Design of a switchable eliminase

Article abstract of DOI:10.1073/pnas.1018191108

The active sites of enzymes are lined with side chains whose dynamic, geometric, and chemical properties have been finely tuned relative to the corresponding residues in water. For example, the carboxylates of glutamate and aspartate are weakly basic in w

Full text of DOI:10.1073/pnas.1018191108

Design of a switchable eliminase
Ivan V. Korendovycha, Daniel W. Kulpa, Yibing Wua, Hong Chengb, Heinrich Rodera,b, and William F. DeGradoa,b,c,1
aDepartment of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104; bFox Chase Cancer Center, Philadelphia, PA 19111; and
cDepartment of Chemistry, University of Pennsylvania, Philadelphia, PA 19104
Edited by David Baker, University of Washington, Seattle, WA, and approved March 10, 2011 (received for review December 23, 2010)
The active sites of enzymes are lined with side chains whose
O
OH
B
dynamic, geometric, and chemical properties have been finely
tuned relative to the corresponding residues in water. For example,
the carboxylates of glutamate and aspartate are weakly basic in
water but become strongly basic when dehydrated in enzymatic
sites. The dehydration of the carboxylate, although intrinsically
thermodynamically unfavorable, is achieved by harnessing the free
energy of folding and substrate binding to reach the required
basicity. Allosterically regulated enzymes additionally rely on the
free energy of ligand binding to stabilize the protein in a catalyti-
cally competent state. We demonstrate the interplay of protein
folding energetics and functional group tuning to convert calmo-
dulin (CaM), a regulatory binding protein, into AlleyCat, an allos-
terically controlled eliminase. Upon binding Ca(II), native CaM
opens a hydrophobic pocket on each of its domains. We computa-
tionally identified a mutant that (i) accommodates carboxylate as
a general base within these pockets, (ii) interacts productively in
the Michaelis complex with the substrate, and (iii) stabilizes the
transition state for the reaction. Remarkably, a single mutation
of an apolar residue at the bottom of an otherwise hydrophobic
cavity confers catalytic activity on calmodulin. AlleyCat showed
the expected pH-rate profile, and it was inactivated by mutation
of its active site Glu to Gln. A variety of control mutants demon-
strated the specificity of the design. The activity of this minimal
75-residue allosterically regulated catalyst is similar to that ob-
tained using more elaborate computational approaches to rede-
sign complex enzymes to catalyze the Kemp elimination reaction.
O
N
O
O
O
N
O
N
O
N
O
N
N
H
H
B
B
protein
protein
protein
Scheme 1. Kemp elimination reaction.
generation of such a site occurs at a thermodynamic cost,
so the starting protein must have sufficient initial stability to
withstand the substitution or, in switchable enzymes, receive
additional stabilization by association with an allosteric ligand.
Several features were considered in choosing the particular
starting scaffold protein for this application: (i) a lack of intrinsic
catalytic activity, (ii) spatial separation between the substrate-
binding and allosteric ligand-binding sites, (iii) the presence of a
cavity of sufficient depth to accommodate the substrate, (iv) high
thermodynamic stability, (v) small size to facilitate NMR as well
as crystallographic studies and to allow facile preparation by
either chemical synthesis or bacterial expression. These criteria
were fulfilled by calmodulin (CaM), a prototypical member
of the EF-hand class of regulatory proteins, and we chose its
C-terminal domain (cCaM, M76-K148) for its stability and high
affinity for Ca2þ (17, 18). Importantly, CaM is known to have no
catalytic activity in the Kemp elimination reaction (19).
Results
protein design ∣ catalysis ∣ pKa
Computational Design. The C-terminal domain of CaM has a cavity
that binds aromatic side chains of peptides, suggesting that it
could accommodate the similarly sized benzisoxazole substrate.
