Chemical Bypass of General Base Catalysis in Hedgehog Protein Cholesterolysis Using a Hyper-Nucleophilic Substrate

Article abstract of DOI:10.1021/jacs.7b05161

Proteins in the hedgehog family undergo self-catalyzed endoproteolysis involving nucleophilic attack by a molecule of cholesterol. Recently, a conserved aspartate residue (D303, or D46) of hedgehog was identified as the general base that activates cholesterol during this unusual autoprocessing event; mutation of the catalyzing functional group (D303A) reduces activity by >10⁴-fold. Here we report near total rescue of this ostensibly dead general base mutant by a synthetic substrate, 3β-hydroperoxycholestane (3HPC) in which the sterol-OH group is replaced by the hyper nucleophilic-OOH group. Other hedgehog point mutants at D303, also unreactive with cholesterol, accepted 3HPC as a substrate with the rank order: WT > D303A ～D303N D303R, D303E. We attribute the revived activity with 3-HPC to the α-effect, where tandem electronegative atoms exhibit exceptionally high nucleophilicity despite relatively low basicity.

Full text of DOI:10.1021/jacs.7b05161

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Communication
Chemical bypass of general base catalysis in hedgehog
protein cholesterolysis using a hyper-nucleophilic substrate.
Daniel Ciulla, Michael Jorgensen, Jose-Luis Giner, and Brian P Callahan
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b05161 • Publication Date (Web): 20 Sep 2017
Downloaded from http://pubs.acs.org on September 20, 2017
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Chemical bypass of general base catalysis in hedgehog protein cholesterꢀ
olysis using a hyperꢀnucleophilic substrate.
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Daniel A. Ciulla1, Michael T. Jorgensen2, JoséꢀLuis Giner2, Brian P. Callahan1*.
Chemistry Department, Binghamton University (SUNY), Binghamton NY 13902
Department of Chemistry, SUNYꢀESF, Syracuse, NY 13210.
Supporting Information Placeholder
lently to a molecule of cholesterol (Scheme 1).8ꢀ10 The reaction
ABSTRACT: Proteins in the hedgehog family undergo selfꢀ
begins with an amide to thioester rearrangement at a conserved
GCF sequence (step 1); this is followed by transesterification to
cholesterol (step 2). Both steps can be brought about in vitro
independent of cofactors or accessory proteins by the Hh precurꢀ
sor’s catalytic Cꢀterminal region, HhC (Scheme 1, green) 9
catalyzed endoproteolysis involving nucleophilic attack by a molꢀ
ecule of cholesterol. Recently, a conserved aspartate residue
(D303, or D46) of hedgehog was identified as the general base
that activates cholesterol during this unusual autoprocessing
event; mutation of the catalyzing functional group (D303A) reꢀ
duces activity by >104 fold. Here we report near total rescue of
this ostensibly dead general base mutant by a synthetic substrate,
3ßꢀhydroperoxycholestane (3HPC) in which the sterol –OH group
is replaced by the hyper nucleophilic –OOH group. Other hedgeꢀ
hog point mutants at D303, also unreactive with cholesterol, acꢀ
cepted 3HPC as
a
substrate with the rank order:
WT>D303A≈D303N>>D303R, D303E. We attribute the revived
activity with 3ꢀHPC to the alphaꢀeffect, where tandem electronegꢀ
ative atoms exhibit exceptionally high nucleophilicity despite
relatively low basicity.
Scheme 1. Hedgehog precursor autoprocessing.
Recently, Xie et al proposed that an aspartic acid residue
(D303, or D46) in the HhC from Drosophila melanogaster funcꢀ
tions as a general base to facilitate attack of cholesterol at the
thioester intermediate.11 Multiple lines of evidence support this
idea: (1) alignment of distantly related HhC sequences reveal
strong conservation of the D303 residue; (2) crystal structure of
an HhC fragment places D303 in proximity to the thioester formꢀ
ing residue, C258; (3) NMR measurements indicate that the pKa
value of the D303 side chain is elevated to 5.6, which would aid
in deprotonating the sterol 3ꢀOH group; finally, (4) an alanine
General base variants of enzymes can present structureꢀfunction
enigmas: while remarkably similar in structure to the native enꢀ
zyme, point mutants often show turnover numbers (kcat) diminꢀ
ished by 104 to 108ꢀfold. For example, general base mutation of
HIV protease (D25N) suppresses catalytic activity to such a deꢀ
gree that the protein can be coꢀcrystallized in “tight” complex
with intact substrate.1 A 108 fold reduction in kcat was reported
with the E104A general base mutant of cytidine deaminase; notꢀ
withstanding, the protein retained nM affinity for a mechanismꢀ
based inhibitor.2 General base mutants of aspartate aminotransꢀ
ferase (K258A) and ketosteroid isomerase (D38G) are among
many other examples that display similar characteristics: structurꢀ
ally sound and catalytically inert.3,4 Toney and Kirsch established
that mutants like these can be “rescued” to varying degrees by
addition in trans of the missing functional group.4 Alternatively,
activity may be restored by appending the catalyzing group to
point mutant at D303 renders HhC unreactive toward cholesterol.
