Unusual properties of the fungal conventional kinesin neck domain from Neurospora crassa

Kallipolitou, Athina; Deluca, Dominga; Majdic, Ulrike; Lakämper, Stefan; Cross, Robert A.; Meyhöfer, Edgar; Moröder, Luis; Schliwa, Manfred; Woehlke, Günther

doi:10.1093/emboj/20.22.6226

Cited by 34 publications

(66 citation statements)

References 50 publications

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“…Protein Expression and Purification-Kinesin was expressed and purified as described (19,20). Briefly, Neurospora crassa kinesin (NcKin) 2 was bacterially expressed using a T7 promoter system, isolated * This work was supported by the Deutsche Forschungsgemeinschaft and the FriedrichBaur-Foundation.…”

Section: Methodsmentioning

confidence: 99%

“…The C-terminal deletion construct NcKin-343 (NcKin residues 1-343; Ref. 19) was used to study the behavior of monomeric constructs, NcKin-436hKT (NcKin residues 1-436 and human kinesin residues 432-546; Ref. 23) that of dimeric kinesins.…”

Section: Methodsmentioning

confidence: 99%

“…10 and 24). The selected wild-type residues were changed to cysteines in Kinesin-1 of N. crassa (NcKin) using a PCR-based mutagenesis protocol (Stratagene QuickChange) on a bacterial expression vector (19). In the N. crassa kinesin sequence, the introduced cysteine residues were located at positions Ser (14).…”

Section: Methodsmentioning

confidence: 99%

“…The basal ATP turnover was assayed in 12A25 by quantification of radioactive inorganic phosphate released from hydrolyzed [␥-32 P]ATP over time (19). Phosphate was separated from nucleotide and protein using charcoal (28).…”

Section: Methodsmentioning

confidence: 99%

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Feedback of the Kinesin-1 Neck-linker Position on the Catalytic Site

Hahlen

Ebbing

Reinders

et al. 2006

Journal of Biological Chemistry

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Kinesin-1 motor proteins step along microtubules by a mechanism in which the heads cycle through microtubule-bound and unbound states in an interlaced fashion. An important contribution to head-head coordination arises from the action of the neck-linker that docks onto the core motor domain upon ATP binding. We show here that the docked neck-linker not only guides the microtubule-unbound head to the next microtubule binding site but also signals its position to the head to which it is attached. Cross-linking studies on mutated kinesin constructs reveal that residues at the interface motor core/docked neck-linker, among them most importantly a conserved tyrosine, are involved in this feedback. The primary effect of the docked neck-linker is a reduced microtubule binding affinity in the ADP state.Kinesin-1 (formerly conventional kinesin) proteins are molecular motors that move along microtubules in a stepwise fashion at the expense of ATP hydrolysis energy. A large body of evidence shows that these motors are dimeric and generate motility by the alternating action of two identical motor domains (1-6). A crucial structural intermediate is a Kinesin-1 molecule that is strongly attached to the filament by one nucleotide-free head, whereas the other one remains in a weakly microtubule-binding ADP state. Upon ATP binding, a very rapid structural change of the neck-linker of the microtubule-bound motor head occurs that allows the partner head to lose its ADP and to bind tightly to the microtubule (7). The neck-linker, a short stretch of ϳ10 amino acid residues that follows immediately C-terminal of the catalytic core motor domain, is thought to exist in at least two different conformations, one that is docked or zippered to the motor core, and a flexible undocked structure (8, 9).The docked neck-linker is visible in some crystal structures (10, 11), and its path along microtubule-bound kinesin has been deduced from cryo-EM (8, 12). According to these data, the neck-linker zippers along a groove in the core motor domain between helix 6 on one end, and loop 10 on the other end where it is held by a meshwork of hydrogen bonds. The nature of the undocked conformation is not clear and might differ in truncated, monomeric constructs (that have been used for many of the structural investigations) and dimeric ones.The conformational change of the neck-linker has been used to explain the mechanism of Kinesin-1 motility. It is thought that necklinker docking allows or causes the second head to bind to a forward microtubule binding site. Interestingly, it is unlikely that the docking process of the neck-linker generates the force exerted by kinesin. The free energy of neck-linker docking is surprisingly low (⌬G ϳϪ1 kJ/mol; Ref. 9), and hardly accounts for the force that can be generated by a single kinesin molecule (5-6 pN, Ref. 13). Estimated from crystal structures, the length of the neck-linker is at least 4 nm. Accordingly, ⌬G will be Ն 12 kJ/mol, which is roughly 10-fold larger than the measured free enthalpy change of neck-l...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Feedback of the Kinesin-1 Neck-linker Position on the Catalytic Site

