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Nucleotide-induced conformations in the neck region of dimeric kinesin.

ABSTRACT: The neck region of kinesin constitutes a key component in the enzyme's walking mechanism. Here we applied cryoelectron microscopy and image reconstruction to investigate the location of the kinesin neck in dimeric and monomeric constructs complexed to microtubules. To this end we enhanced the visibility of this region by engineering an SH3 domain into the transition between neck linker and neck coiled coil. The resulting chimeric kinesin constructs remained functional as verified by physiology assays. In the presence of AMP-PNP the SH3 domains allowed us to identify the position of the neck in a well defined conformation and revealed its high flexibility in the absence of nucleotide. We show here the double-headed binding of dimeric kinesin along the same protofilament, which is characterized by the opposite directionality of neck linkers. In this configuration the neck coiled coil appears fully zipped. The position of the neck region in dimeric constructs is not affected by the presence of the tubulin C-termini as confirmed by subtilisin treatment of microtubules prior to motor decoration.

SUBMITTER: Skiniotis G

PROVIDER: S-EPMC152908 | biostudies-literature | 2003 Apr

REPOSITORIES: biostudies-literature

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Nucleotide-induced conformations in the neck region of dimeric kinesin.

Skiniotis Georgios G Surrey Thomas T Altmann Stephan S Gross Heinz H Song Young-Hwa YH Mandelkow Eckhard E Hoenger Andreas A

The EMBO journal 20030401 7

The neck region of kinesin constitutes a key component in the enzyme's walking mechanism. Here we applied cryoelectron microscopy and image reconstruction to investigate the location of the kinesin neck in dimeric and monomeric constructs complexed to microtubules. To this end we enhanced the visibility of this region by engineering an SH3 domain into the transition between neck linker and neck coiled coil. The resulting chimeric kinesin constructs remained functional as verified by physiology a ...[more]

PMID: 12660159

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Project description:Identifying conformational changes in kinesin family motors associated with nucleotide and microtubule (MT) binding is essential to determining an atomic-level model for force production and motion by the motors. Using the mobility of nucleotide analog spin probes bound at the active sites of kinesin family motors to monitor conformational changes, we previously demonstrated that, in the ADP state, the open nucleotide site closes upon MT binding [Naber, N., Minehardt, T. J., Rice, S., Chen, X., Grammer, J., Matuska, M., et al. (2003). Closing of the nucleotide pocket of kinesin family motors upon binding to microtubules. Science, 300, 798-801]. We now extend these studies to kinesin-1 (K) and ncd (nonclaret disjunctional protein) motors in ATP and ATP-analog states. Our results reveal structural differences between several triphosphate and transition-state analogs bound to both kinesin and ncd in solution. The spectra of kinesin/ncd in the presence of SLADP•AlFx/BeFx and kinesin, with the mutation E236A (K-E236A; does not hydrolyze ATP) bound to ATP, show an open conformation of the nucleotide pocket similar to that seen in the kinesin/ncd•ADP states. In contrast, the triphosphate analogs K•SLAMPPNP and K-E236A•SLAMPPNP induce a more immobilized component of the electron paramagnetic resonance spectrum, implying closing of the nucleotide site. The MT-bound states of all of the triphosphate analogs reveal two novel spectral components. The equilibrium between these two components is only weakly dependent on temperature. Both components have more restricted mobility than observed in MT-bound diphosphate states. Thus, the closing of the nucleotide pocket when the diphosphate state binds to MTs is amplified in the triphosphate state, perhaps promoting accelerated ATP hydrolysis. Consistent with this idea, molecular dynamics simulations show a good correlation between our spectroscopic data, X-ray crystallography, and the electron microscopy of MT-bound triphosphate-analog states.

Project description:Kinesin motor domains couple cycles of ATP hydrolysis to cycles of microtubule binding and conformational changes that result in directional force and movement on microtubules. The general principles of this mechanochemical coupling have been established; however, fundamental atomistic details of the underlying allosteric mechanisms remain unknown. This lack of knowledge hampers the development of new inhibitors and limits our understanding of how disease-associated mutations in distal sites can interfere with the fidelity of motor domain function. Here, we combine unbiased molecular-dynamics simulations, bioinformatics analysis, and mutational studies to elucidate the structural dynamic effects of nucleotide turnover and allosteric inhibition of the kinesin-5 motor. Multiple replica simulations of ATP-, ADP-, and inhibitor-bound states together with network analysis of correlated motions were used to create a dynamic protein structure network depicting the internal dynamic coordination of functional regions in each state. This analysis revealed the intervening residues involved in the dynamic coupling of nucleotide, microtubule, neck-linker, and inhibitor binding sites. The regions identified include the nucleotide binding switch regions, loop 5, loop 7, ?4-?5-loop 13, ?1, and ?4-?6-?7. Also evident were nucleotide- and inhibitor-dependent shifts in the dynamic coupling paths linking functional sites. In particular, inhibitor binding to the loop 5 region affected ?-sheet residues and ?1, leading to a dynamic decoupling of nucleotide, microtubule, and neck-linker binding sites. Additional analyses of point mutations, including P131 (loop 5), Q78/I79 (?1), E166 (loop 7), and K272/I273 (?7) G325/G326 (loop 13), support their predicted role in mediating the dynamic coupling of distal functional surfaces. Collectively, our results and approach, which we make freely available to the community, provide a framework for explaining how binding events and point mutations can alter dynamic couplings that are critical for kinesin motor domain function.

Dataset Information

Nucleotide-induced conformations in the neck region of dimeric kinesin.

Publications

Nucleotide-induced conformations in the neck region of dimeric kinesin.

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