Site-specific N- and C-terminal labeling of a single polypeptide using sortases of different specificity

Article abstract of DOI:10.1021/ja902681k

(Figure Presented) The unique reactivity of two sortase enzymes, SrtA _staph from Staphylococcus aureus and SrtA_strep from Streptococcus pyogenes, is exploited for site-specific labeling of a single polypeptide with different labels a

Full text of DOI:10.1021/ja902681k

Published on Web 07/17/2009
Site-Specific N- and C-Terminal Labeling of a Single Polypeptide Using
Sortases of Different Specificity
John M. Antos, Guo-Liang Chew, Carla P. Guimaraes, Nicholas C. Yoder, Gijsbert M. Grotenbreg,
Maximilian Wei-Lin Popp, and Hidde L. Ploegh*
Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142
Received April 3, 2009; E-mail: ploegh@wi.mit.edu
Methods for site-specific modification of proteins remain in high
demand. The transpeptidation reaction catalyzed by sortase A from
Staphylococcus aureus (SrtAstaph) allows site-specific derivatization
of proteins with virtually any type of functional material.1 Target
proteins are engineered to contain the SrtAstaph recognition site
(LPXTG) near their C terminus, thus allowing a transacylation
reaction in which the residues C-terminal to threonine are exchanged
for a synthetic oligoglycine peptide (Scheme 1). While the range
of applications for this technology has expanded considerably,1 the
ligation chemistry itself has seen relatively few modifications or
improvements. Since nearly all Gram-positive bacteria possess
sortases, many with reactivity distinct from SrtAstaph, there is an
exciting opportunity to develop complementary strategies for protein
engineering using other members of this enzyme family.2,3 Here
we present a strategy for placing discrete labels at both termini in
the same polypeptide through the use of multiple sortases. We first
describe the ability of SrtAstaph to append labels at the protein N
terminus and then demonstrate how this can be used in conjunction
with the activity of sortase A from Streptococcus pyogenes (SrtAstrep
)
to yield dual-labeled proteins.
Scheme 1. C-terminal Labeling Using SrtAstaph
Figure 1. N-terminal labeling using SrtAstaph. (a) SrtAstaph catalyzes a
transacylation reaction using labeled LPRT methyl esters as substrates. The
labeled LPRT fragment is transferred to proteins containing N-terminal
glycines in a site-specific fashion. (b) FITC (1) and biotin (2) LPRT methyl
esters for N-terminal transacylation. (c) CtxB derivatives (50 µM) were
treated with 500 µM 1 and 50 µM SrtAstaph for 2 h at 37 °C in 50 mM Tris
(pH 7.5), 150 mM NaCl, and 10 mM CaCl2. Reactions were analyzed by
SDS-PAGE with visualization by coomassie staining and fluorescent gel
scanning.
With regard to N-terminal labeling mediated by SrtAstaph, we
reasoned that labeled synthetic peptides containing the LPXTG
recognition motif, or structural analogues thereof, could generate
the requisite acyl-enzyme intermediate necessary for transpepti-
dation. In combination with protein nucleophiles containing one
or more N-terminal glycines, this should result in transfer of the
label to the protein N terminus (Figure 1a). We synthesized FITC
(1) and biotin (2) derivatives of an LPRT peptide in which the
glycine of the normal LPXTG motif was replaced by a methyl ester
(Figure 1b and Figure S1 in the Supporting Information). The use
of an ester derivative rather than the entire LPXTG motif was
motivated by our concern that the glycine residue released after
LPXTG cleavage might compete with the protein nucleophile,
potentially complicating the desired N-terminal-labeling reaction.
In contrast, transacylation with 1 and 2 would generate MeOH, a
poor nucleophile for transacylation compared with glycine. It should
be noted that concurrently with the work described here, it was
we prepared a control construct containing an N-terminal alanine
residue. In the presence of SrtAstaph, we observed robust labeling
of G3-CtxB and G5-CtxB using 500 µM 1 for 2 h at 37 °C, with no
apparent labeling of the alanine-containing control (Figure 1c).
Electrospray ionization mass spectrometry (ESI-MS) revealed
quantitative labeling of G3-CtxB and G5-CtxB, with no modification
observed for G1-CtxB and AG4-CtxB (Figure S2). Similar experi-
ments with biotinylated derivative 2 and G5-CtxB yielded compa-
rable results, as verified by ESI-MS and streptavidin immunoblot
(Figure S3). In all cases, residual labeling of SrtAstaph itself,
attributable to the formation of a covalent acyl-enzyme intermedi-
ate, was detected. N-terminal transpeptidation was also successful
for two additional protein substrates, eGFP with five N-terminal
glycines and UCHL3 containing a single N-terminal glycine (Figure
S4).
demonstrated that labeled LPETGG peptides are viable tools for
4
N-terminal labeling using SrtAstaph
.
With 1 and 2 in hand, we expressed a series of model proteins
containing N-terminal glycine residues. We prepared variants of
the cholera toxin B subunit (CtxB) with one, three, or five
N-terminal glycines. In order to verify the selectivity for glycine,
With the ability of SrtAstaph to append labels at either terminus,
we pursued the possibility of installing two modifications within
9
10800 J. AM. CHEM. SOC. 2009, 131, 10800–10801
10.1021/ja902681k CCC: $40.75  2009 American Chemical Society

