pocket by the neck linker and the C terminus of α6. Density corresponding to the docked conformation of the neck linker is also evident
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Fig3: Experimental observation of elongation by helix α6 following ATP analog binding, in 8 Å cryo-EM maps. a Cryo-EM density map of microtubule-attached, no-nucleotide kinesin, showing a shortened “sausage” of density for α6 that corresponds to a non-helical conformation of the C terminus as seen in crystal structures of "ADP-like" kinesin. b Density map of microtubule-attached, ADP•Al•Fx kinesin showing elongation of α6, corresponding to occupation of the docking pocket by the neck linker and the C terminus of α6. Density corresponding to the docked conformation of the neck linker is also evident

Mentions: On the opposite side of the motor from the nucleotide cleft (right-hand side in Fig. 2), ATP-triggered leftward tilting by the seesaw opens up a second hydrophobic pocket (this may be thought of as a "docking pocket") into which the neck linker can bind (Fig. 2a and b, right-hand side). An important observation from the kinesin-1 cryo-EM structures was that the C terminus of helix 6 (to which the neck linker attaches) also contributes to the docking interaction (Sindelar and Downing 2010), as has been observed in crystal structure comparisons of "ADP-like" and "ATP-like" kinesin conformations (Khalil et al. 2008). As shown in Fig. 3, the cryo-EM maps indicate that α6 lengthens by at least one turn during the docking transition, which would insert a conserved hydrophobic element from α6 (A321 in human conventional kinesin-1) into the docking pocket, as seen in crystal structures that exhibit a docked neck linker. The neck linker itself also contributes a highly conserved hydrophobic side chain (I325 in human kinesin-1 construct) into the docking pocket in the ATP-bound motor state. The resulting hydrophobic interactions are likely to contribute a sizable component of free energy stabilizing the docked motor conformation, owing to the substantial depth, size, and conserved hydrophobic nature of the docking pocket. The docked neck linker conformation is stabilized by additional interactions with kinesin’s N-terminal "cover strand" (Khalil et al. 2008), although cover-strand interactions do not appear to be directly modulated by the switch II helix. The overall view of kinesin’s microtubule-attached docking transition that emerges is that tight coupling between ATP binding and neck linker docking occurs through seesaw-mediated changes in the hydrophobic pockets present on either side of the motor domain. As described above, however, when kinesin is detached from the microtubule the left-hand side of the seesaw would no longer be regulated by bound nucleotide, accounting for the observed nonproductive behavior of the motor in the absence of microtubules.Fig. 3


A seesaw model for intermolecular gating in the kinesin motor protein.

Sindelar CV - Biophys Rev (2011)

Experimental observation of elongation by helix α6 following ATP analog binding, in 8 Å cryo-EM maps. a Cryo-EM density map of microtubule-attached, no-nucleotide kinesin, showing a shortened “sausage” of density for α6 that corresponds to a non-helical conformation of the C terminus as seen in crystal structures of "ADP-like" kinesin. b Density map of microtubule-attached, ADP•Al•Fx kinesin showing elongation of α6, corresponding to occupation of the docking pocket by the neck linker and the C terminus of α6. Density corresponding to the docked conformation of the neck linker is also evident
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3117274&req=5

