In Situ Cyclization of Native Proteins: Structure-Based Design of a Bicyclic Enzyme

Article abstract of DOI:10.1002/anie.201804506

Increased tolerance of enzymes towards thermal and chemical stress is required for many applications and can be achieved by macrocyclization of the enzyme resulting in the stabilizing of its tertiary structure. Thus far, macrocyclization approaches utilize a very limited structural diversity, which complicates the design process. Herein, we report an approach that enables cyclization through the installation of modular crosslinks into native proteins composed entirely of proteinogenic amino acids. Our stabilization procedure involves the introduction of three surface-exposed cysteine residues, which are reacted with a triselectrophile, resulting in the in situ cyclization of the protein (INCYPRO). A bicyclic version of sortase A was designed that exhibits increased tolerance towards thermal as well as chemical denaturation, and proved to be efficient in protein labeling under denaturing conditions. In addition, we applied INCYPRO to the KIX domain, resulting in up to 24 °C increased thermal stability.

Full text of DOI:10.1002/anie.201804506

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Accepted Article
Title: In Situ Cyclization of Native Proteins: Structure-Based Design of
a Bicyclic Enzyme
Authors: Marta Pelay-Gimeno, Tanja Bange, Sven Hennig, and Tom N.
Grossmann
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http://dx.doi.org/10.1002/ange.201804506

Angewandte Chemie International Edition
10.1002/anie.201804506
In Situ Cyclization of Native Proteins: Structure-Based Design of a
Bicyclic Enzyme
Marta Pelay-Gimeno, Tanja Bange, Sven Hennig, and Tom N. Grossmann*
[
*] Dr. Marta Pelay-Gimeno, Dr. Sven Hennig, Prof. Dr. T. N. Grossmann (E-mail: t.n.grossmann@vu.nl)
Department of Chemistry & Pharmaceutical Sciences
VU University Amsterdam
De Boelelaan 1108, 1081 HZ, Amsterdam (The Netherlands)
Dr. Tanja Bange
Department of Mechanistic Cell Biology
Max-Planck Institute of Molecular Physiology
Otto-Hahn-Str. 11, 44227 Dortmund (Germany)
Dr. Tanja Bange
Department for Systems Chronobiology
LMU Munich
Goethe-Str. 31, 80336 Munich (Germany)
[
**] We thank A. Musacchio (MPI Dortmund) for SrtA expression construct and D. M. Krüger (CGC Dortmund)
for the discussion of SrtA variants. We thank F. Müller (MPI Dortmund) for technical support in mass
spectrometry. We thank the Deutsche Forschungsgemeinschaft (DFG; Emmy Noether program GR3592/2-1)
and the European Reasearch Council (ERC; ERC starting grant, no. 678623), and we are grateful for support
by AstraZeneca, Bayer CropScience, Bayer HealthCare, Boehringer Ingelheim, Merck KGaA and the Max
Planck Society.
Keywords: INCYPRO, Protein engineering, Protein labeling, Sortase A, Tertiary structure stabilization
Abstract: Increased tolerance of enzymes towards thermal and chemical stress is required for many applications
and can be achieved by macrocyclization of the enzyme resulting in the stabilizing of its tertiary structure. So far,
macrocyclization approaches utilize a very limited structural diversity which complicates the design process. Here,
we report an approach that enables cyclization via the installation of modular crosslinks into native proteins
composed entirely of proteinogenic amino acids. Our stabilization procedure involves the introduction of three
surface exposed cysteines which are reacted with a triselectrophile resulting in the in situ cylization of the protein
(INCYPRO). A bicyclic version of Sortase A was designed exhibiting increased tolerance towards thermal as well
as chemical denaturation, and proved efficient in protein labeling under denaturing conditions. In addition, we
applied INCYPRO to the KIX domain resulting in up to 24 °C increased thermal stability.