9
In Figure 1 (AꢀC) and Table 1 (left column) we summarize the
diminished cholesterolysis activity of HhC D303A. For quantitaꢀ
tive analysis, we used an optical assay that monitors reaction kiꢀ
netics continuously by loss of Förster resonance energy transfer
(FRET) from an engineered Hh precursor (Figure 1A).12 The
FRETꢀactive precursor, CꢀHꢀY, is a translational fusion of Cyan
fluorescent protein, HhC, and Yellow fluorescent protein. FRET
from CꢀHꢀY is lost as autoprocessing liberates Cꢀsterol from HꢀY.
Results are verified with a gelꢀbased assay, where precursor and
products separate by molecular weight allowing reaction progress
to be visualized directly (Figure 1B). Plots of initial velocity,
determined using the FRET assay, as a function of cholesterol
5
substrate itself . Here we present a nonꢀclassical approach to
chemical rescue, one that sidesteps the general base imperative
altogether and instead restores activity through the use of a hyperꢀ
nucleophilic substrate.
Hedgehog proteins, extracellular signaling ligands of imꢀ
portance to embryogenesis and cancer,6 undergo a specialized
endoproteolytic lipidation called cholesterolysis as part of their
complex biosynthesis7. In cholesterolysis, a precursor form of
hedgehog (Hh) separates into Nꢀ and Cꢀterminal polypeptides, and
the departing Nꢀterminal fragment (HhN) becomes linked covaꢀ
concentration
were
fit
by
1
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FIGURE 1. Activation of general base mutant D3030A with an αꢀeffect substrate. (A) Continuous assay to measure kinetics of Hh activity
by using a FRETꢀactive precursor (CꢀHꢀY). (B) Kinetics of precursor processing with wildꢀtype and D303A constructs (1x10ꢀ7 M) +/ꢀ
cholesterol (5x10ꢀ5 M), monitored by FRET (left) and by SDSꢀPAGE (right). (C) Initial velocity of wildꢀtype and D303A HhC constructs
plotted as a function of increasing cholesterol concentration. Trend line for wildꢀtype was drawn using the fitted KM value of 1 µM. (D)
Bond line formulas and structural overlay of cholesterol and 3ꢀβ hydroperoxycholestane (3HPC). (E) Kinetics of precursor processing
with wildꢀtype and D303A HhC constructs +/ꢀ 3HPC (50 µM), monitored by FRET (left) and by SDSꢀPAGE (right). (F) Initial velocity of
wildꢀtype and D303A constructs plotted as a function of increasing 3HPC. Trend lines were drawn using the fitted KM values of 1.5 x10ꢀ6
M for wildꢀtype, and 3.5 x10ꢀ6 M for D303A.
nonꢀlinear regression to a MichealisꢀMenten equation to obtain
Km values (Figure 1C). First order rate constants for cholesterolyꢀ
sis (kmax) were calculated at saturating substrate concentration by
fitting the entire reaction progress curve to an exponential decay.
cholesterol are similar except for a saturated stanol ring system
and the –OOH substituent in place of the sterol –OH (Figure 1 D).
The analog 3HPC was obtained in 3 steps starting from cholesꢀ
tanone and separated from its Cꢀ3 epimer by preparative TLC and
HPLC. Stereochemistry of the hydroperoxy group was confirmed
by 1H NMR using the chemical shift and splitting pattern of the ꢀ
CHꢀOOH (19 and Supporting Information).
In contrast to HhC wildꢀtype (HhC WT), the D303A mutant
exhibits an autoprocessing rate with cholesterol that is reduced by
a factor of >300 fold (Table 1) with cholesterol added at concenꢀ
trations 50ꢀfold above the KM value of HhC WT. A limiting
value of kmax/KM for the D303A is thereby reduced by ≥ 104 for
cholesterol, equivalent to 5.4 kcal/mol in free energy. The very
large deleterious effect of removing the D303 carboxyl group is
within range of general base mutations mentioned earlier. Despite
inactivity toward cholesterol, the D303A variant continues to
generate internal thioester in amounts comparable to wild type
HhC, and similar behavior was observed with D303E, R, & N
variants.11 Thus, general base mutants of HhC can bring about
step 1 (Scheme 1) but autoprocessing advances no further with
substrate cholesterol.
