Na +-coordinating residues in DISP1 are conserved in PTCH1 and other metazoan RND family members, suggesting that Na + flux powers their conformationally driven activities. The bound and unbound DISP1 conformations display distinct Na +-site occupancies, which suggests a mechanism by which transmembrane Na + flux may power extraction of the lipid-linked Hedgehog signal from the membrane.
#Flux power series
Variability analysis reveals that ShhN binding is restricted to one extreme of a continuous series of DISP1 conformations. Interaction of the amino-terminal signalling domain of the Sonic hedgehog protein (ShhN) with DISP1 occurs via an extensive buried surface area and contacts with an extended furin-cleaved DISP1 arm. DISP1-NNN and variants that disrupt single Na + sites retain binding to, but are impaired in export of the lipid-modified Hedgehog protein to the SCUBE2 acceptor. The transmembrane channel and Na + binding are disrupted in DISP1-NNN, a variant with asparagine substitutions for three intramembrane aspartate residues that each coordinate and neutralize the charge of one of the three Na + ions. We find that the rate of Hedgehog export is dependent on the Na + gradient across the plasma membrane. Here we determine a cryo-electron microscopy structure of the mouse protein Dispatched homologue 1 (DISP1), revealing three Na + ions coordinated within a channel that traverses its transmembrane domain. Dispatched protein, which is related to the NPC1 and PTCH1 cholesterol transporters 1,2 and to H +-driven transporters of the RND family 3,4, enables tissue-patterning activity of the lipid-modified Hedgehog protein by releasing it from tightly -localized sites of embryonic expression 5-10. 9 Departments of Urology, and Developmental Biology, Stanford University School of Medicine, Stanford, CA, USA.
8 Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA. 7 Howard Hughes Medical Institute, University of California, San Francisco, CA, USA. 6 Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA.1 Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA. 5 Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, China.4 Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA.3 Howard Hughes Medical Institute, Neuroscience Department, The Scripps Research Institute, La Jolla, CA, USA.2 Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA.1 Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA.
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