1
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Angewandte Chemie International Edition
10.1002/anie.201804506
[
1]
Enzymes are an essential component of most biotechnological and biomedical processes but their scope of
application is hampered by a limited stability under often desired harsh conditions (e.g. elevated temperature or
presence of denaturants). Consequently, the stabilization of protein structures is essential when developing suitable
enzymes. The complexity of interactions in protein tertiary structures and the sensitivity of enzymatic activity on
sequence alterations render enzyme stabilization very challenging. A minimal invasive strategy involves the use of
covalent protein modifications (e.g. pegylation or glycosylation) being mainly applied to increase biostability for
[2]
therapeutic applications. Alternatively, enzyme stabilization can be achieved via sequence variation applying
[
3]
directed evolution, consensus-based mutagenesis or computational approaches which can be complimented by the
[
4]
introduction of non-proteinogenic amino acids. These approaches aim for improved protein core interactions,
structure rigidification, and/or surface charge redistribution, and often require multiple rounds of optimization to
achieve relevant stabilization.
The stabilization of enzymes has also been achieved by the introduction of intramolecular crosslinks. Early
[
5]
examples involve the installation of additional disulfide bridges which was later complemented by disulfide
[
6]
mimics being insensitive to reducing environments. Stabilization via disulfides is challenging, in particular when
replacing residues in the protein core, as this can cause the loss of non-covalent interactions thereby reducing the
[
7]
benefit of crosslinking. For that reason, the incorporation of disulfides in flexible protein regions and on the
protein surface proved more successful. However, the short distance of the disulfide bridge limits its
[8]
[9]
applicability. Alternatively, the crosslinking of protein termini via lactam formation was applied requiring a
suitable spatial alignment of N- and C-terminus. To reduce these structural prerequisites, the incorporation of non-
[
10]
natural amino acids was pursued to enable crosslinking with an appropriately aligned cysteine side chain.
However, the necessity of amber stop codon suppression for the introduction of these non-natural amino acids
complicates protein expression. In addition, the screening of linker libraries is hampered since incorporation of
[
11]
such modified amino acids requires the evolution of a corresponding tRNA synthetase. Taken together, enzyme
stabilization endeavors would greatly benefit from an approach that allows the installation of modular crosslinks
into proteins only consisting of natural amino acids.
Here we report a structure-based stabilization strategy involving the in situ cyclization of proteins (INCYPRO)
composed entirely of proteinogenic amino acids (Figure 1a). Using an initial set of mono-cyclic enzyme variants,
we evaluated the feasibility of crosslinking two intradomain cysteines using a biselectrophile. Subsequently, a
triselectrophilic crosslinker was employed to generate a bicyclic enzyme with high tolerance towards thermal and
chemical stress.
We chose Staphylococcus aureus Sortase A (SrtA, aa 60–206) as the target of our stabilization efforts. SrtA is a
[12]
transpeptidase and an important biomolecular tool allowing specific labeling of proteins. Labeling efficiency
drops when higher temperatures or denaturants are used limiting its applicability. To stabilize SrtA, we considered
a crosslinking strategy that has previously been applied to constrain peptides and involves the use of
[
13]
biselectrophiles that target pairs of cysteines. Different from these examples, SrtA already contains a cysteine
(C184) which is crucial for catalytic activity and may undergo undesired reactions with the electrophile. For that
2
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reason, we first tested four electrophiles (1 – 4, Figure 1b), previously used for cysteine labeling, regarding their
propensity to react with cysteine C184. Reaction conditions suitable for preparative scale protein modification led
to substantial modification of C184 when incubated with maleimide (1) and 2-bromoacetamide (2), but not for 2-
chloroacetamide (3) and acrylamide (4) (Figure S1, Table S1). These differences in the labeling potential are in line
[
14]
with their electrophilicity (1 > 2 > 3 ≈ 4).
Noteworthy, C184 is shielded by active site residues reducing its
[15]
general accessibility.
As thiols tend to show reversible addition to acrylamides, we finally chose 2-chloro-
acetamide (3) as the electrophile for our crosslinkers which should enable selective labeling of introduced, solvent
exposed cysteines. A set of biselectrophilic linkers with 8 – 17 bridging atoms (b1 – b6, Figure 2a) spanning a
broad range of distances (up to 21 Å, Figure S2) was designed.
Figure 1. a) Macrocyclization strategy towards stabilized protein tertiary structures using a modular bis- or triselectrophilic
crosslink; b) Electrophiles (maleimide 1, 2-bromoacetamide 2, 2-chloroacetamide 3, acrylamide 4) considered for crosslinking
of accessible cysteines.
