Binding of ncd to microtubules induces a conformational change near the junction of the motor domain with the neck.
We have covalently attached an electron paramagnetic resonance (EPR) spin probe to Cys-670 of the motor domain of ncd (nonclaret disjunctional protein) in order to investigate conformational changes associated with the chemomechanical cycle. Spin-labeling is highly specific and does not affect ncd function as monitored by either the binding affinity to microtubules or the rate of ATP hydrolysis. The EPR spectra can be deconvoluted into two components, one that is highly mobile with respect to the protein and one that is strongly immobilized. In the absence of microtubules, the relative proportions of these two components varied with temperature, showing that the transition between them involves a large change in enthalpy (DeltaH degrees = -75 kJ/mol). This result implies that the two populations represent very different protein conformations. Binding to microtubules results in virtually all probes shifting into the immobilized component, independent of the nucleotide bound. Superposition of the structures of ncd and myosin subfragment 1 reveals that the labeled cysteine is very close to the region which is homologous to the helix containing the two reactive sulfhydryls in myosin and is approximately 10 A from the junction of the motor domain with the remainder of the molecule. We conclude that the binding of ncd to microtubules results in a conformational change in this region which may be involved in the working power stroke.
Naber
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N
Department of Biochemistry and Biophysics, Cardiovascular Research Institute, University of California, San Francisco 94143, USA. NABER@NIIHAU.UCSF.EDU
Cooke
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R
Pate
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E
D000595:Amino Acid Sequence; D000818:Animals; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D004330:Drosophila; D029721:Drosophila Proteins; D004578:Electron Spin Resonance Spectroscopy; D016547:Kinesins; D008868:Microtubule Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D009218:Myosins / Q000737:chemistry; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013113:Spin Labels; D013696:Temperature
D029721:Drosophila Proteins; D008868:Microtubule Proteins; D013113:Spin Labels; C066696:ncd protein, Drosophila; D009218:Myosins; D016547:Kinesins; D003545:Cysteine