C O M M U N I C A T I O N S
the same protein. Attempts to execute this type of transformation
using SrtAstaph alone were unsuccessful, as intramolecular transpep-
tidation between N-terminal glycines and the C-terminal LPXTG
motif was unavoidable in most cases. Therefore, we considered
the possibility of using a second, distinct sortase, an idea that has
been suggested but never reduced to practice.1,5 We initially sought
to use sortase B (SrtB) from either Staph. aureus or Bacillus
anthracis as enzymes with recognition sequences (NPQTN and
observed to ligate both glycine and alanine nucleophiles (data not
shown). Substrates also contained an LPXTG motif at the C
terminus to allow a first round of labeling with SrtAstrep. For both
eGFP and UCHL3, C-terminal labeling using 3 and SrtAstrep resulted
in >90% conversion to the desired adduct, as revealed by ESI-MS
(Figure S6). SrtAstrep was quenched by the addition of MTSET
followed by removal of His6-tagged SrtAstrep using Ni-NTA.
Residual 3 was then removed using a disposable desalting column.
Thrombin cleavage proceeded in quantitative fashion using com-
mercial thrombin agarose resin (Figure S6). The exposed N-terminal
glycines were then labeled by treatment with 500 µM 1 and 50
NPKTG, respectively) orthogonal to that of SrtAstaph.
6,7 Both SrtB
enzymes were easily produced in Escherichia coli and purified to
homogeneity. We reproduced the reported in vitro enzyme activity
using a FRET-based assay to measure cleavage of short peptides
substrates.6,7 However, to date we have failed to obtain transpep-
tidation with either SrtB on protein substrates modified with the
appropriate recognition sequences on a time scale or with yields
that compare favorably with SrtAstaph (data not shown).
We ultimately arrived at a successful orthogonal strategy using
SrtAstrep, which recognizes the same LPXTG sequence used by
SrtAstaph but can accept alanine-based nucleophiles.8 This leads to
the formation of an LPXTA sequence at the site of ligation, a motif
refractory to cleavage by SrtAstaph.9 This allows SrtAstaph to act on
the N terminus without affecting the C-terminal modification
10
µM ∆59-SrtAstaph for ∼1 h at 37 °C. ESI-MS of crude reaction
mixtures showed the dual-labeled material as the major component,
with only minor amounts of byproduct (Figure S6). A final
separation by anion-exchange chromatography yielded dual-labeled
eGFP and UCHL3 with excellent purity, as determined by both
SDS-PAGE and ESI-MS (Figure 2c,d and Figure S6). In the case
of UCHL3, we observed some additional low-intensity bands in
the fluorescent gel scan (Figure 2d). However, quantitative densi-
tometric analysis of coomassie-stained gels indicated purity in
excess of 95% for both dual-labeled eGFP and UCHL3.
In summary, we have developed a strategy for placing different
chemical labels at the two ends of the same polypeptide using two
sortase enzymes with unique reactivities. We anticipate that this
method will be applicable to the preparation of protein conjugates
for refolding studies or for the construction of protein sensors, where
