Butelase-mediated synthesis of protein thioesters and its application for tandem chemoenzymatic ligation

Article abstract of DOI:10.1039/c5cc07227a

Using a recently discovered peptide ligase, butelase 1, we developed a novel method to access protein thioesters in good yield. We successfully combined it with native chemical ligation and sortase-mediated ligation in tandem for protein C-terminal labeling and dual-terminal labeling to exploit the orthogonality of these three ligation methods.

Full text of DOI:10.1039/c5cc07227a

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DOI: 10.1039/C5CC07227A
Journal Name
COMMUNICATION
Butelase-mediated synthesis of protein thioesters and its
application for tandem chemoenzymatic ligation
Received 00th January 20xx,
Accepted 00th January 20xx
Yuan Cao, Giang K T Nguyen, James P Tam* and Chuan-Fa Liu*
DOI: 10.1039/x0xx00000x
www.rsc.org/
1
1
12
Using a recently discovered peptide ligase, butelase 1, we Other methods using subtiligase
or SrtA 3to prepare
1
developed a novel method to access protein thioesters in good peptide/protein thioesters have also been developed.
yield. We successfully combined it with native chemical ligaiton
and sortase-mediated ligation in tandem for protein C-terminal discovered. Butelase 1 performs the cyclization step in the
labeling and dual-terminal labeling to exploit the orthogonality of cyclotide biosynthesis in the medicinal plant Clitoria ternatea. It
Recently a novel Asn/Asp(Asx)-peptide ligase, butelase 1, was
1
4
these three ligation methods.
recognizes a –N(D)HV motif at the C-terminus and cleaves the N(D)-
H bond to conjugate with an incoming N-terminal amino group
Over the past three decades, considerable efforts have been
devoted to developing chemoselective peptide ligation strategies
(Xaa) to form an Asx-Xaa at the ligation junction. Butelase 1 has
1
been shown to achieve success in both inter- and intramolecular
peptide ligations with a high catalytic efficiency and nearly
that operate in aqueous conditions and without the use of a
protection group scheme, to enable protein synthesis and site-
specific tagging of macromolecules for various biochemical
1
4
“
traceless” feature.
Herein we report a butelase-mediated ligation (BML) method to
2
applications. The chemical strategies thus developed often exploit
prepare protein thioesters (Fig. 1B) with high efficiency. We further
coupled this method with NCL and sortase-mediated ligation (SML)
for labelling proteins to show that these three ligation strategies are
orthogonal to each other.
the unique bifunctional moiety of a peptide’s N-terminal amino-acid
residue that has a nucleophilic side chain such as Cys or Ser/Thr to
react with another peptide containing a C-terminal "activated"
moiety. These include, in chronological order, pseudo-proline
3
4
5
A. Intein-mediated protein thioester preparation
ligation, native chemical ligation, thioacid capture ligation, and
6
HS
serine/threonine ligation, although the last one requires a non-
aqueous solvent system. The most successful of these is native
chemical ligation (NCL) to which the key is the use of a peptide C-ter
thioester of aryl or alkyl nature for mediating the ligation reaction
Protein
NH
Intein
O
4
, 7
with a cysteinyl moiety.
Besides being used in NCL, peptide
RSH
thioesters are also building blocks in the more classic peptide
segment condensation method via silver ion activation. Thus, an
HS
8
Protein
SR
+
H
2
N
Intein
efficient and convenient method to prepare a peptide/protein
thioester is always desirable and continues to be well sought after.
Whereas peptide thioesters can be synthesized directly by Boc
O
7
b, c, 8
9
B. This work: butelase-mediated ligation for protein thioester preparation
chemistry
or indirectly by Fmoc chemistry, protein thioesters
1
0
O
are obtained mostly through the intein technology. This method
requires the fusion expression of the target protein with an
engineered intein and the addition of an external thiol to release
Protein
N
V
+
2
H N
H
SR
butelase 1
Protein
10d, e
H V
the protein thioester from the immobilized intein (Fig. 1A).
O
N
G
SR
a.
School of Biological Sciences, Nanyang Technological University, 60 Nanyang
Fig. 1 Protein thioester preparation using (A) intein-mediated
technology and (B) butelase-mediated ligation.
Drive, Singapore 637551.E-mail: jptam@ntu.edu.sg (J. P. Tam), cfliu@ntu.edu.sg
(
C.-F. Liu)
†
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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at pH 4, 7.5 and 8. This result indicates that butelase 1 performs the
ligation optimally under mildly acidic pH conditions, a feature
Table 1 Ligation between a model peptide and different glycine
thioesters
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DOI: 10.1039/C5CC07227A
desirable for the stability of thioesters. For convenience, we used
the glycine thioester 1b in our following work. We further prepared
a series of peptides 4a-f with a common sequence YXNHV varying
the residue at P2 position (X = Val, Leu, Phe, Ser, Nle, D-Ala). All six
peptides, including two peptides containing unnatural amino acids,
ligated with excessive glycine thioester 1b at pH 6 to afford the
O
O
Butelase 1
YKNHV
2
+
H N
YKNG
SR
2
SR
pH 6, 42 o
C
3
a-d
1a-d
Glycine thioester
R
Yield [%]
A
1
1
1
a
b
c
R1
>95
>95
R2
R3
>95
>95
1d
R4
All reactions were carried out in the same condition: 50 µM peptide,
2
.2 mM glycine thioester, 100 nM butelase 1, pH 6, 20 mM
o
phosphate buffer, 42 C. Yields were measured after 2 h when
reactions reached equilibrium.
