Efficient N-terminal labeling of proteins by use of sortase

Article abstract of DOI:10.1002/anie.201204538

"Sorting out" N-terminal labeling: The reversibility of transpeptidase reactions makes protein N-terminal labeling challenging. Depsipeptide substrates for sortase A release alcohol by-products, which are poor nucleophiles for the reverse reaction, during ligation. Proteins with an unhindered N-terminal glycine residue can be labeled efficiently with only a minimal excess of the labeling reagent (see scheme).

Full text of DOI:10.1002/anie.201204538

Angewandte
Chemie
DOI: 10.1002/anie.201204538
Protein Modification
Efficient N-Terminal Labeling of Proteins by Use of Sortase**
Daniel J. Williamson, Martin A. Fascione, Michael E. Webb,* and W. Bruce Turnbull*
Protein labeling is a pivotal technique in molecular and cell
biology. Strategies include derivatization of cysteine resi-
dues,[1] labeling lysine or N-terminal amino groups with
activated esters, periodate or PLP-mediated oxidation of the
N-terminus for oxime ligation,[2] and native-chemical liga-
tion.[3] Each of these methods has its own associated
challenges: Selective labeling of a single cysteine residue
frequently requires rounds of site-directed mutagenesis to
introduce the labeling site and/or remove other cysteine
residues, and selective labeling of the N-terminal amino group
requires careful control of pH to ensure lysine residues are
not also modified.[4] Other modern methods for chemo-
selective labeling often require the introduction of specific
recognition sequences[5] or nonnatural amino acids into the
protein to be labeled.[6] In most cases, a substantial excess of
the labeling reagent is necessary to ensure complete con-
version to the product. We report a method for chemo-
selective N-terminal labeling of recombinant proteins in
quantitative yield using depsipeptide substrates for the trans-
peptidase sortase A. The method does not require engineer-
ing of the protein sequence beyond that typically used in
contemporary recombinant protein purification strategies.
Unlike previous approaches,[7] the method requires only
a single N-terminal glycine residue in a sterically unhindered
position, a minimal excess of the labeling reagent and
substoichiometric quantities of transpeptidase.
certain constraints: the LPXTG sequence must be engineered
into the protein and excess nucleophilic labeling reagent is
required to push the equilibrium toward formation of product
as the transpeptidase reaction is reversible (Scheme 1a).
Scheme 1. Sortase A ligation employing a) peptide and b) depsipeptide
substrates.
Sortase A (SrtA) catalyzes the reversible attachment of
virulence factors to the cell walls of Gram positive bacteria by
C-terminal modification of proteins at an LPXTG recognition
sequence.[8] The enzyme catalyzes the covalent attachment of
the LPXT motif to a cysteine residue in the catalytic site to
form a thioester intermediate. An N-terminal oligoglycine
motif in the peptidoglycan can then react with this inter-
mediate to covalently attach the substrate to the cell wall.
SrtA has been exploited extensively for the C-terminal
modification of proteins.[7b,9] However, this method has
N-terminal labeling of proteins using SrtA has the
potential advantage that only a single N-terminal glycine is
required on the protein.[10] Since many commercial expression
plasmids incorporate protease recognition sequences which
yield an N-terminal glycine after cleavage, such an approach
is potentially widely applicable. However, reversibility of the
SrtA-catalyzed reaction is a greater problem for N-terminal
labeling of proteins.
We chose to investigate whether the reaction could be
made irreversible by using depsipeptide substrates (Sche-
me 1b). We anticipated that the hydroxyacetyl byproduct
would not be a substrate for the reverse reaction, thus
rendering the labeling reaction irreversible. A similar strategy
has been applied previously to subtiligase,[11] but overall yields
were dependent on specific recognition sequences in both
peptides and an excess of depsipeptide was necessary to drive
the reaction to completion. Ploegh and co-workers have
reported the use of a methyl ester substrate with SrtA;[7a]
however, their method employed stoichiometric quantities of
SrtA and excess methyl ester.
[*] D. J. Williamson, Dr. M. A. Fascione, Dr. M. E. Webb,
Dr. W. B. Turnbull
School of Chemistry and Astbury Centre for Structural Molecular
Biology, University of Leeds, Leeds LS2 9JT (UK)
E-mail: m.e.webb@leeds.ac.uk
w.b.turnbull@leeds.ac.uk
Homepage: http://www.chem.leeds.ac.uk/MEW/
[**] We thank Dr. C. Neylon for providing an expression construct for
sortase, Prof. K. Drickamer for providing a plasmid for expressing
the ManBP variant, and Dr. T. Edwards and Dr. H. Jenkins for
a sample of the pumilio protein. This work was supported by an
EPSRC studentship for D.J.W. and also research grants EP/
