Demonstration of Coiled-Coil Interactions within the Kinesin Neck Region Using Synthetic Peptides

Tripet, Brian; Vale, Ronald D.; Hodges, Robert S.

doi:10.1074/jbc.272.14.8946

Cited by 78 publications

(107 citation statements)

References 57 publications

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“…However, the charged residue found at the d position in the EKEK sequence (see Fig. 1 A) acts in an extremely destabilizing manner within a canonical coiled coil (9,21). Consequently, the observed overall stability of segment I may only be achieved if the low stability of the EKEK region is overcompensated by the hydrophobic collar.…”

Section: Resultsmentioning

confidence: 99%

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Single molecule mechanics of the kinesin neck

Bornschlögl

Woehlke

Rief

2009

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

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Structural integrity as well as mechanical stability of the parts of a molecular motor are crucial for its function. In this study, we used high-resolution force spectroscopy by atomic force microscopy to investigate the force-dependent opening kinetics of the neck coiled coil of Kinesin-1 from Drosophila melanogaster. We find that even though the overall thermodynamic stability of the neck is low, the average opening force of the coiled coil is >11 pN when stretched with pulling velocities >150 nm/s. These high unzipping forces ensure structural integrity during motor motion. The high mechanical stability is achieved through a very narrow N-terminal unfolding barrier if compared with a conventional leucine zipper. The experimentally mapped mechanical unzipping profile allows direct assignment of distinct mechanical stabilities to the different coiled-coil subunits. The coiled-coil sequence seems to be tuned in an optimal way to ensure both mechanical stability as well as motor regulation through charged residues.atomic force microscopy ͉ neck coiled coil ͉ unzipping ͉ molecular motor

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Section: Resultsmentioning

confidence: 99%

“…Kinesin-1's neck coiled coil contains highly unconventional sequence elements (9,10) that seem to be conserved in Kinesin-1 motor proteins from bilateral animals ( Fig. 1A) (11).…”

mentioning

confidence: 99%

Single molecule mechanics of the kinesin neck

Bornschlögl

Woehlke

Rief

2009

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

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“…Recent work with peptides from the neck region has established that such uncoiling of the NH 2 -terminal half of the neck can occur (4,5). Although the exact oligomeric state of the complex between the tail and head/neck is not known, the interaction between monomeric DKH365 and GST893-975 suggests that formation of a 4-helix bundle is not necessary, at least for the NH 2 -terminal half of the neck.…”

Section: Discussionmentioning

confidence: 99%

“…There is a likely hinge at position ϳ400 that marks the boundary between the neck and the stalk. Peptides from the neck (4,5) and stalk (6) have been demonstrated to interact in a coiled-coil manner by several criteria. The crystal structure of a dimeric head plus neck construct has been recently determined (7) and it directly demonstrates the coiled-coil interactions in the neck region.…”

mentioning

confidence: 99%

Formation of the Compact Confomer of Kinesin Requires a COOH-terminal Heavy Chain Domain and Inhibits Microtubule-stimulated ATPase Activity