Four glycine thioesters Gly-COSR 1a-d (Table 1) were first used to
demonstrate butelase-mediated thioester synthesis. Since these
simple glycyl thioesters were easily synthesized from Boc-Gly-OH
and commercially available thiols, one could afford to use them in
large excess (44 fold to the acyl peptide substrate, Table 1) to
outcompete the reverse reaction, essentially overcoming the
reversibility problem of BML. As a result, all four glycine thioesters
ligated efficiently with the model peptide YKNHV 2 in the presence
of butelase 1 to afford the corresponding peptide thioesters YKNG-
COSR 3a-d in near quantitative yields (Table 1, Fig. S1†) as analysed
by HPLC. To determine the pH effect of this enzymatic ligation,
reaction between 1b and 2 was performed in different pH
conditions: pH 4, 5, 6, 6.5, 7.5 and 8. We found that the reaction
proceeded efficiently at pH 5, 6 and 6.5 (>90%), and less efficiently
B
C
Table 2 Ligation between 1b and different peptides
O
O
Butelase 1
+
H N
YXNG
S
YXNHV
2
S
pH 6, 42 o
C
4a-f
1b
5a-f
Peptide
X
Yield [%]
>95
4a
4b
4c
4d
Val
Leu
Ser
93
81
>95
>95
Phe
Nle
4e
Fig. 2 Butelase-mediated thioester synthesis of (A) ubiquitin, observed ESI-
MS for the starting material 6 and product 7 are 9982.5 Da (calc. 9982.3 Da)
and 9066 Da (calc. 9066.2 Da), respectively. (B) GFP, observed ESI-MS for
the starting material 8 and product 10 are 29724 Da (calc. 29725.7 Da) and
29631 Da (calc. 29632.6 Da), respectively. (C) DARPin(ERK), observed ESI-MS
for the starting material 9 and product 11 are 20215 Da (calc. 20214.4 Da)
and 20122 Da (calc. 20121 Da), respectively.
4
f
D
-Ala
91
All reactions were carried out in standard condition: 50 µM peptide,
mM glycine thioesters, 100 nM butelase 1, 20 mM phosphate
buffer, pH 6, 42 C. All yields were measured after 95 min when
reactions reached equilibrium.
2
o
2
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A
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DOI: 10.1039/C5CC07227A
H
His6
+ G
COSR
G
Ub
N
V
14
1b
BML
G
+
C
G
Ub
N
COSR
15
12
O
O
NCL
P
T
L
E
G
C
G
+
G
Ub
N
O
16
17a-b
SML
P
T
G
L
E
G
Ub
18a-b
N
C
Fig. 3 C-terminal biotin labeling of ubiquitin via NCL of ub
thioester with 12. Observed ESI-MS for the biotin-labeled
7
ubiquitin 13 is 9848 Da (calc. 9848.4 Da).
B
respective products YXNG-COSR 5a-f in good yields after 95 min
(Table 2). This result clearly showed the generality of our method
and that butelase 1 tolerates a variety of amino acids N-terminal to
the NHV tripeptide recognition motif. Clearly, an advantage of this
approach is that a large excess of the Gly-COSR can be used to force
the reaction to completion in a short duration.
We also tested the thioesters of four other amino acids (Ala,
Leu, Ile, Phe) in butelase-mediated ligation with model peptide 2.
Surprisingly, only alanine thioester gave some modest ligation
product, while the other amino thioesters did not work in the
ligation reaction (data not shown). So, a limitation of this method is
that, among the amino-acid thioesters, only glycinyl thioesters are
efficient substrates of butelase 1.
To show the compatibility of BML with NCL, the peptide
thioester YKNG-COSR 3b was ligated with a cysteinyl peptide
CGYKNHV to afford YKNGCGYKNHV in quantitative yield under
standard NCL conditions (Fig. S2†).
To show the utility of this approach for proteins, we prepared
an engineered ubiquitin 6 with the NHV recognition motif and a
C
His -tag for affinity purification at its C-terminus. Reaction of 6 (100
6
µM) with glycine thioester 1b (2 mM, 20 eq.) in the presence of 200
nM butelase 1 (0.002 eq.) afforded the protein thioester product 7
in >90% yield after 2 h (Fig. 2A). We also prepared two other
1
5
proteins: GFP 8 and an ERK-ankyrin repeat protein (DARPin(ERK))
, both engineered to have a C-ter NHV motif. Using the same
9
reaction, we obtained the thioester product 10 and 11 in ca. 85%
yield in short time (Fig. 2B and C). These results show again the high
catalytic efficiency of butelase 1. Since the –NHV motif is very small,
any protein tagged with it can be easily expressed as shown in the
three protein examples here. These features make our approach a
very robust and convenient method to prepare protein thioesters.
