The Mechanism of Cholesterol Modification of Hedgehog Ligand

Abstract

Hedgehog (Hh) proteins are important components of signal transduction pathways involved in animal development, and their defects are implicated in carcinogenesis. Their N-terminal domain (HhN) acts as a signaling ligand, and their C-terminal domain (HhC) performs an autocatalytic function of cleaving itself away, while adding a cholesterol moiety to HhN. HhC has two sub-domains: a hedgehog/intein (hint) domain that primarily performs the autocatalytic activity, and a sterol-recognition region (SRR) that binds to cholesterol and properly positions it with respect to HhN. The three-dimensional details of this autocatalytic mechanism remain unknown, as does the structure of the precursor Hh protein. In this study, a complete cholesterol-bound precursor form of the drosophila Hh precursor is modeled using known crystal structures of HhN and the hint domain, and a hypothesized similarity of SRR to an unrelated but similar-sized cholesterol binding protein. The restrained geometries and topology switching (RGATS) strategy is then used to predict atomic-detail pathways for the full autocatalytic reaction starting from the precursor and ending in a cholesterol-linked HhN domain and a cleaved HhC domain. The RGATS explicit solvent simulations indicate the roles of individual HhC residues in facilitating the reaction, which can be confirmed through mutational experiments. These simulations also provide plausible structural models for the N/S acyl transfer intermediate and the product states of this reaction. This study thus provides a good framework for future computational and experimental studies to develop a full structural and dynamic understanding of Hh autoprocessing.

Dual roles of the sterol recognition region in Hedgehog protein modification

“…The SRR, which spans 50-100 residues in different species, bears no clear homology to known proteins in any domain of life. While a recent computational study posits an intriguing connection between the SRR of D. melanogaster and the cryptogein protein of P. cryptogea, 23 this Fig. 1 The SRR is a helix-loop-helix motif.…”

Cholesterol and Hedgehog Signaling: Mutual Regulation and Beyond

“…All HH family members share the same tertiary structure and follow the same way of maturation in HH-producing cells ( Valentini et al, 1997 ). After translation, initially synthesized HH pro-proteins undergo multiple post-translational processing modifications to obtain activated HH proteins and subsequently to be released from the producing cells ( Banavali, 2020 ). As depicted in Figure 1 , after the signal sequence is removed, the HH molecule (∼45 kDa) first undergoes a cleavage catalyzed by its own C-terminal domain that is between the conserved glycine and cysteine residues, generating two polypeptides including the N-terminal polypeptide (HH-Np) of ∼19 kDa that contains the whole functions of the signaling in both vertebrates and invertebrates, and the C-terminal polypeptide (HH-Cp) of ∼25 kDa that has no function other than catalyzing the autoproteolytic cleavage.…”

Hedgehog proteins create a dynamic cholesterol interface

“…Although the Hh cholesterolysis reaction was identified over 20 years ago [11], the structural details of Hog-promoted cholesterol ligation remain vague. In 2020, a first computational study by Banavali advanced a model of the full-length D. melanogaster (fly) protein using a cholesterol-binding bacterial cryptogein as a template for the SRR (S1 Fig) [34]. This study provided a detailed picture of the Hint fold active site during the cholesterolysis process, and suggested SRR residues that might influence the efficiency of the reaction.…”