General Base Swap Preserves Activity and Expands Substrate Tolerance in Hedgehog Autoprocessing

Abstract

Hedgehog (Hh) autoprocessing converts Hh precursor protein to cholesterylated Hh ligand for downstream signaling. A conserved active-site aspartate residue, D46, plays a key catalytic role in Hh autoprocessing by serving as a general base to activate substrate cholesterol. Here we report that a charge-altering Asp-to-His mutant (D46H) expands native cholesterylation activity and retains active-site conformation. Native activity toward cholesterol was established for D46H in vitro using a continuous FRET-based autoprocessing assay and in cellulo with stable expression in human 293T cells. The catalytic efficiency of cholesterylation with D46H is similar to that with wild type (WT), with k max/K M = 2.1 × 103 and 3.7 × 103 M–1 s–1, respectively, and an identical pK a = 5.8 is obtained for both residues by NMR. To our knowledge this is the first example where a general base substitution of an Asp for His preserves both the structure and activity as a general base. Surprisingly, D46H exhibits increased catalytic efficiency toward non-native substrates, especially coprostanol (>200-fold) and epicoprostanol (>300-fold). Expanded substrate tolerance is likely due to stabilization by H46 of the negatively charged tetrahedral intermediate using electrostatic interactions, which are less constrained by geometry than H-bond stabilization by D46. In addition to providing fundamental insights into Hh autoprocessing, our findings have important implications for protein engineering and enzyme design.

Hedgehog proteins create a dynamic cholesterol interface

“…Intriguingly, while we placed no constraints on the distance between the G197 oxyanion and the acidic proton of D243, these two atoms engaged in a 1.7 Å hydrogen bond over the course of equilibration (S6 Fig). This finding agrees with recent biochemical studies showing that a neutral D243 side chain stabilizes the oxyanion in during adduction, providing key insight into the mechanism of catalysis [53,54].…”

“…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 ).…”

Hedgehog Autoprocessing: From Structural Mechanisms to Drug Discovery

“…The general base-swap D303H has been shown to preserve both structure and activity while also expanding substrate space in Hh autoprocessing ( Zhao et al, 2019 ). Using X-ray crystallography, D303H Hint domain ( Figure 6 , cyan ) was found to have an almost identical 3D structure compared to WT ( Figure 6 , red ) with an RMSD of 0.4 Å. NMR pH titration showed that H303 has the same p Ka as D303.…”

“…Patched activation relieves its inhibition on Smoothened (SMO), another membrane protein, which turns on glioma-associated oncogene transcription factor (GLI) family of transcription factors to activate Hh signaling. Hh autoprocessing is important because it is at the origin of canonical Hh signaling, where it precedes all downstream signaling events ( Porter et al, 1996a ; Hall et al, 1997 ; Jiang and Paulus, 2010 ; Xie et al, 2014 ; Xie et al, 2015 ; Xie et al, 2016 ; Zhang et al, 2019 ; Zhao et al, 2019 ; Smith et al, 2020 ), and is unique to Hh proteins. Although it lies at the very origin of Hh signaling, there are only a few structural/mechanistic studies ( Owen et al, 2015a ; Owen et al, 2015b ; Callahan and Wang, 2015 ; Bordeau et al, 2016 ; Ciulla et al, 2018 ; Ciulla et al, 2019 ; Zhang et al, 2019 ; Zhao et al, 2019 ; Smith et al, 2020 ), compared to the great number of studies of downstream components, such as PTCH ( Ingham et al, 1991 ; Chen and Struhl, 1996 ; Sidransky, 1996 ; Kallassy et al, 1997 ; Xie et al, 1997 ; Zedan et al, 2001 ; Shao et al, 2006 ; Lorberbaum et al, 2016 ; Tukachinsky et al, 2016 ; Zhang et al, 2018 ; Abd Elrhman and Ebian, 2019 ; Kinnebrew et al, 2021 ), SMO ( Xie et al, 2014 ; Owen et al, 2015a ; Owen et al, 2015b ; Callahan and Wang, 2015 ; Xie et al, 2015 ; Xie et al, 2016 ; Zhang et al, 2019 ; Zhao et al, 2019 ; Smith et al, 2020 ), and GLI ( Lauth et al, 2007 ; Kim et al, 2010 ; Maun et al, 2010 ; Beauchamp et al, 2011 ; Pan et al, 2012 ; Long et al, 2016 ; Xiao et al, 2017 ; Kowatsch et al, 2019 ; Liu et al, 2019 ; Quagli...…”

Intein Inhibitors as Novel Antimicrobials: Protein Splicing in Human Pathogens, Screening Methods, and Off-Target Considerations

“…The conserved Asp likely coordinates the binding of cholesterol to the SRR with the first step of autoprocessing, and activates the hydroxyl group of cholesterol to attack the thioester (Hall et al, 1997;Xie et al, 2016). Surprisingly, substitution of this Asp with His, a nominal charge reversal substitution (D46H in Drosophila HINT), can allow for a wider array of molecules, including coprostanol and epicoprostanol, to serve as the sterol substrate (Zhao et al, 2019). This is likely due to the more favorable, less spatially restrictive electrostatic stabilization of transition state provided by the H46 variant.…”