Suitable positions for the introduction of cysteine pairs in SrtA were selected to evaluate their suitability for tertiary
structure stabilization. We considered surface residues not involved in substrate recognition and selected pairs that
are located in two different secondary structure elements while still being in spatial proximity (distance <20 Å,
based on NMR structure, PDB ID 1ija). Based on these criteria, six SrtA variants (S1 – S6, Figure 2b, Figure S3)
were designed, expressed in E. coli and purified. Subsequently, crosslinking reactions with all biselectrophiles were
performed showing various degrees of conversion. Formation of the cyclization product was confirmed by MS and
SDS-PAGE (Figure S4 and S5). While we observed high conversions for S1, S3, S4 and S6 with all crosslinkers,
S2 and S5 showed overall high heterogeneity (Figure S4). After the reaction, protein samples were dialyzed to
remove unreacted biselectrophiles. Initially, apparent melting temperatures (T
variants (as obtained after dialysis) were determined via changes in tryptophan fluorescence (Figure 2c, Figure S6).
Compared to SrtA (T = 59.4 °C), all non-crosslinked variants show a lower thermal stability except for S3
ΔT = +2.9 °C). Enzyme crosslinking results in strong stabilization of the cyclic S3 versions (ΔT ≥ +10.1 °C)
m
) of all unmodified and crosslinked
m
(
m
m
3
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while more moderate effects were observed for the remaining variants. The most stable versions per variant are
S1-b1 (ΔT = +2.8 °C), S2-b2 (ΔT = +0.4 °C), S4-b3 (ΔT = +4.4 °C), S5-b5 (ΔT = +3.4 °C) and S6-b1 (ΔT
3.9 °C).
m
m
m
m
m
=
+
SrtA is a transpeptidase which cleaves its peptide recognition motif (LPXTG, Figure 2d) resulting in the formation
of a thioester intermediate. This intermediate is preferably attacked by the N-terminus of oligo-glycine to form a
new peptide bond (Figure 2d). In absence of a suitable nucleophile, water will attack and hydrolyze the thioester
(Figure 2d). To investigate enzymatic activity, a previously reported probe system was applied in which a
fluorophore/quencher pair is separated upon SrtA processing (Figure S7). For activity screening, we chose the
[
16]
hydrolysis reaction at 65 °C, where wildtype SrtA shows strongly reduced performance (4 % residual activity,
Figure S7). Relative to SrtA (v = 1, Figure 2e), a number of crosslinked enzymes show increased activity.
r
Surprisingly, the thermostable cyclic versions of S3 provide reduced enzymatic activity (Figure 2e). In contrast,
crosslinked versions of S4 and S5 exhibit robust activity enhancements (>2-fold, light and dark red, Figure 2e). The
overall highest increase in activity was observed for S4-b3, which is 3.4-fold more active than SrtA. Taken together,
observed improvements in activity at 65 °C are moderate indicating that mono-cyclization may not be sufficient to
convey enough stabilization of the tertiary structure.
Figure 2. a) Biselectrophiles (b1 – b6) used for the generation of mono-cyclic enzymes; b) NMR structure of SrtA (PDB: 1ija)
with positions of cysteine variations highlighted. Cysteine pairs (same color) and their positions are shown (for details see
Figure S3); c) Heat map representation of apparent T
m
-values (heating rate 1 °C/min) for linear and crosslinked SrtA variants
-values see Figure S6); d) Mechanism of SrtA-mediated transpeptidation reaction
recognition motif: LPXTG); e) Heat map representation of enzymatic activity (v , relative to wildtype SrtA) of linear and
-values see Table S2) (buffer for 2c and 2e:
, 2 mM TCEP, for 3e: including 0.01 % Tween 20).