We observed robust autoprocessing of HhC WT and D303A
with 3HPC in a set of in vitro experiments, summarized in Figure
1 (EꢀF) and Table 1 (right column). Assays using 3HPC were
carried out as before using the optical system for determining
kinetic parameters and gelꢀbased assay for orthogonal readout.
Results of SDSꢀPAGE show that the product polypeptide derived
from reaction with 3HPC migrates with an Rf value similar to
product from the cholesterol reaction; both appear larger than the
Rf of the hydrolysis product (Figure 1E, gel, compare C and Cꢀ
3HPC), which is consistent with steroylation. 10,12 20 We note that
selected carboxylic acid hydrolases accept hydrogen peroxide as
an alternative substrate in a soꢀcalled perhydrolysis side reaction
αꢀEffect nucleophiles, where the attacking atom is adjacent (i.e.
21
.
Comparison of the specificity constants (kmax/KM) derived
alpha) to a second electronegative atom, are known for reacting at
13ꢀ15
from the FRET assay indicate a modest 3ꢀfold preference for choꢀ
lesterol over 3HPC by HhC WT, diminished only by the kmax
value (Table 1). Substrate activity of 3HPC remained strong usꢀ
ing the general base D303A mutant (Figure 1E, red traces): the
kmax value is within 10% of wildꢀtype, and the apparent KM value
is elevated by only 2ꢀfold. In comparison with cholesterol, the t1/2
value for autoprocessing by D303A is accelerated from >19 h to
10 minutes, equivalent to a rate enhancement of >1000x.
exceptionally fast rates.
Large positive deviations from
linear free energy relationships are observed with αꢀeffect nucleoꢀ
16,17
philes like hydrazine, hydroxylamine and hydroperoxides.
For example, methyl peroxide anion (CH3OOꢀ) cleaves an aryl
ester at a rate 4x faster than methoxide (CH3Oꢀ) despite having a
pKa value (11.5) 4 units lower than methanol (15.6).18 Said anꢀ
other way, methyl peroxide reacts as if it were ~10,000x more
basic than its measured pKa value.
With the potential for augmented acidity and nucleophilicity,
we prepared 3ßꢀhydroperoxy cholestane (3HPC) as a potential
substrate for the Hh D303A mutant. Structures of 3HPC and
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Corresponding Author
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Brian Callahan, callahan@binghamton.edu
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Notes
The authors declare no competing financial interests.
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ACKNOWLEDGMENT
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We acknowledge generous support from the National Cancer
Institute (Grant R01 CA206592) and Department of Defense
(Grant W81XWHꢀ14ꢀ1ꢀ0155). We also acknowledge the upgrade
of the 600 MHz NMR spectrometer at SUNYꢀESF under NSF
grant CHEꢀ1048516 and NIH grant S10 OD012254.
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TABLE 1. Substrate activity of cholesterol and 3HPC with wildꢀ
type and D303 mutant HhC.
REFERENCES
(1)
(2)
(3)
PrabuꢀJeyabalan, M.; Nalivaika, E.; Schiffer, C. A. J
Mol Biol 2000, 301, 1207.
Carlow, D. C.; Smith, A. A.; Yang, C. C.; Short, S. A.;
Wolfenden, R. Biochemistry 1995, 34, 4220.
Lamba, V.; Yabukarski, F.; Pinney, M.; Herschlag, D. J
Am Chem Soc 2016, 138, 9902.
To explore the specificity using 3HPC we surveyed three other
seemingly inert D303 point mutants: charged reversed, D303R;
isosteric, charge neutralized, D303N; and charge conserved,
11
D303E.
In the absence of steric or electrostatic clashes, it
seemed reasonable to expect that 3HPC would rescue in a manner
similar to the D303A HhC mutant. In fact, comparable activity
was observed for D303N, with an apparent kmax value within 2ꢀ
fold of D303A. Rate constants with D303R and D303E were
slowed by ~ 10ꢀfold compared with D303A. We speculate based
on these results that at the transition state for transesterification
(Scheme 1, step 2) the attacking atom of the sterol and the side
chain of residue 303 are narrowly separated, consistent with genꢀ
eral base catalysis. Larger sideꢀchains introduced by D303R and E
substitution could sterically inhibit approach of the attacking –
OOH group of 3HPC, leading to slower reaction rates. Full exꢀ
planation of the variation awaits complete structural analysis of
HhC.
