A Single Aspartate Coordinates Two Catalytic Steps in Hedgehog Autoprocessing

Abstract

Hedgehog (Hh) signaling is driven by the cholesterol-modified Hh ligand, generated by autoprocessing of Hh precursor protein. Two steps in Hh autoprocessing, N–S acyl shift and transesterification, must be coupled for efficient Hh cholesteroylation and downstream signal transduction. In the present study, we show that a conserved aspartate residue, D46 of the Hh autoprocessing domain, coordinates these two catalytic steps. Mutagenesis demonstrated that D46 suppresses non-native Hh precursor autoprocessing and is indispensable for transesterification with cholesterol. NMR measurements indicated that D46 has a pKa of 5.6, ~2 units above the expected pKa of aspartate, due to a hydrogen-bond between protonated D46 and a catalytic cysteine residue. However, the deprotonated form of D46 side chain is also essential, because a D46N mutation cannot mediate cholesteroylation. On the basis of these data, we propose that the proton shuttling of D46 side chain mechanistically couples the two steps of Hh cholesteroylation.

Hedgehog proteins create a dynamic cholesterol interface

“…For these studies, we used the D . melanogaster Hog protein (244–471), which is amenable to in vitro expression [ 12 , 53 ]. To facilitate purification and limit off-target crosslinking by photocholesterol, we introduced relatively small (11 kDa) N- and C-terminal SUMO proteins, yielding a SUMO-Hh(244–471)-SUMO (“fHog”) fusion protein.…”

“…Importantly, formation of a covalent bond to the Hh protein requires the C3-OH of cholesterol to approach the C197-C198 thioester at an appropriate angle for orbital overlap. To guide cholesterol relocation, we used a modest force constant (0.48 kcal.mol -1 .Å -2 ) to place a distance constraint of 4 Å between C3-OH and D243 in the Hint fold active site (see Materials and Methods ) [ 12 , 53 , 54 ]. A movie of cholesterol’s migration to the active site during constrained dynamics analysis shows that conformational changes in the loop and HWY hinge at the back of the cholesterol pocket orient cholesterol for attack ( S2 Movie ).…”

“…To generate sufficient quantities of functional Hog protein for crosslinking/mass spectrometry analysis, we designed and expressed an active Hog construct in E. coli. For these studies, we used the D. melanogaster Hog protein (244-471), which is amenable to in vitro expression [12,53]. To facilitate purification and limit off-target crosslinking by photocholesterol, we introduced relatively small (11 kDa) N-and C-terminal SUMO proteins, yielding a SUMO-Hh(244-471)-SUMO ("fHog") fusion protein.…”

Addressable Cholesterol Analogs for Live Imaging of Cellular Membranes

“…Cholesterol is an essential component of biological membranes involved in a variety of cellular processes, which range from signal transduction and membrane trafficking to the regulation of bacterial and viral infections. Membrane cholesterol mediates its effects by cholesterol binding proteins, cholesteroylation of proteins and, in particular, by changing properties of the lipid bilayer, e.g., membrane stiffness, permeability, and domain formation (Lingwood et al, 2011;Schwarzer et al, 2014;Xie et al, 2016). Cholesterol-rich nano-and microdomains are of particular relevance as they have been implicated in the control of many of the processes mentioned above (Lingwood and Simons, 2010).…”

The Mechanism of Cholesterol Modification of Hedgehog Ligand

“…These caveats do leave room for improvements in the precursor model based on biochemical studies. One example is the Asp303 residue, which is suggested by NMR and mutagenesis experiments to play a direct role in both the N/S acyl shift and transesterification steps of hedgehog autoprocessing but does not seem to play any significant role in the RGATS simulations started from the present precursor model.…”