(75 µM, for melting curves and apparent T
m
(
r
crosslinked SrtA variants at 65 °C (10 µM enzyme, 10 µM fluorescent probe, for v
r
2
0 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM CaCl
2
4
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To achieve stronger stabilization effects, we aimed for bicyclization of the enzyme. Notably, the two best
performing SrtA variants S4 and S5 (light and dark blue, Figure 2b) share one variation site (aa 149). Thus, we
decided to introduce their three cysteine variations simultaneously (aa 111, 149 and 177) resulting in variant S7
(Figure 3a), which can form a bicyclic protein upon reaction with a triselectrophile. In analogy to bicyclic
[
17]
[18]
3
peptides and mini-proteins , we selected a C -symmetric core for our crosslinker, which was modified with
three 2-chloroacetamide groups (t1, Figure 3b). Triselectrophile t1 involves 13 bridging atoms thereby lying
between the preferred crosslink ranges of S4 (b3/b4: 10/11 atoms) and S5 (b5/b6: 14/17 atoms). The crosslinking
reaction of S7 and t1 proceeds efficiently and provides stapled enzyme S7-t1 (Figure 3b). Analytical HPLC-MS
analysis indicates quantitative conversion of S7, clearly showing the formation of a product with the expected
molecular weight (Figure S8). High-resolution MS analysis of tryptic fragments confirms the modification of the
three introduced cysteines also verifying the unmodified state of C184 (Figure S9, S10 and S11). Importantly, S7-
t1 exhibits strongly increased thermal stability (T
m
= 70.6 °C, Figure 3c) being considerably higher than that of
= +6.8°C). Next, we determined the
SrtA (ΔT = +11.2 °C) and of the most active mono-cyclic protein S4-b3 (ΔT
m
m
enzymatic activity of S7-t1 at 65 °C (Figure 3d). In line with its superior thermal stability, we observe a strongly
increased enzymatic activity at 65 °C when compared to SrtA (8.7-fold) and S4-b3 (2.6-fold, Figure 3d).
Figure 3. a) NMR structure of SrtA (PDB: 1ija) with positions of cysteine variations in S7 highlighted; b) Chemical structure
of triselectrophile t1 and Coomassie-stained SDS-PAGE gel showing protein bands after incubation with t1 (50 µM S7, 1 mM
t1, 50 mM HEPES, pH 8.5, 150 mM NaCl, 5 mM CaCl
2
, 2 mM TCEP); c) Melting curves of SrtA, S4-b3 and S7-t1 including
apparent T -values (heating rate 1 °C/min); d) Fluorescent readout of enzymatic activity at 65 °C (10 µM enzyme, 10 µM
m
2
fluorescent probe) (buffer for 3c and 3d: 20 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM CaCl , 2 mM TCEP with 0.01 %
Tween 20 for d).
5
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So far, we evaluated enzyme activity under hydrolytic conditions. Envisioning the application of S7-t1 for protein
labeling, we next investigated transpeptidation at 65 °C with above described fluorescent probe but now in the
presence of the nucleophile triglycine (transfer, Figure 2d). Using HPLC-MS as readout (Figure 4a), we again
observed only very low substrate conversion with SrtA (dark grey) similar to a treatment without any enzyme (light
grey). In the presence of S7-t1 (red, Figure 4a), the signal of the starting material (●), was greatly diminished and
two new peaks appeared. Based on the MS (Figure S12), one peak was assigned to the C- (▲) and the other one to
the N-terminal fragment (■), which appears to be ligated to triglycine. Importantly, a signal for the hydrolysis
product (Dabcyl-QALPET) was not detected verifying the correct functionality of S7-t1. To assess if protein
unfolding under elevated temperature is reversible, we compared the enzymatic activity of SrtA and S7-t1 at 37 °C
before and after heating (85 °C, Figure S13). Notably, the transpeptidase activity of both enzymes is not affected by
the heating/cooling cycle indicating reversible unfolding.