We computationally scanned the entire cavity using a three-step
procedure to identify a site where a Glu or Asp would be accom-
modated within the pocket projecting the carboxylate toward
the target C–H bond of the substrate (Fig. 1). Although the re-
placement of a hydrophobic side chain with Glu or Asp should
lead to some destabilization, it is important that they not entirely
disrupt the structure of the protein. Thus, in the first step, indi-
vidual Glu and Asp substitutions were first examined in all low-
energy rotamers, allowing variation in the conformation of the
remaining wild-type residues (Fig. S1). The substrate must also
be bound productively in the Michaelis complex. It was therefore
docked into the single-site mutants remaining after the first step
of screening by using an automated procedure (see Materials and
Methods) to determine whether the benzisoxazole C–H hydrogen
would be appropriately positioned in the Michaelis complex.
he design of enzyme-like catalytic proteins is a challenging
Tendeavour that has been approached using combinatorial
(1) and computational strategies (2), as well as by following
simple chemical principles (3, 4). The Kemp elimination (5)
(Scheme 1) is an extensively studied benchmark for catalyst
design (6–8) as a model for enzymatic C–H bond abstraction.
Previously, computational methods were employed to alter the
active sites of natural enzymes to catalyze this reaction (7). Mu-
tation of a dozen or more side chains resulted in catalysts that
showed rate enhancements of 104 to 105 relative to a given
reference rate. Here, by combining a few physical principles with
modern computational tools, we show that a single mutation
can confer similar catalytic efficiency into a much simpler and
allosterically switchable scaffold that previously had no catalytic
activity. This approach circumvents often contentious questions
concerning residual activities of the enzymatic framework or the
appropriate reference background rate for expression of a “rate
enhancement” while also suggesting a practical approach to the
design of catalytically amplified sensors.
Here we applied a minimalist approach to protein design
(9–11), which emphasizes the use of the simple scaffolds and
the minimal number of mutations required to achieve a given
activity. Dehydrated carboxylates are excellent catalysts of the
Kemp elimination (12) due to their basicity toward carbon acids.
Thus, an effective catalyst might be engineered by appropriately
placing a single carboxylate at the bottom of a hydrophobic
pocket, which would destabilize the anionic form and increase
its basicity (13–16) and ability to deprotonate C–H bonds. The
Author contributions: I.V.K. and W.F.D. designed research; I.V.K., D.W.K., Y.W., and H.C.
performed research; I.V.K., H.R., and W.F.D. analyzed data; and I.V.K. and W.F.D. wrote
the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The NMR structure of AlleyCat has been deposited in the Protein
Data Bank, www.pdb.org (PDB ID code 2kz2). Chemical shifts were deposited in the
BioMagResBank http://www.bmrb.wisc.edu/ (accession code 16994).
1To whom correspondence should be addressed: E-mail: wdegrado@mail.med.upenn.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1018191108/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1018191108
PNAS ∣ April 26, 2011 ∣ vol. 108 ∣ no. 17 ∣ 6823–6827

Fig. 1. Summary of the computational procedure. A) Identification of potential points to introduce a catalytic residue into cCaM (coordinates from X-ray
structure 1EXR). (B) Examination of the Glu residues in the identified positions/docking of the substate. (C) Transition state docking based on a search of a
superrotamer library for the transition state.
Finally, a “superrotamer” library (20–22), in which the Glu car-
boxylate was virtually fused to the substrate in the transition state
geometry (23), was evaluated at each position (Fig. S2 and
Table S1). Only one position, F92, could accommodate an Asp
or Glu in the calcium bound state as well as productively interact
with the substrate in the transition state. In particular, Glu gave
the lower computed energy, it better positioned its sidechain in a
low-energy rotameric configuration to interact with incoming
substrate, and it is intrinsically more basic than Asp. Thus, cCaM
F92E, which we named AlleyCat (for ALLostEricallY Controlled
cATalyst), was strongly predicted to be optimal of the possible
mutations.
To test the specificity of the design, we also prepared a total
of 13 mutants in which each of the hydrophobic residues in
the binding pocket was individually mutated to Asp and Glu
(Fig. S3). The active site Glu92 residue was also mutated to Gln,
which eliminates the carboxylate, or His, which substitutes the
carboxylate for an imidazole base.