Fig3: Experimental observation of elongation by helix α6 following ATP analog binding, in 8 Å cryo-EM maps. a Cryo-EM density map of microtubule-attached, no-nucleotide kinesin, showing a shortened “sausage” of density for α6 that corresponds to a non-helical conformation of the C terminus as seen in crystal structures of "ADP-like" kinesin. b Density map of microtubule-attached, ADP•Al•Fx kinesin showing elongation of α6, corresponding to occupation of the docking pocket by the neck linker and the C terminus of α6. Density corresponding to the docked conformation of the neck linker is also evident
Mentions: On the opposite side of the motor from the nucleotide cleft (right-hand side in Fig. 2), ATP-triggered leftward tilting by the seesaw opens up a second hydrophobic pocket (this may be thought of as a "docking pocket") into which the neck linker can bind (Fig. 2a and b, right-hand side). An important observation from the kinesin-1 cryo-EM structures was that the C terminus of helix 6 (to which the neck linker attaches) also contributes to the docking interaction (Sindelar and Downing 2010), as has been observed in crystal structure comparisons of "ADP-like" and "ATP-like" kinesin conformations (Khalil et al. 2008). As shown in Fig. 3, the cryo-EM maps indicate that α6 lengthens by at least one turn during the docking transition, which would insert a conserved hydrophobic element from α6 (A321 in human conventional kinesin-1) into the docking pocket, as seen in crystal structures that exhibit a docked neck linker. The neck linker itself also contributes a highly conserved hydrophobic side chain (I325 in human kinesin-1 construct) into the docking pocket in the ATP-bound motor state. The resulting hydrophobic interactions are likely to contribute a sizable component of free energy stabilizing the docked motor conformation, owing to the substantial depth, size, and conserved hydrophobic nature of the docking pocket. The docked neck linker conformation is stabilized by additional interactions with kinesin’s N-terminal "cover strand" (Khalil et al. 2008), although cover-strand interactions do not appear to be directly modulated by the switch II helix. The overall view of kinesin’s microtubule-attached docking transition that emerges is that tight coupling between ATP binding and neck linker docking occurs through seesaw-mediated changes in the hydrophobic pockets present on either side of the motor domain. As described above, however, when kinesin is detached from the microtubule the left-hand side of the seesaw would no longer be regulated by bound nucleotide, accounting for the observed nonproductive behavior of the motor in the absence of microtubules.Fig. 3

Bottom Line: Recent structural observations of kinesin-1, the founding member of the kinesin group of motor proteins, have led to substantial gains in our understanding of this molecular machine.The new structural information revises or replaces key details of earlier models of kinesin's ATPase cycle that were based principally on crystal structures of free kinesin, and demonstrates that high-resolution characterization of the kinesin-microtubule complex is essential for understanding the structural basis of the cycle.I discuss the broader implications of the seesaw mechanism within the cycle of a fully functional kinesin dimer and show how the seesaw can account for two types of "gating" that keep the ATPase cycles of the two heads out of sync during processive movement.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, SHMC-E25, 333 Cedar Street, New Haven, CT 06520-8024 USA.

ABSTRACT
Recent structural observations of kinesin-1, the founding member of the kinesin group of motor proteins, have led to substantial gains in our understanding of this molecular machine. Kinesin-1, similar to many kinesin family members, assembles to form homodimers that use alternating ATPase cycles of the catalytic motor domains, or "heads", to proceed unidirectionally along its partner filament (the microtubule) via a hand-over-hand mechanism. Cryo-electron microscopy has now revealed 8-Å resolution, 3D reconstructions of kinesin-1•microtubule complexes for all three of this motor's principal nucleotide-state intermediates (ADP-bound, no-nucleotide, and ATP analog), the first time filament co-complexes of any cytoskeletal motor have been visualized at this level of detail. These reconstructions comprehensively describe nucleotide-dependent changes in a monomeric head domain at the secondary structure level, and this information has been combined with atomic-resolution crystallography data to synthesize an atomic-level "seesaw" mechanism describing how microtubules activate kinesin's ATP-sensing machinery. The new structural information revises or replaces key details of earlier models of kinesin's ATPase cycle that were based principally on crystal structures of free kinesin, and demonstrates that high-resolution characterization of the kinesin-microtubule complex is essential for understanding the structural basis of the cycle. I discuss the broader implications of the seesaw mechanism within the cycle of a fully functional kinesin dimer and show how the seesaw can account for two types of "gating" that keep the ATPase cycles of the two heads out of sync during processive movement.

No MeSH data available.