Hedgehog
proteins, a family of vital cell signaling factors, are
expressed in precursor form, which requires specialized autoprocessing,
called cholesterolysis, for full biological activity. Cholesterolysis
occurs in cis through the action of the precursor’s
C-terminal enzymatic domain, HhC. In this work, we describe HhC activator
compounds (HACs), a novel class of noncovalent modulators that induce
autoprocessing infidelity, diminishing native cholesterolysis in favor
of precursor autoproteolysis, an otherwise minor and apparently nonphysiological
side reaction. HAC-induced autoproteolysis generates hedgehog protein
that is cholesterol free and hence signaling deficient. The most effective
HAC has an AC50 of 9 μM, accelerates HhC autoproteolytic
activity by 225-fold, and functions in the presence and absence of
cholesterol, the native substrate. HACs join a rare class of “antagonists”
that suppress native enzymatic activity by subverting mechanistic
fidelity.
The enzymatic agent of hedgehog protein cholesterolysis accommodates substrate sterols with undersized, oversized and distorted ring systems relative to cholesterol.
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.
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