Stock

Guerrero²,

Cobb

et al. 1999

Journal of Biological Chemistry

146

145

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Full-length Drosophila kinesin heavy chain from position 1 to 975 was expressed in Escherichia coil (DKH975) and is a dimer. The sedimentation coefficient of DKH975 shifts from 5.4 S at 1 M NaCl to ϳ6.9 S at <0.2 M NaCl. This transition of DKH975 between extended and compact conformations is essentially identical to that for the heavy chain dimer of bovine kinesin (Hackney, D. D., Levitt, J. D., and Suhan, J. (1992) J. Biol. Chem. 267, 8696 -8701). Thus the capacity for undergoing the 7 S/5 S transition is an intrinsic property of the heavy chains and requires neither light chains nor eukaryotic post-translational modification. DKH960 undergoes a similar transition, indicating that the extreme COOH-terminal region is not required. More extensive deletions from the COOH-terminal (DKH945 and DKH937) result in a shift in the midpoint for the transition to lower salt concentrations. DKH927 and shorter constructs remaining extended even in the absence of added salt. Thus the COOH-terminal ϳ50 amino acids are required for the formation of the compact conformation. Separately expressed COOH-terminal tail segments and NH 2 -terminal head/neck segments interact in a salt-dependent manner that is consistent with the compact conformer being produced by the interaction of domains from these regions of the heavy chain dimer. The microtubule-stimulated ATPase rate of DKH975 in the compact conformer is strongly inhibited compared with the rate of extended DKH894 (4 s ؊1 and 35 s ؊1 , respectively, for k cat at saturating microtubules).Kinesin is an ATP-dependent motor protein that is involved in movement of membranous vesicles along MTs 1 (see Refs. 1 and 2). The NH 2 -terminal ϳ340 amino acids of the heavy chain forms a globular motor domain (head) that has MT-stimulated ATPase activity (see Fig. 2B). The motor domain is followed by a long central coiled-coil stalk region and a small nonhelical domain at the COOH-terminal. The central region contains several positions at which the coiled-coil propensity is low and these likely represent hinges in the stalk. The first coiled-coil region that extends from the motor domain is designated the neck region. Constructs that contain the head and the COOHterminal part of the coiled-coil neck form dimers (3). There is a likely hinge at position ϳ400 that marks the boundary between the neck and the stalk. Peptides from the neck (4, 5) and stalk (6) have been demonstrated to interact in a coiled-coil manner by several criteria. The crystal structure of a dimeric head plus neck construct has been recently determined (7) and it directly demonstrates the coiled-coil interactions in the neck region.Native kinesin is a heterotetramer composed of a dimeric heavy chain core with two light chains attached in the COOHterminal region (8, 9). At high salt kinesin exist in an extended conformation with an s 20,w value of ϳ6 S, but adopts a more compact conformation at low salt concentration with an s 20,w value of ϳ9 S. This global conformational transition is readily reversible and can be observed b...

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“…The neck and its link to the head are important for the determination of the directionality of movement (Henningsen and Schliwa, 1997;Case et al, 1997;Endow and Waligora, 1998). The neck of animal kinesins has been found to form a coiled-coil (Kozielski et al, 1997;Morii et al, 1997;Tripet et al, 1997) and to be involved in kinesin's processivity (Romberg et al, 1998), while both the neck and hinge have been shown to play a role in mechanochemical coupling (Grummt et al, 1998).…”

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confidence: 99%

Universal and Unique Features of Kinesin Motors: Insights from a Comparison of Fungal and Animal Conventional Kinesins

Kirchner¹,

Woehlke²,

Schliwa³

1999

Biological Chemistry

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Kinesins are microtubule motors that use the energy derived from the hydrolysis of ATP to move unidirectionally along microtubules. The founding member of this still growing superfamily is conventional kinesin, a dimeric motor that moves processively towards the plus end of microtubules. Within the family of conventional kinesins, two groups can be distinguished to date, one derived from animal species, and one originating from filamentous fungi. So far no conventional kinesin has been reported from plant cells. Fungal and animal conventional kinesins differ in several respects, both in terms of their primary sequence and their physiological properties. Thus all fungal conventional kinesins move at velocities that are 4 -5 times higher than those of animal conventional kinesins, and all of them appear to lack associated light chains. Both groups of motors are characterized by a number of group-specific sequence features which are considered here with respect to their functional importance. Animal and fungal conventional kinesins also share a number of sequence characteristics which point to common principles of motor function. The overall domain organization is remarkably similar. A C-terminal sequence motif common to all kinesins, which constitutes the only region of high homology outside the motor domain, suggests common principles of cargo association in both groups of motors. Consideration of the differences of, and similarities between, fungal and animal kinesins offers novel possibilities for experimentation (e. g., by constructing chimeras) that can be expected to contribute to our understanding of motor function.

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Demonstration of Coiled-Coil Interactions within the Kinesin Neck Region Using Synthetic Peptides

Cited by 78 publications

References 57 publications

Single molecule mechanics of the kinesin neck

Single molecule mechanics of the kinesin neck

Formation of the Compact Confomer of Kinesin Requires a COOH-terminal Heavy Chain Domain and Inhibits Microtubule-stimulated ATPase Activity

Universal and Unique Features of Kinesin Motors: Insights from a Comparison of Fungal and Animal Conventional Kinesins

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