F92D was 16-fold less active, F92H showed very low activity
(at least 20-fold lower than that of F92E); the remaining 11 con-
trol mutants had activities that were not above the background
and at least 25-fold lower than AlleyCat. AlleyCat is active for
over 40 turnovers and shows no evidence of product inhibition.
The initial rate is linearly dependent on substrate concentration
at low substrate concentrations and shows the onset of apparent
saturation near 1 mM (Fig. S4), as has been observed for other
designed proteins (7). However, because the activity coefficient
(γ) of this poorly soluble substrate is likely to deviate from unity
at high concentrations, we restricted our analysis of the data to
the linear region to obtain kcat∕KM rather than the individual
parameters.
The pH-rate profile and a mutational analysis support the
expected mechanism, in which Glu92 with an elevated pKa acts
as a general base. The pH-rate profile is typical of a single ioniz-
able group; the value of kcat∕Km for the deprotonated form is
5.8 ꢀ 0.3 M−1 s−1, with an apparent pKa ¼ 6.9 ꢀ 0.1 (Fig. 2C).
The protonated form showed no activity over background. Also,
the wild-type protein and the mutant F92Q were at least 50-fold
less active than AlleyCat, providing strong evidence that the
Characterization of AlleyCat’s Activity. AlleyCat exhibited Kemp
elimination activity in the presence of 10 mM Ca2þ, while
A
B
C
D
Fig. 2. (A) Comparison of the benzisoxazole elimination activity of AlleyCat (circles) with cCaM-F92Q (diamonds), ½Proteinꢁ ¼ 16 μM, ½Substrateꢁ ¼ 0.5 mM. (B)
Dependence of the activity of AlleyCat on [Ca2þ] at pH 6.9, solid line shows the simulated two-site binding isotherm with an average Kd of 10−5 M. (C) pH profile
of activity of AlleyCat. (D) Chemical (guanidinium hydrochloride, GdmCl) denaturation profiles of AlleyCat (circles) and cCaM (triangles).
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Korendovych et al.

measured pKa was associated with the Glu92 (Fig. 2A). Finally, to
assure that the activity was not associated with a trace contami-
nant, we examined a variety of different constructs containing
F92E in either the full-length protein or the isolated C-terminal
domain, with or without a His-tag and purified using different
procedures. Under the same buffer and substrate conditions
(see Materials and Methods) the activity of these constructs was
constant within 20%, indicating the activity was indeed associated
with the C-terminal domain of CaM.
Discussion
These results illustrate how our emerging understanding of the
delicate interplay between protein folding energetics, allosteric
ligand binding, and functional group tuning (25, 26) can be com-
bined with computational methods to design an allosterically
regulated switch-like protein catalyst. By linking binding at one
site to catalysis at a second site it has been possible to create a
highly amplified sensor. Thus by using different natural or engi-
neered proteins that bind other ligands it might be possible to
design a variety of catalytically amplified sensors.
Allosteric Regulation and Ca2þ-Dependent Conformational Change.
AlleyCat is allosterically regulated by Ca2þ. Its catalytic activity
of the protein is strictly Ca2þ-dependent, showing less than 25-
fold lower activity in the absence of Ca2þ. The activity versus
[Ca2þ] profile conforms well to a tight two-site binding isotherm
with a Kd < 10 μM (Fig. 2B). The CD spectrum of CaM shows an
increase in the magnitude of the ellipticity at 222 nm (θ222) upon
binding of Ca2þ. The binding of Ca2þ to AlleyCat triggered an
even larger increase in the magnitude of θ222; the value of θ222
The primary goal of this work was to link a ligand-driven con-
formational change to the creation of a novel catalytic activity,
which could easily be measured in vitro. The catalytic activity
of AlleyCat was sufficient for this purpose, but how does it com-
pare to earlier catalysts of Kemp elimination? A frequently used
comparison is the ratio of the value of kcat∕KM for AlleyCat ver-
sus the second order rate constant for acetate in water, which
amounts to 2.8 × 105 in this case. The value of kcat∕KM for Alley-
Cat (5.8 ꢀ 0.3 M−1 s−1) is within the same range as observed for
catalytic antibodies (8, 28, 29) (kcat∕KM ¼ 1.4–5;500 M−1 s−1).