(4)
(5)
(6)
Toney, M. D.; Kirsch, J. F. Science 1989, 243, 1485.
Carter, P.; Wells, J. A. Science 1987, 237, 394.
Briscoe, J.; Therond, P. P. Nat Rev Mol Cell Biol 2013,
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(10)
(11)
(12)
In summary, a synthetic Hh substrate bearing an αꢀeffect funcꢀ
tional group, noteworthy for reacting at rates disproportionately
faster than expected from its pKa value, supports near wildꢀtype
activity of a HhC general base mutant (D303A). Thus, a functionꢀ
ing general base appears critical for HhC to activate the –OH
group of cholesterol, but superfluous with the αꢀeffect –OOH
group of 3HPC. These results are consistent with a nonꢀclassical
mode of chemical rescue, distinct from convention where the
missing catalytic group is returned to the mutant enzyme.22 The
present study does evoke rescueꢀtype experiments on glucosidases
and ribozymes where general acid mutants were revived by subꢀ
strates bearing hyperꢀlabile leaving groups.23,24 To the extent that
any rescue approach succeeds, the restored activity seems to point
toward a key structural feature of enzyme and enzymeꢀlike active
sites. While active sites almost certainly provide a template for
the transition state 25, that template is to some degree elastic even
improvisatory.
(13)
(14)
Edwards, J. O. P., R. G. J Am Chem Soc 1962, 84, 16.
Jencks, W. P.; Carriuolo, J. J Am Chem Soc 1960, 82,
1778.
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Herschlag, D.; Jencks, W. P. J Am Chem Soc 1990, 112,
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Kalia, J.; Raines, R. T. Chembiochem 2006, 7, 1375.
Jencks, W. P.; Gilchrist, M. J Am Chem Soc 1968, 90,
2622.
(19)
(20)
Caglioti, L.; Gasparrini, F.; Misiti, D.; Palmieri, G.
Tetrahedron 1978, 34, 135.
An anonymous reviewer suggested two alternative
mechanisms to explain the observed cleavage of CꢀHꢀY
with added 3HPC: first, 3HPC could react transiently
with the internal thioester followed by rapid hydrolysis;
and second, the –OOH group of 3HPC could activate an
adventitious water molecule for thioester cleavage. A
third possibility involves oxidative cleavage of the
internal thioester. We could not identify by mass
spectrometry covalent peroxy esterified protein (Cꢀ
3HPC), therefore these pathways remain viable
alternatives.
ASSOCIATED CONTENT
Supporting Information
Detailed methods describing protein purification, activity assays,
and chemical synthesis, along with results from mutant studies.
The Supporting Information is available free of charge on the
ACS Publications website.
AUTHOR INFORMATION
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(21)
Kirk, O.; Conrad, L.S. Angew Chem Int Ed Engl 1999,
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38, 977.
(22)
(23)
(24)
Peracchi, A. Current Chemical Biology 2008, 2, 32.
Das, S. R.; Piccirilli, J. A. Nat Chem Biol 2005, 1, 45.
Rakic, B.; Withers, S. G. Australian Journal of
Chemistry 2009, 62, 510.
(25)
Wolfenden, R. Annu Rev Biophys Bioeng 1976, 5, 271.
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SCHEME 1
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HhC
(24 kDa)
HhN
(20 kDa)
Hh Precursor
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O
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HS
O
HS
S
C258
N
N
N
HH
HH
H
sterol
2
D303
-
-
-
H
O
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FIGURE 1
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A.
B.
C.
C-H-Y
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400 nm
D303A
-WT+
- +
cholesterol
chol
X
+
WT (-)
C-H-Y
H-Y
2.2
1.2
0.2
D303A (-)
D303A (+)
540 nm
WT
D303A
O
WT (+)
X
C
+
C-chol
0
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Coomassie blue
C-sterol
20 40 60 80 100
Cholesterol (µM)
H-Y
time (m)
3HPC
WT
- +
D303A
- +
D.
E.
F.
HO
3HPC
WT (-)
2.2
1.2
0.2
C-H-Y
H-Y
D303A (-)
HOO
WT
D303A
WT (+)
C
D303A (+)
C-3HPC
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40
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Coomassie blue
20 40 60 80 100
3HPC (µM)
time (m)
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HO
HOO
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10-1
10-2
10-3
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