Next, we showed the prepared protein thioester was useful for
NCL. So, a cysteinyl peptide CK(biotin)LKVA 12 was ligated with the
ubiquitin thioester 7 under standard NCL conditions to afford, in
near quantitative yield as observed by HPLC analysis (Fig. 3), the
product ubiquitin 13 containing now a biotin probe at its C-ter end.
To show that SML can also be used in conjunction with the
above two reactions for a three-step tandem ligation, another
recombinant ubiquitin 14 was prepared. It contained a –NHV motif
Fig. 4 (A) Schematic illustration for the dual-termini labelling of ubiquitin.
B) Sortase-mediated N-terminal labeling of the Ub-biotin 16 with Nle-
YLPET-glc-G 17a. Observed ESI-MS for the final dual-terminal labeled
product 18a is 10680 Da (calc. 10680.2 Da). (C) Observed ESI-MS of the
dual-labeled fluor-Ub-biotin 18b is 10926 Da (calc. 10925.7 Da ) obtained
via SML of 16 with fluor-YLPET-glc-G 17b .
(
a glycine residue for SrtA recognition. One of the practical
applications of this convergent methodology could be dual-
terminus modification of proteins (Fig. 4A). This attempt was
1
6
previously done by using two sortases with different activities. In
our case, the C-terminal labeling of 14 was first conducted using the
above two-step process: BML with glycine thioester 1b followed by
NCL with biotinyl peptide 12. 85% and quantitative HPLC
followed by a His -tag at the C-terminus. At N-terminus, it displayed
6
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View Article Online
DOI: 10.1039/C5CC07227A
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Science, 1994, 266, 776-779.
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G. The use of the ester derivative of the SrtA motif LPXTG was 6. X. Li, H. Y. Lam, Y. Zhang and C. K. Chan, Org. Lett., 2010, 12
based on a previous report that it could significantly improve the 1724-1727.
SML yield due to irreversibility of the reaction. We placed a short 7. (a) T. Wieland, E. Bokelmann, L. Bauer, H. U. Lang and H. Lau,
,
,
17
sequence –GSGSGS- between the N-terminal Gly residue and the
main body of ubiquitin for better accessibility of its N-terminus by
SML. A depsipeptide Nle-YLPET-glc-G 17a was first used for
demonstration. The SML reaction gave a near quantitative yield of
Liebigs Ann. Chem., 1953, 583, 129-149; (b) J. P. Tam, Y. A.
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0
8a after 2.5 h with two molar equivalents of the depsipeptide and 8. S. Aimoto, Biopolymers, 1999, 51, 247-265.
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,
,
ratio of SrtA, which is a significant drawback as compared to BML
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simple treatment with Ni-NTA removed the His-tagged SrtA and 10. (a) K. V. Mills, M. A. Johnson and F. B. Perler, J. Biol. Chem.
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and C-ter ends, respectively (Fig. 4C).
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T. C. Evans, Jr., J. Benner and M. Q. Xu, Protein Sci., 1998, 7,
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Andrea C. DeDent and Olaf
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substrates of different sizes. This method requires the substrate
protein to have only a small tripeptide motif NHV at the C-terminus
and renders little change on the protein sequence after ligation as it
leaves behind only a dipeptide trace -NG. We have further shown
Schneewind.,
(b) H. Ton-That, G. Liu, S. K. Mazmanian, K. F. Faull and O.
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and SML for sequential ligation to achieve bi-directional and
orthogonal labelling of ubiquitin in a model system. These results
demonstrate butelase 1 as a versatile tool for protein manipulation.
We foresee that, like other immerging methods, BML will offer
numerous future opportunities in biotechnology.
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1
8
We thank Dr. T. Cornvik at Nanyang Technological University for 14. G. K. Nguyen, S. Wang, Y. Qiu, X. Hemu, Y. Lian and J. P. Tam,
providing the plasmids of some of the model proteins. This work Nat. Chem. Biol., 2014, 10, 732-738.
was supported by Singapore National Research Foundation- 15. H. K. Binz, M. T. Stumpp, P. Forrer, P. Amstutz and A.
Competitive Research Programme Grant (NRF-CRP8-2011-05).
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(
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. (a) C. P. Hackenberger and D. Schwarzer, Angew. Chem., Int.
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4
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DOI: 10.1039/C5CC07227A
O
O
butelase 1
Protein
N
V
+ H N
Protein
N
2
H
G
SR
SR
H V
Recombinant proteins with a C-terminal thioester are readily prepared through the catalysis of
butelase 1 – a powerful peptide ligase.