This is a significant accomplishment given that AlleyCat is switch-
able, its molecular weight is less than one tenth that of IgG anti-
bodies, and AlleyCat was designed by a very simple, reproducible
automated procedure.
It is of more interest to compare the present strategy, which
incorporated elements of the minimalist design, fundamental
physical principles, and fully automated computational design to
previous work of Houk, Tawfik, Baker, and coworkers (7). These
researchers employed computational algorithms to redesign
enzymatic scaffolds, which are arguably more favorable platforms
for catalysis than the binding protein used here. Of 59 designed
proteins, which were created by substitution of 10–15 aminoacid
residues in each design, eight were active for the Kemp elimina-
tion, showing kcat∕KM ranging from 5.9 M−1 s−1 (for protein
KE71) to 163 M−1 s−1 (for protein KE59). These investigators
emphasized the importance of structural studies to guide future
computational studies, mechanistic studies, and comparison with
other designs. Of the 59 proteins, the X-ray structure of one,
KE07, was determined, providing an interesting comparison with
AlleyCat, which has been also structurally characterized. Alley-
Cat has an activity similar to that of KE07 on a molar basis and
ca. 2.5-fold greater activity on a weight basis. However, mutating
the catalytic acid and base of KE07 led to only modest twofold to
threefold increases or decreases in activity, suggesting that a step
other than proton transfer is rate-determining or, possibly, that
the complexity of the design led to a protein that employed an
unanticipated mechanism. Although some of the other designs
such as KE59 showed the expected response to mutations, their
structures were not experimentally determined. By contrast, the
simplicity of the minimalist approach not only allowed the design
of a switchable catalyst, but the small scaffold and minimal num-
ber of mutations facilitated mechanistic determination. AlleyCat
was similar for cCaM and AlleyCat in the presence of Ca2þ
,
but in the absence of Ca2þ AlleyCat appeared significantly less
structured. This finding is consistent with the expectation that
the F92E mutation destabilizes the protein, resulting in a partially
folded apo state. Although the calcium bound state was essen-
tially fully folded we expected that its thermodynamic stability
might be lower than that of the wild type, because studies of
natural and designed proteins show that enzymes often sacrifice
thermodynamic stability to place their functional residues in
nonoptimal environments (15, 24); for example, the degree of
perturbation of a side chain’s pKa can be related to the corre-
sponding decrease in the overall free energy of folding (25, 26).
The high apparent pKa of Glu92 is consistent with this hypothesis
and predicts that AlleyCat should be thermodynamically less
stable than the corresponding wild-type domain. Indeed, the
wild-type protein is 3.1 ꢀ 0.6 kcal∕mol more stable than AlleyCat
in the presence of saturating concentrations of Ca2þ at pH 6.9
(Fig. 2D).
Structural Characterization. The structure of AlleyCat was deter-
mined by heteronuclear NMR, using 1,205 distance and 140
dihedral restraints (average 16.2 restraints/residue, Fig. 3). The
computed ensemble of structures (backbone rmsd 1.05 Å) is as
well defined as previous NMR structures of cCaM (computed
from a similar number of restraints). The centroid is in good
agreement with the starting model and the NMR structure of
wild-type cCaM (27) (rmsd 1.35 Å). Thus, the mutation has
not caused large rearrangements in the backbone. The Glu92 side
chain is located at the bottom of the hydrophobic pocket sur-
rounded by apolar residues, where it is well positioned to serve
as a general base. Also, the Glu92 carboxylate is well separated
from the closest calcium atom, confirming the spatial segregation
and distinct roles of the allosteric versus the catalytic sites.
Fig. 3. NMR structure of AlleyCat. (A) Representation of protein’s surface showing E92 pointing into the hydrophobic binding pocket; carbons and the
backbone atoms are shown in green. (B) The 20 conformers with the lowest energy representing the solution structure shown after superposition of the
backbone heavy atoms N, Cα, and C′ atoms of the secondary structures. (C) “Sausage” representation of backbone and best-defined side chains: A spline
function was drawn through the mean positions of Cα atoms with the thickness proportional to the mean global displacement of Cα atoms in the 20 conformers
after superposition as in B. Calcium atoms are shown in yellow.
