Role of the kinesin neck linker and catalytic core in microtubule-based motility.
Kinesin motor proteins execute a variety of intracellular microtubule-based transport functions [1]. Kinesin motor domains contain a catalytic core, which is conserved throughout the kinesin superfamily, followed by a neck region, which is conserved within subfamilies and has been implicated in controlling the direction of motion along a microtubule [2] [3]. Here, we have used mutational analysis to determine the functions of the catalytic core and the approximately 15 amino acid 'neck linker' (a sequence contained within the neck region) of human conventional kinesin. Replacement of the neck linker with a designed random coil resulted in a 200-500-fold decrease in microtubule velocity, although basal and microtubule-stimulated ATPase rates were within threefold of wild-type levels. The catalytic core of kinesin, without any additional kinesin sequence, displayed microtubule-stimulated ATPase activity, nucleotide-dependent microtubule binding, and very slow plus-end-directed motor activity. On the basis of these results, we propose that the catalytic core is sufficient for allosteric regulation of microtubule binding and ATPase activity and that the kinesin neck linker functions as a mechanical amplifier for motion. Given that the neck linker undergoes a nucleotide-dependent conformational change [4], this region might act in an analogous fashion to the myosin converter, which amplifies small conformational changes in the myosin catalytic core [5,6].
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Departments of Pharmacology, Biochemistry and Biophysics, University of California, San Francisco 94143, USA.
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D000251:Adenosine Triphosphatases / Q000378:metabolism; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D006801:Humans; D016547:Kinesins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism / Q000502:physiology*; D008958:Models, Molecular; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence / Q000379:methods
D020409:Molecular Motor Proteins; D000251:Adenosine Triphosphatases; D016547:Kinesins