Yohendran Baskaran1,#, Khay Chun Ang1,2,,#, Praju Vikas Anekal1, Wee Lee Chan1, Jonathan M. Grimes3,4, Ed Manser1,5,6*, Robert C. Robinson1,2
1 Institute of Molecular and Cell Biology, Proteos, 61 Biopolis Drive Singapore 138673
2 Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597
3 Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK
4 Diamond Light Source Ltd., Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom
5 Institute of Medical Biology, A*STAR, Biopolis, Singapore 138648
6 Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597.
* corresponding author.
# These authors contributed equally to this work
Published in Nature Communications on 26 November 2015.
(Nature Communications 6: 8681; DOI: 10.1038/ncomms9681)
PAK4 is a metazoan-specific kinase acting downstream of Cdc42. Here we describe the structure of human PAK4 in complex with Inka1, a potent endogenous kinase inhibitor. Using single mammalian cells containing crystals 50 mm in length, we have determined the in cellulo crystal structure at 2.95 Å resolution, which reveals the details of how the PAK4 catalytic domain binds cellular ATP and the Inka1 inhibitor. The crystal lattice consists only of PAK4– PAK4 contacts, which form a hexagonal array with channels of 80 Å in diameter that run the length of the crystal. The crystal accommodates a variety of other proteins when fused to the kinase inhibitor. Inka1–GFP was used to monitor the process crystal formation in living cells. Similar derivatives of Inka1 will allow us to study the effects of PAK4 inhibition in cells and model organisms, to allow better validation of therapeutic agents targeting PAK4.
Incorporation of GFP into PAK4cat: Inka crystals (left). In cellulo crystals in a cell containing two crystals when GFP-inka is transfected with PAK4cat observed by confocal microscopy with actin (red) and nucleus (blue). Crystal packing of the PAK4cat (cyan/yellow) crystals (right) which form a channel in the presence of Inka1 (red).
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