Figure 4. a) HPLC chromatograms (440 nm) of transpeptidation reaction (12 h at 65 °C) with fluorescent probe (●) in absence
of enzyme (light grey), with SrtA (dark grey) or with S7-t1 (red). Product formation (▲, ■) was only observed in presence of
S7-t1 (50 µM enzyme, 10 µM probe, 2.5 mM GGG, for MS spectra of signals see Figure S12); b) Temperature dependence of
enzymatic activity (v
50 mM NaCl, 5 mM CaCl
p<0.001, ns: not significant); c) Relative enzymatic activity (v
r
, relative to SrtA at 37 °C) (10 µM enzyme, 10 µM probe, 2.5 mM GGG, buffer: 20 mM HEPES, pH 7.5,
, 2 mM TCEP, 0.01 % Tween 20). Values are mean of triplicate (+/- 1 σ, * p<0.05, ** p<0.01, ***
, relative to SrtA in absence of GdnHCl) at 37 °C for various
1
2
r
concentrations of GdnHCl (for details see supporting methods); d) Coomassie-stained SDS-PAGE gel showing the soluble α-
Syn fractions before fibril formation (A) and after re-solubilization in absence (B) and presence (C) of GdnHCl (1 M); e)
Fluorescent readout (λem = 520 nm) of α-Syn labeling using soluble fractions before fibril formation A (w/o GdnHCl) and after
re-solubilization B (w/o GdnHCl) and C (1 M GdnHCl) with either SrtA or S7-t1 (for details see Figure S14 and S15).
6
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Next, we determined the thermal activity profile of the transpeptidation reaction (Figure 4b). Between 37 °C and
5
5
5 °C, the activity of SrtA (grey) and S7-t1 (red) is similar exhibiting only low temperature dependence. Above
5 °C, both enzymes experience a loss in activity which is very severe for SrtA resulting in almost complete
inactivation at 65 °C (Figure 4b). For S7-t1, the activity reduction is much smaller with residual activities of 63 %
at 65 °C) and 27 % (at 70 °C) relative to 37 °C. Compared to SrtA, S7-t1 shows a ~10 °C increased tolerance
(
towards thermal stress which correlates well with its +11.2 °C higher apparent melting temperature. Enhanced
thermal stability often goes in hand with a resistance towards denaturants such as guanidinium hydrochloride
(
GdnHCl). For that reason, the impact of GdnHCl on transpeptidase activity was investigated (Figure 4c), revealing
low dependence of SrtA and S7-t1 on the denaturant concentration up to 0.5 M. Between 0.75 and 1.5 M, S7-t1 is
significantly more active than SrtA. Most notably at 1 M GdnHCl, SrtA does not yield any product (v < 1 %,
r
Figure 4c) while S7-t1 still provides 40 % residual activity (compared to absence of GdnHCl). At higher GdnHCl
concentrations (≥ 2 M) both enzymes are inactive.
So far, we have applied S7-t1 for the labeling of a short test peptide. Next, we were interested if S7-t1 is also useful
for protein labeling in particular under conditions where wildtype SrtA does not provide sufficient activity. For that
purpose, we chose α-synuclein (α-Syn) as target. α-Syn comprises 140 amino acids and can form pathogenic fibrils
[19]
which are associated with the onset of various neurodegenerative diseases including Parkinson’s. α-Syn fibrils
[
20]
can be solubilized using GdnHCl.
We designed an α-Syn version with a C-terminal SrtA-recognition motif
(Figure S14) to allow labeling. Following expression and purification, soluble α-Syn (A) was subjected to fibril
[20]
formation and ultra-centrifugation. Insoluble fibrils were washed and treated with buffer either lacking (B) or
[
20]
containing (C) GdnHCl (1 M). When comparing the resulting soluble fractions (B and C) with the purified and
soluble form of α-Syn (A, Figure 4d), we observed re-solubilization only in the presence (C) but not in absence (B)
of GdnHCl. To investigate protein labeling, these soluble samples (A, B, C) were incubated with either SrtA or S7-
t1, and a fluorescent substrate (Figure S14). We then performed analysis via SDS-PAGE employing a fluorescence
imager for readout. For soluble α-Syn prior fibril formation (A) and therefore in absence of GdnHCl, SrtA and S7-
t1 result in intense bands indicating efficient protein labeling (Figure 4e, Figure S15). As expected, under re-
solubilization conditions lacking GdnHCl (B) and therefore also lacking soluble α-Syn, we did not observe any
fluorescent signal (B, Figure 4e, Figure S15). On the contrary, for re-solubilization with GdnHCl (1 M), α-Syn
labeling occurs but only with S7-t1 and not with wildtype SrtA (C). Notably, differences in the fluorescent band
intensities for S7-t1 (A vs. C, Figure 4e, Figure S15) correlate well with the amount of α-Syn in the soluble
fractions (A vs. C, Figure 4d) indicating good labeling efficiencies for S7-t1 in the presence of GdnHCl.
