Major Finding: A previously unknown sterol binding pocket was found in the crystal structure of active Smoothened.
Mechanism: Sterol binding stabilizes Smoothened's active state, yielding increased Hedgehog-pathway signaling.
Impact: Analysis of the structure suggests a way to overcome Smoothened-inhibitor resistance.
Malfunctions in Hedgehog signaling are involved in several types of cancer, and some new and investigational therapies target the Hedgehog-pathway GPCR Smoothened (SMO). Deshpande and colleagues determined the 2.8-Å crystal structure of mouse SMO bound to the agonist SAG21k and a nanobody that stabilizes the active state. The structure revealed that binding of the nanobody stabilized a conformation typically associated with GPCR activation, likely representing SMO's active state. Transmembrane helix 6 was displaced outward and transmembrane helix 5 was shifted inward relative to their positions in the inactive human SMO crystal structure. Additionally, a sterol molecule—probably cholesterol—bound a previously uncharacterized sterol binding pocket deep within the seven-transmembrane (7TM) pocket on the intracellular side. Molecular dynamics simulations of active SMO in the absence of the agonist and the nanobody with cholesterol bound in the 7TM sterol-binding site showed that cholesterol binding at the site was stable and that the active conformation was maintained without the agonist or nanobody when cholesterol was present. Mutations that prevented sterol binding in the 7TM sterol-binding pocket ablated SMO-dependent GLI activation by both the agonist and native Sonic hedgehog, and this activation was also decreased by mutating a residue in the 7TM that hydrogen bonds to cholesterol. In cells and in vitro, cholesterol binding within the 7TM was required for sterol-mediated SMO activation. The structure also contained a channel leading to the newly discovered sterol binding site. This tunnel is occluded by many SMO antagonists, suggesting they may work, at least in part, by blocking sterol access to the site. The antagonist SANT-1 is unique in that its binding site overlaps substantially with this sterol binding pocket, implying it may be less prone to acquired SMO-inhibitor resistance because mutations that destabilize SANT-1 binding would have a strong propensity to disrupt sterol binding as well.
Notes
Note: Research Watch is written by Cancer Discovery editorial staff. Readers are encouraged to consult the original articles for full details. For more Research Watch, visit Cancer Discovery online at http://cancerdiscovery.aacrjournals.org/CDNews.
- ©2019 American Association for Cancer Research.
Volume 9, Issue 9