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extinction coefficient (15;800 cm−1 M−1) is taken from ref. 5. Kinetic para-
meters were obtained by fitting the data to the Michaelis–Menten equation
fv0 ¼ kcat½Eꢁ0½Sꢁ0∕ðKM þ ½S0ꢁÞg. kcat∕KM values were obtained by fitting the
linear portion of the plot to v0 ¼ ðkcat∕KMÞ½Eꢁ0½Sꢁ0. The following buffers
(at 25 mM) were used for the pH profile studies: citrate (pH 4.0–5.5), MES
(pH 5.5–6.5), HEPES (pH 6.5–8.0), bicine (8.0–9.0). kcat∕KM values were
uses E92 as the catalytic base as inferred from the rate/pH profile
and the F92Q mutation.
Our computational design strategy differs significantly from
previous studies (7), which focused exclusively on optimizing
interactions to satisfy a potential function developed to predict
thermodynamic stability of a protein. As a result the carboxylate
would be stabilized by hydrogen-bonded interactions to minimize
the overall energy. However, catalysis often requires destabilizing
specific groups to enhance their chemical reactivity. In this case,
strong desolvation of a carboxylate in an environment replete of
H-bond donors enhances its basicity, as was also inferred based
on activity-enhancing substitutions observed during the directed
evolution of KE07 (7, 30). Secondly, it is important to consider
the ground state Michaelis complex to assume that the preferred
bound orientation is competent for catalysis. Thus, it is likely that
simple considerations such as these could enhance future design
work, allowing more sophisticated computations to reach their
full potential.
It is likely that the binding site of AlleyCat could be improved
to optimize geometric complementarity with the substrate, which
might lead to an enhanced KM, better dehydration of the
substrate, and increased basicity of the active site Glu. Random
mutagenesis might also be used to improve activity without any
preconceived notion of the requirements for activity. For exam-
ple, directed evolution of KE07 led to a 200-fold increase in
activity (30). Thus, it will be particularly interesting to determine
the extent to which catalytic efficiency of AlleyCat can be
increased, given that it is smaller than almost all enzymes and
lacked catalytic activity in the starting protein.
obtained from fitting to kcat∕KM ¼ ðkcat∕KMÞprotonated þ ðkcat∕KMÞdeprotonated
×
10−pKa∕ð10−pH þ 10−pKaÞ, where pKa is the apparent pKa value of the active
residue.
Chemical Denaturation Fits. The chemical denaturation data were fit to (32):
MRE ¼ fðMREf þ yf ½DꢁÞ
þ ðMREu þ yu½DꢁÞ expððΔG-m½DꢁÞ∕RTÞg∕f1
þ expððΔG-m½DꢁÞ∕RTÞg
Where MRE is observed mean residue ellipticity; MREf and MREu are mean
residue ellipticities in the folded and unfolded states, respectively; [D] con-
centration of denaturant; ΔG is free energy of unfolding, yf and yu are slopes
for the folded and unfolded states, respectively. Experimental conditions:
4 mM HEPES (pH 6.9), 30 mM NaCl, 2 mM CaCl2. The fit yields the following
thermodynamic parameters for unfolding: ΔG ¼ 15 ꢀ 2 kJ∕mol and m ¼
7.0 ꢀ 0.8 kJ∕mol ꢂ M for AlleyCat; ΔG ¼ 23 ꢀ 1 kJ∕mol and m ¼ 5.3ꢀ
0.3 kJ∕mol ꢂ M for cCaM. The ΔΔG value determined from these parameters
is 3.1 ꢀ 0.6 kcal∕mol (the value was determined at the midpoint between the
respective Cm to avoid a long extrapolation to ½GdmClꢁ ¼ 0).
Calmodulin Starting Structure Computational Modeling. The sequence
SLMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDEEVDE MIREA-
DIDGDGQVNYEEFVQMMTAK was modeled onto the structure of C-terminal
domain of CaM (PDB ID code 1QS7) by maintaining the backbone and side
chains of the common amino acids and building the amino acids that varied
by finding the lowest energy (CHARMM force field) (33) conformation.