Hahlen

Ebbing

Reinders

et al. 2006

Journal of Biological Chemistry

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“…The neck linker of conventional kinesin has been shown to be a key motion-transducing element (13,34,(37)(38)(39)(40)(41). ATP binding promoted neck linker docking onto the catalytic core in the direction toward the plus end of the microtubule, and ATP hydrolysis returned the neck linker to a more mobile conformation.…”

mentioning

confidence: 99%

Microtubule−Kinesin Interface Mutants Reveal a Site Critical for Communication

et al. 2004

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Strict coordination of the two motor domains of kinesin is required for driving the processive movement of organelles along microtubules. Glutamate 164 of the kinesin heavy chain was shown to be critical for kinesin function through in vivo genetics in Drosophila melanogaster. The mutant motor E164K exhibited reduced steady-state ATPase activity and higher affinity for both ATP and microtubules. Moreover, an alanine substitution at this position (E164A) caused similar defects. It became stalled on the microtubule and was unable to bind and hydrolyze ATP at the second motor domain. Glu 164 , which has been conserved through evolution, is located at the motor-microtubule interface close to key residues on helix α12 of β-tubulin. We explored further the contributions of Glu 164 to motor function using several site-directed mutant proteins: E164K, E164N, E164D, E164Q, and D165A. The results indicate that the microtubule-E164K complex can only bind and hydrolyze one ATP. ATP with increased salt was able to dissociate a population of E164K motors from the microtubule but could not dissociate E164A. We tested the basis of the stabilized microtubule interaction with E164K, E164N, and E164A. The results provide new insights about the motormicrotubule interface and the pathway of communication for processive motility.Kinesin uses the energy of ATP hydrolysis to generate unidirectional processive movement along microtubules (1-4). The key to understanding the processivity of kinesin is to define the transition states and structural requirements for the coordination of the two catalytic motor domains with each other and with the microtubule. The nucleotide binding motifs of kinesin show both structural and sequence homology with G-proteins, myosins, and other kinesin superfamily members in amino acid sequence and in structural organization (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). In addition, these diverse enzymes exhibit a conserved mechanism in which the nucleotide state at the † This work was supported by National Institutes of Health Grants GM54154 (S.P.G.) and GM46295 (W.M.S.), National Institute of Arthritis and Musculoskeletal and Skin Diseases Career Development Award K02-AR47841 (S.P.G.), a predoctoral fellowship from the American Heart Association, Indiana Affiliate, Inc. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript active site is translated, through switch I and switch II, into a series of conformational changes that are critical for function (18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34) (reviewed in refs 17,35, and 36).The neck linker of conventional kinesin has been shown to be a key motion-transducing element (13,34,(37)(38)(39)(40)(41). ATP binding promoted neck linker docking onto the catalytic core in the direction toward the plus end of the microtubule, and ATP hydrolysis returned the neck linker to a more mobile conformation. The neck linker docking site includes parts of the motor domain near loop L12 (microtubule binding face) and sw...

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Synthesis and conformational characterization of peptides related to the neck domain of a fungal kinesin

Deluca

Woehlke

Moröder

2003

Journal of Peptide Science

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The Y362K mutation in the neck domain of conventional kinesin from Neurospora crassa provokes a significant reduction of the rate of movement along microtubules. Since the alpha-helical coiled-coil structure of the neck region is implicated in the mechanism of the processive movement of kinesins, a series of peptides related to the heptad region 338-379 of the wild-type and the variant fungal kinesinswere synthesized as monomers and as N-terminal disulfide dimers, crosslinked to favour self-association into coiled-coil structures entropically. A comparison of the dichroic properties of the peptides and the effects of trifluoroethanol and peptide concentration clearly confirmed the strong implication of the single point mutation in destabilizing the intrinsic propensity of the peptides to fold into the supercoiled conformation. That there is a correlation between the stability of the coiled-coil and rate of movement of the kinesin is confirmed.

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Unusual properties of the fungal conventional kinesin neck domain from Neurospora crassa

Cited by 34 publications

References 50 publications

Feedback of the Kinesin-1 Neck-linker Position on the Catalytic Site

Feedback of the Kinesin-1 Neck-linker Position on the Catalytic Site

Microtubule−Kinesin Interface Mutants Reveal a Site Critical for Communication

Synthesis and conformational characterization of peptides related to the neck domain of a fungal kinesin

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