Identification of Kinesin Neck Region as a Stable α-Helical Coiled Coil and Its Thermodynamic Characterization

Morii, Hisayuki; Takenawa, Tatsuyuki; Arisaka, Fumio; Shimizu, Takashi

doi:10.1021/bi962392l

Cited by 60 publications

(84 citation statements)

References 33 publications

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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.…”

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confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

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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“…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).…”

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confidence: 99%

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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“…Khc 74 over a large deletion [Df(2R)Jp6] caused paralysis and 50% lethality by mid-third instar with Figure S1 (Hackney 2007;Kull et al 1996;Morii et al 1997;Sindelar 2011;Vale and Fletterick 1997). c The stage in which 50% of Khc mutant /Df(2R)Jp6 animals had died (n $ 100).…”

Section: Extragenic Influences On Phenotype Severitymentioning

confidence: 99%

Three Routes to Suppression of the Neurodegenerative Phenotypes Caused by Kinesin Heavy Chain Mutations

et al. 2012

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Kinesin-1 is a motor protein that moves stepwise along microtubules by employing dimerized kinesin heavy chain (Khc) subunits that alternate cycles of microtubule binding, conformational change, and ATP hydrolysis. Mutations in the Drosophila Khc gene are known to cause distal paralysis and lethality preceded by the occurrence of dystrophic axon terminals, reduced axonal transport, organelle-filled axonal swellings, and impaired action potential propagation. Mutations in the equivalent human gene, Kif5A, result in similar problems that cause hereditary spastic paraplegia (HSP) and Charcot-Marie-Tooth type 2 (CMT2) distal neuropathies. By comparing the phenotypes and the complementation behaviors of a large set of Khc missense alleles, including one that is identical to a human Kif5A HSP allele, we identified three routes to suppression of Khc phenotypes: nutrient restriction, genetic background manipulation, and a remarkable intramolecular complementation between mutations known or likely to cause reciprocal changes in the rate of microtubule-stimulated ADP release by kinesin-1. Our results reveal the value of large-scale complementation analysis for gaining insight into protein structure-function relationships in vivo and point to possible paths for suppressing symptoms of HSP and related distal neuropathies.T O transport organelles and other cargo complexes through cytoplasm over long distances, molecular motor proteins use paired, force-producing subunits to walk along microtubules. This is especially important for the development and maintenance of cells in which biosynthetic processes are asymmetrically located, such as neurons with long axons (Saxton and Hollenbeck 2012). Mutations in microtubule motor proteins are known to cause hereditary forms of spastic paraplegia (HSP) (Reid et al. 2002;Blackstone et al. 2011) and Charcot-Marie-Tooth type 2 (CMT2) (Goizet et al. 2009;Crimella et al. 2011) distal neuropathies and are suspected of contributing to the progression of other neurodegenerative diseases, including amyotrophic lateral sclerosis, Alzheimer's, Huntington's, and Parkinson's (De Vos et al. 2008;Morfini et al. 2009;Perlson et al. 2010). Defining the basic mechanisms of motor-driven cytoplasmic transport in axons and understanding how defects in them contribute to neurodegeneration are important areas for investigation.The structure and mechanochemistry of kinesin-1, a major axonal transport motor, have been the subject of intense study. The holoenzyme is centered around kinesin heavy chain (Khc, or Kif5 in humans), composed of an N-terminal globular head (the motor domain) connected to a long a-helical stalk that terminates in a small C-terminal globular tail (Vale et al. 1985;Yang et al. 1989). Two Khc heads, dimerized via coiled-coil interactions of their stalks, alternate cycles of ATP binding-hydrolysis-release and microtubule binding-release to move stepwise toward microtubule plus ends (reviewed by Vale and Milligan 2000;Sindelar 2011), which in axons are oriented away from the cell...

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Identification of Kinesin Neck Region as a Stable α-Helical Coiled Coil and Its Thermodynamic Characterization

Cited by 60 publications

References 33 publications

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

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

Single molecule mechanics of the kinesin neck

Three Routes to Suppression of the Neurodegenerative Phenotypes Caused by Kinesin Heavy Chain Mutations

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