Materials and Methods
5-nitrobenzisoxazole has been prepared according to the literature
procedure (19).
Calmodulin Mutagenesis and Expression. The C-terminal portion of chicken
calmodulin gene (31) (M76-K148) was cloned into pEXP5-NT/TOPO vector
(Invitrogen) giving the gene corresponding to: MSGSHHHHHHGSSGEN-
Docking. AutoDock version 4.0 (34) was used to dock 5-nitrobenzisoxazole
into CaM (maximum rotational/translational step sizes 50°, 2 Å; population
size 150; 50,000 generations). The docking parameters were systematically
varied in independent runs all achieving approximately the same docked
pose.
LYFQSLMKDTDSEEEIREAFRVFDKDGNGYISAA
ELRHVMTNLGEKLTDEEVDE-
MIREADIDGDGQVNYEEFVQMMTAK. Mutagenesis was done with PfuTurbo
DNA polymerase (Stratagene) using standard protocols. The proteins were
expressed in Escherichia coli BL21(DE3) pLysS cells (Novagen) at 37° (cells were
harvested after 1.5 h after induction) and purified over a Ni-NTA column
(Qiagen).
Modelling the Structure of Mutants. First, 100 side chain conformations (rota-
mers) for the asparate/glutamate position and 50 rotamers for any residue
within 8 Å of the Asp/Glu were built using the Molecular Software Libraries
(MSL, manuscript in preparation). Then, the pairwise energy table (CHARMM
force field) was computed. The global minimum rotamer configuration was
found using a cyclic simulated annealing Monte Carlo protocol, assuming the
reference state energy to be similar. The final models were processed
through a 100-step constrained CHARMM minimization using the Adopted
Basis Newton–Raphson algorithm.
The active mutant was additionally purified over DEAE column (Amer-
sham Biosystems) on an ÄKTA FPLC system (Amersham Biosystems). To assess
the effect of the His-tag, it was removed using TEV protease (Promega), and
the protein purified as described in SI Text. Under standard conditions
(500 μM substrate, 20 mM HEPES (pH 6.9); 150 mM NaCl, 1 mM CaCl2) activity
(kcat∕KM) of the protein with His-tag removed was approximately 95% of the
starting His-tagged protein. Full-length CaM F92E mutant was also expressed
and purified on phenyl sepharose (31). The ratio of kcat∕KM for this protein
relative to AlleyCat was 1.2. Thus, the catalytic activities of a variety proteins
bearing the F92E mutation were essentially the same irrespective of the
nature of the N-terminal domain, purification method, or the presence of a
His-tag sequence. Identity of the proteins was confirmed by mass spectrome-
try. Isotopically labeled samples for NMR studies were expressed on M9
minimal medium containing 15NH4Cl and 13C uniformly labeled glucose
(Cambridge Isotopes).
Superrotamers.
A superrotamer of glutamate-5-nitrobenzisoxazole was
created based on QM calculations (7, 23). The superrotamer models were
generated using the same protocol as specified above with the number of
rotamers doubled. Partial charges were derived for 5-nitrobenzisoxazole
using AM1-BCC (35). The bonded energy parameters for 5-nitrobenzisoxa-
zole not present in CHARMM, were replaced with the most chemically similar
terms.
Kinetic Measurements. Kinetic measurements were done on a SpectraMax M5
plate reader (Molecular Devices) monitoring absorbance at 380 nm at 22 °C
using at least three independent measurements. In a typical experiment 2 μL
of freshly prepared 5-nitrobenzisoxazole stock solution in acetonitrile was
added to 200 mL of buffered (10–25 mM) protein (1–25 μM) solution so
the final concentration of substrate ranges from 50 μM to 1 mM. Product’s
ACKNOWLEDGMENTS. We thank A. Joshua Wand for providing a plasmid
containing the gene of chicken calmodulin. We thank David Baker and
Dan Tawfik for providing genes of KE07. We thank Guy Montelione for
providing NMR time and Kathleen Molnar for assistance with mass spec-
trometry.
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