measuring conformational changes by FRET is a common mode
of detection. In more general terms, this work begins to explore
the range of protein modifications that can be accessed using
alternative sortases. The number of sortases that have been produced
in recombinant form with retention of activity is continually
increasing, and we are exploring the use of these unique enzymes
as tools for protein engineering.
installed with SrtAstrep
.
Acknowledgment. This work was supported by grants from
the National Institutes of Health (R01-AI057182, R01-AI033456,
R21-EB008875). J.M.A. and N.C.Y. acknowledge Clay Postdoctoral
Fellowships. C.P.G. thanks the Fundacao para a Ciencia e Tecno-
logia (Portugal). G.-L.C. was supported by MIT’s Undergraduate
Research Opportunities Program (UROP). The authors thank Mark
J. Banfield and colleagues for providing the expression plasmid
for SrtAstrep and Olaf Schneewind for providing the SrtB constructs.
Supporting Information Available: Full experimental details and
characterization data for peptide derivatives and protein conjugates.
This material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) Tsukiji, S.; Nagamune, T. ChemBioChem 2009, 10, 787–798.
(2) Marraffini, L. A.; Dedent, A. C.; Schneewind, O. Microbiol. Mol. Biol.
ReV. 2006, 70, 192–221.
Figure 2. Site-specific N- and C-terminal labeling using multiple sortases.
(a) Tetramethylrhodamine-labeled dialanine nucleophile (3) for SrtAstrep
mediated transpeptidation. (b) Strategy for the installation of discrete labels
at both termini of the same protein using ∆59-SrtAstaph and SrtAstrep. (c, d)
SDS-PAGE characterization with fluorescent gel scanning of dual-labeled
(c) eGFP and (d) UCHL3.
-
(3) Pallen, M. J.; Lam, A. C.; Antonio, M.; Dunbar, K. Trends Microbiol. 2001,
9, 97–102.
(4) Yamamoto, T.; Nagamune, T. Chem. Commun. 2009, 1022–1024.
(5) Popp, M. W.; Antos, J. M.; Grotenbreg, G. M.; Spooner, E.; Ploegh, H. L.
Nat. Chem. Biol. 2007, 3, 707–708.
(6) Maresso, A. W.; Chapa, T. J.; Schneewind, O. J. Bacteriol. 2006, 188,
8145–8152.
(7) Mazmanian, S. K.; Ton-That, H.; Su, K.; Schneewind, O. Proc. Natl. Acad.
Sci. U.S.A. 2002, 99, 2293–2298.
(8) Race, P. R.; Bentley, M. L.; Melvin, J. A.; Crow, A.; Hughes, R. K.; Smith,
W. D.; Sessions, R. B.; Kehoe, M. A.; McCafferty, D. G.; Banfield, M. J.
J. Biol. Chem. 2009, 284, 6924–6933.
(9) Kruger, R. G.; Otvos, B.; Frankel, B. A.; Bentley, M.; Dostal, P.;
McCafferty, D. G. Biochemistry 2004, 43, 1541–1551.
(10) ∆59-SrtAstaph is a truncated form of SrtAstaph that has identical reactivity.
Our final strategy for dual-terminus labeling is outlined in Figure
2b. We first synthesized a tetramethylrhodamine-labeled peptide
(3) containing two N-terminal alanine residues to serve as the
nucleophile for SrtAstrep-mediated protein ligation (Figure 2a and
Figure S5). We prepared two model substrates (eGFP and UCHL3)
containing masked N-terminal glycines that are exposed only upon
thrombin cleavage. Masking was required because SrtAstrep was
JA902681K
9
J. AM. CHEM. SOC. VOL. 131, NO. 31, 2009 10801