To assess the broader applicability of protein stabilization via bicyclization, we chose the KIX domain from the
human CREB binding protein as a second example (Figure 5a). KIX is an adaptor domain with multiple protein
binding partners, such as mixed-lineage leukemia (MLL), and is composed of a central three α-helix bundle (α
1
2
, α ,
α
). The junction between this bundle and the C-terminal 310 helix (G ) is crucial for structural integrity (Figure
3 1
[
21]
5
a). Thus, we focused on this area for tertiary structure stabilization searching for three positions suitable for
7
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cysteine incorporation. Based on our experiences during SrtA stabilization, the following guidelines were applied:
i) Solvent accessible residues were considered, that are (ii) located in three distinct secondary structures, while (iii)
(
facing the same side of the protein and (iv) spanning a triangle with side lengths between 6 and 17 Å (Cα-Cα
distance). Based on these criteria, we selected H594, L599 and R646 for cysteine introduction resulting in KIX
variant K1 (Figure 5a, Figure S16).
Figure 5. a) NMR structure of KIX (PDB: 2agh) with positions of cysteine variations in K1 highlighted. The secondary
m
structural elements have been named; b) Melting curves of KIX, K1-t1 and K1-t2 including apparent T -values.
For crosslinking, we chose triselectrophile t1 (n = 2, Figure 3b) and a shorter version t2 (n = 1) since we noticed
that the distances between the three variation sites in K1 (7.8, 10.0 and 11.5 Å, Figure S16) are shorter than in S7
(8.5, 12.4 and 15.7 Å, Figure S8). The crosslinking with both triselectrophiles proceeds efficiently as confirmed by
SDS-PAGE (Figure S17) and HPLC-MS analysis (Figure S18). To evaluate if crosslinking affects the tertiary
structure, we compared the affinity of KIX and both bicyclic variants (K1-t1 and K1-t2) to its binding partner
MLL (for sequence see Figure S19). Using a fluorescence polarization assay, similar binding affinities were
observed for KIX, K1-t1 and K1-t2 (K
apparent melting temperatures (Figure 5b) to find a strongly increased thermal stability for K1-t1 and K1-t2
ΔT = +20.6 °C and +24.6 °C, respectively) when compared to KIX. Notably, both triselectrophiles have a similar
d
= 0.6, 0.9 and 0.9 µM, respectively, Figure S19). Then, we determined
(
m
stabilizing effect with the shorter crosslink t2 performing best. Based on these results, we were also interested to
evaluate the effect of triselectrophile t2 on SrtA variant S7. Again, the crosslinking reaction proceeds efficiently
resulting in bicyclic enzyme S7-t2 (Figure S20). Notably, we observe a similar thermal stabilization for S7-t2
(
ΔT
m m
= +11.5 °C, Figure S20) as for S7-t1 (ΔT = +11.2 °C), indicating tolerance towards minor variations in the
length of the crosslink.
In summary, we here report the in situ cyclization of proteins (INCYPRO) enabling a structure-based stabilization
of recombinant proteins being entirely composed of proteinogenic amino acids. The use of synthetic electrophiles
for protein cyclization gives straight-forward access to diverse crosslink architectures with tunable length and
flexibility. We applied INCYPRO to generate bicyclic SrtA variant S7-t1 exhibiting strongly increased tolerance
towards thermal and chemical denaturation. Importantly, S7-t1 proves efficient in labeling α-Syn in presence of
1
M GdnHCl. Under these conditions, wildtype SrtA did not show enzymatic activity. From our findings with SrtA,
we derived guidelines for the bicyclization and stabilization of proteins and applied them to the KIX domain. A
8
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three cysteine KIX variant was designed and reacted with two different C
3
-symmetric triselectrophiles to provide
bicyclic KIX versions with up to 24 °C increased thermal stability. In the future, we envision the use of
crosslinking agents that allow the introduction of an additional functionality such as an affinity handle (e.g. for
[22]
[23]
enzyme purification/recycling or proximity-assisted enzyme activity ). Taken together, the presented protein
stabilization technology holds the potential to give rapid access to novel stabilized enzymes providing the
opportunity for a simultaneous incorporation of additional functions.
[
[
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