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bioRxiv · 10.1101/2023.10.17.562760

Allosteric regulation of a molecular motor through de novo protein design

Cross, J. A.·Dawson, W. M.·Shulka, S. R.·Weijman, J. F.·Mantell, J.·Dodding, M. P.·Woolfson, D. N.
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Abstract

Many enzymes are allosterically regulated. Our ability to manipulate these structural changes is limited. Here we install an allosteric switch into the kinesin-1 microtubule motor in vitro and in cells. Kinesin-1 is a heterotetramer that accesses open active and closed auto-inhibited states. The equilibrium between these centres on a flexible elbow within a complex coiled-coil architecture. We target the elbow to engineer a closed state that can be opened with a de novo designed peptide. The alternative states are modelled computationally and confirmed by biophysical measurements and electron microscopy. In cells, peptide-driven activation increases kinesin transport, demonstrating a primary role for conformational switching in regulating motor activity. The designs are enabled by our understanding of ubiquitous coiled-coil structures, opening possibilities for controlling other protein activities. One Sentence SummaryDe novo peptide and protein design are used to engineer an allosteric switch into kinesin-1 motors in vitro and directly in cells.

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Cross, J. A., Dawson, W. M., Shulka, S. R., Weijman, J. F., Mantell, J., Dodding, M. P., Woolfson, D. N.. 2023-10-17. Allosteric regulation of a molecular motor through de novo protein design. https://doi.org/10.1101/2023.10.17.562760

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