The cation-π interaction in cysteine-rich domain of Smoothened is critical for its cholesterylation and function

Acta Biochim Biophys Sin (Shanghai). 2022 Aug 25;54(8):1171-1179. doi: 10.3724/abbs.2022090.

Abstract

The Hedgehog (Hh) signaling pathway is critical for embryonic development and tissue renewal. The G protein-coupled receptor (GPCR)-like protein Smoothened (SMO) is the central signal transducer in the Hh pathway. Cholesterol binds and then covalently links to the D95 residue of cysteine-rich domain (CRD) of human SMO. The cholesterylation of CRD is critical for SMO activation. SMO cholesterylation is a Ca 2+-boosted autoreaction that requires the formation of an ester bond between the side chains of D95 and Y130 as an intermediate. It is unknown whether other residues of SMO are involved in the esterification between D95 and cholesterol. In this study, we find that the SMO-CRD(27-192) can undergo cholesterylation. In addition to D95 and Y130, the residues critical for cholesterol modification include Y85, T88, T90, W109, W119, K133, E160 and F166. T88, W109, W119 and F166 also seem to be involved in protein folding. Notably, we find that Y85 and K133 form a cation-π interaction whose disruption abolishes cholesterylation and ciliary localization of SMO. This study highlights the mechanism and function of cholesterol modification of SMO.

Keywords: Hedgehog; Smoothened; cation-π interaction; cholesterylation; cysteine-rich domain.

MeSH terms

  • Cations
  • Cholesterol / metabolism
  • Cysteine*
  • Esters
  • Hedgehog Proteins* / genetics
  • Hedgehog Proteins* / metabolism
  • Humans
  • Receptors, G-Protein-Coupled / genetics
  • Receptors, G-Protein-Coupled / metabolism
  • Smoothened Receptor / genetics
  • Smoothened Receptor / metabolism

Substances

  • Cations
  • Esters
  • Hedgehog Proteins
  • Receptors, G-Protein-Coupled
  • Smoothened Receptor
  • Cholesterol
  • Cysteine

Grants and funding

This work was supported by the grants from the National Natural Science Foundation of China (Nos. 92153301, and 32021003) and Ministry of Science and Technology of China (No. 2018YFA0800700). B.-L.S. acknowledges the support from the Tencent Foundation through the XPLORER PRIZE.