Selected highlights:
1. Structural characterization of the Hh signal transducer, oncoprotein and GPCR Smoothened (SMO) ((Byrne Nature 2016, Luchetti Elife 2016):
SMO belongs to the Frizzled-class GPCRs composed of an extracellular (CRD) and transmembrane (TMD) domain. We determined the SMO crystal structure, the first structure of any GPCR with an ectodomain, and identified two separable ligand-binding sites, one in the TMD and one in the CRD. The TMD-binding site binds to synthetic ligands like the anticancer drug vismodegib. We showed that cholesterol occupies the CRD-binding site and activates Hh signalling. We also determined the structure of SMO with vismodegib. Vismodegib-binding transmits a conformational change to the CRD resulting in loss of cholesterol-binding, thus revealing how GPCRs are controlled by ligand-regulated interactions between their extracellular and transmembrane domains. Several SMO residues mutated in vismodegib-resistant cancers are located close to the vismodegib-binding site, explaining loss of binding and resistance observed in the clinic. Whereas the CRD-binding site is essential for signalling, mutations in the TMD site blocking vismodegib-binding display normal Hh activity. Our identification of a ligand-binding site in the CRD essential for SMO activation opens new therapeutic avenues especially for cancers resistant to conventional therapy.
2. Development of an effective and innovative mammalian expression system for structural studies (Elegheert Nature Protocols 2019):
For efficient production of our target proteins and complexes, we improved our transient transfection protocols that have been traditionally used in our laboratory. We designed and implemented a lentiviral plasmid suite for constitutive or inducible large-scale production of soluble and membrane proteins in HEK293 cell lines. Inducible protein expression also allows full control over expression parameters and leads to milligram-scale quantities of target proteins and complexes from litre-scale suspension cultures (e.g. for the GPCR SMO, the 12 TM helix-containing Hh receptor Patched-1 (PTCH1) and some of our binary and ternary Hh signal transduction complexes). A feature of our vector suite is the bicistronic expression of fluorescent marker proteins for enrichment of co-transduced cells using cell sorting. This strategy conveniently allows the rapid production of HEK293 cell lines that stably co-express multiple proteins of interest, and as such is suitable for protein complexes.
3. Structural studies on Hh receptor-ligand complexes (Rudolf Nature Chem Biol 2019):
HH ligands are covalently coupled to two lipids - a palmitoyl group at the N-terminus and a cholesteryl group at the C-terminus. While the palmitoyl group plays a role in inactivating PTCH1, the main Hh receptor, the function of the cholesterol modification has remained mysterious. Using structural and biochemical studies, along with the re-assessment of prior cryo-EM structures, we find that the HH-attached cholesterol binds the first extracellular domain of PTCH1 and promotes its inactivation, thus triggering Hh signalling. Molecular dynamics simulations show that this interaction leads to the closure of a tunnel through PTCH1 that serves as the putative conduit for sterol transport. Thus, SHH inactivates PTCH1 by grasping its extracellular domain with two lipidic pincers, the N-terminal palmitate and the C-terminal cholesterol, which are both inserted into the PTCH1 protein core. Since PTCH1 is thought to function as a cholesterol transporter, we suggest that Hh ligands evolved the ability to inhibit PTCH1 by linking its substrate cholesterol to a protein chain that arrests the transport cycle.
