A conserved tyrosine in the neck of a fungal kinesin regulates the catalytic motor core

Schäfer, Friederike; Deluca, Dominga; Majdic, Ulrike; Kirchner, Joachim; Schliwa, Manfred; Moröder, Luis; Woehlke, Günther

doi:10.1093/emboj/cdg036

Cited by 20 publications

(28 citation statements)

References 29 publications

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“…The microtubule-induced mant-ADP release was measured by mixing mant-ADP charged kinesin and microtubules in the presence of unlabeled ATP, either manually in a spectrophotometer (Aminco-Bowman AB2) or in a stopped-flow instrument (Biologics SF-3) under conditions published before (29).…”

Section: Methodsmentioning

confidence: 99%

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%

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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“…One conspicuous residue is tyrosine Y362 in the center of the neck coiled-coil domain. We showed that it is required for proper folding of the ␣-helical coiled-coil, but also for down-regulation of the motor activity, presumably in the inactivated, cargo-free state (Schafer et al, 2003). To explain how this residue inhibits the motor activity under repressing conditions, but allows full function under activating conditions, we hypothesized that the neck can switch between an ␣-helical coiled-coil conformation (active) and another, unknown conformation that allows interaction between Y362 and the core motor domain (inactivated).…”

Section: Introductionmentioning

confidence: 91%

“…To introduce cross-links between the neck domains of the two polypeptide chains of a kinesin dimer, proline 342 was exchanged to cysteine, because this residue is expected to be in the first a-position of the ␣-helical neck coiled-coil (Schafer et al, 2003). Such an arrangement is known to form interstrand disulfide bonds readily (Zhou et al, 1993).…”

Section: Cloning Of Constructsmentioning

confidence: 99%

“…Expression and purification of NcKin constructs was accomplished as described (Schafer et al, 2003). Briefly, 4 g of Escherichia coli cells were suspended in buffer A (20 mM Na-phosphate, pH 7.4, 50 mM NaCl, 5 mM MgCl 2 , 1 mM EGTA, 1 mM dithiothreitol [DTT], 10 M ATP, protease inhibitor mix (Roche Diagnostics, Penzberg, Germany), sonified, and centrifuged (35 min at 100,000 ϫ g).…”

Section: Expression and Purification Of Kinesin Constructsmentioning

confidence: 99%

“…The resulting mutant, NcKin hinge W384F was monomeric and showed a decelerated ATPase rate (21 Ϯ 6 vs. 66 Ϯ 21 s Ϫ1 for wild type, Table 1). This slow ATPase rate is typical for constructs that are inhibited by the residue Y362 in the neck domain (Schafer et al, 2003) and therefore suggests that the absence of W384 mimics the inactive state of NcKin where the coiled-coil conformation of the neck is disrupted.…”

Section: Conservation and Importance Of W384mentioning

confidence: 99%

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The Complex Interplay between the Neck and Hinge Domains in Kinesin-1 Dimerization and Motor Activity

Bathe

Hahlen

Dombi

et al. 2005

MBoC

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Kinesin-1 dimerizes via the coiled-coil neck domain. In contrast to animal kinesins, neck dimerization of the fungal kinesin-1 NcKin requires additional residues from the hinge. Using chimeric constructs containing or lacking fungalspecific elements, the proximal part of the hinge was shown to stabilize the neck coiled-coil conformation in a complex manner. The conserved fungal kinesin hinge residue W384 caused neck coiled-coil formation in a chimeric NcKin construct, including parts of the human kinesin-1 stalk. The stabilizing effect was retained in a NcKinW384F mutant, suggesting important -stacking interactions. Without the stalk, W384 was not sufficient to induce coiled-coil formation, indicating that W384 is part of a cluster of several residues required for neck coiled-coil folding. A W384-less chimera of NcKin and human kinesin possessed a non-coiled-coil neck conformation and showed inhibited activity that could be reactivated when artificial interstrand disulfide bonds were used to stabilize the neck coiled-coil conformation. On the basis of yeast two-hybrid data, we propose that the proximal hinge can bind kinesin's cargo-free tail domain and causes inactivation of kinesin by disrupting the neck coiled-coil conformation.

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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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A conserved tyrosine in the neck of a fungal kinesin regulates the catalytic motor core

Cited by 20 publications

References 29 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

The Complex Interplay between the Neck and Hinge Domains in Kinesin-1 Dimerization and Motor Activity

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

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