G043302/1, EP/I013083/1 and BB/G004145/1. W.B.T. thanks the
Royal Society for a University Research Fellowship.
We first chose to establish an assay to evaluate the
efficiency of the SrtA reaction with various peptide sub-
strates. Three classes of labeling substrate 1, 2 and 3 were
synthesized (Figure 1a); these peptides share a common
YALPET sequence followed by a single glycine or glycine-
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201204538.
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Scheme 2. Synthetic strategy for preparation of depsipeptides for use
in SrtA-mediated ligation.
SrtA-mediated ligation of each depsipeptide with
GGSEFG peptide
(Figure 2). When one equivalent of either depsipeptide 8 or
4 was followed by using HPLC
Figure 1. a) Model reactions to investigate ligation efficiency for a vari-
ety of peptide substrates. Peptide 1 was not turned over by SrtA.
b) Time-course of SrtA-mediated ligation of 3 and 4 to generate 5.
Increasing the ratio of the 3 to 4 is sufficient to drive the reaction to
~
*
^
completion.
, , and denote the ratio of 3 to 4 as indicated. The
reaction was monitored by HPLC using 11 mm SrtA to catalyze the
reaction of 500 mm 3 with the corresponding ratio of the nucleophilic
acyl acceptor 4 in 50 mm HEPES, 5 mm CaCl2 150 mm NaCl pH 7.5.
amide, or two glycines, respectively. All three peptides were
tested as substrates for ligation to a short peptide with the
sequence GGSEFG 4 using SrtA in aqueous buffer (Fig-
ure 1a). HPLC analysis using an authentic sample of product
5 as a standard showed that only peptides 2 and 3 act as
substrates for SrtA, as indicated previously.[12] Neither
reaction achieved complete conversion to product 5 as this
peptide is itself a substrate for SrtA and can thus react with
glycine amide 6 or diglycine 7 to reform the starting peptides.
The reaction mixture therefore comes to equilibrium after
50% conversion (Figure 1b). Increasing the ratio of the
“nucleophilic” peptide 4 to the “electrophilic” acyl donor
peptides 2 and 3 leads to an increase in conversion but the
system still goes to equilibrium (Figure 1b and Supporting
Information).
Figure 2. Time-course of SrtA-mediated ligation of ligation of 3, 9 and
13 with 4 at a 1:1 ratio at 378C. 500 mm 4 and 500 mm 3, 9 or 13 with
11 mm SrtA in 50 mm HEPES, 5 mm CaCl2 150 mm NaCl pH 7.5.
Depsipeptides 8 and 9 were synthesized by Fmoc solid
phase peptide synthesis (SPPS) using Fmoc-protected TG
depsipeptide 12 (Scheme 2). First, alkylation of Fmoc tert-
butyl-protected threonine 10 with benzyl bromoacetate in the
presence of tetrabutylammonium iodide yielded the tri-
protected depsipeptide 11. Hydrogenolysis of the benzyl
group gave carboxylic acid 12 which was activated using
HCTU for attachment to NovaGel Rink amide resin or
glycine-loaded 2-chlorotrityl resin. Standard Fmoc-SPPS
protocols were used to extend these depsipeptides to provide
compounds 8 and 9 in 87% and 67% yield, respectively.
9 was used, 4 was almost quantitatively transformed to the
ligation product 5 (Figure 2 and Supporting Information).
The presence of 2 mol% SrtA in the reaction mixture
prevents the reaction going to completion as the equilibrium
between the product and thioester intermediate remains
significant. In contrast, the corresponding methyl ester 13
reacted slowly under these conditions, leading to only 30%
product formation after 8 h. When the amount of depsipep-
tide 9 was increased to 1.5 equiv, transformation of 4 to 5
became rapid and quantitative (see Supporting Information).
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As depsipeptides proved effective for labeling model
peptides, we sought to apply the method to labeling proteins.
SrtA-mediated ligations are typically carried out using
peptides containing an N-terminal oligoglycine motif; how-
ever, this is not frequently observed in proteins. A single N-
terminal glycine is, however, commonly produced in recombi-
nant proteins after cleavage with proteases such as thrombin
or tobacco etch virus (TEV) protease. Depsipeptide 14
containing a dansyl lysine residue was synthesized using the
same methodology as for 8 and 9. Initial labeling experiments
were performed using a variant of the human mannose
binding protein (ManBP).[13] This trimeric protein has a single
N-terminal glycine at the end of a three-stranded a-helical
bundle. The substrate protein was labeled quantitatively
within 4 h when incubated with 1.5 equiv of the depsipeptide
labeling reagent per protomer (Figure 3). The degree of
labeling was confirmed by electrospray mass spectrometry
(ESMS) of the reaction mixture, which indicated complete
conversion to the labeled protein (Figure 3c). A small
quantity of acylated SrtA can also be observed in the SDS-
PAGE gels of the crude reaction mixtures, corresponding to
10 mol% SrtA present in the reaction mixture. SrtA can be
easily removed by affinity purification to yield a pure reaction
product. ManBP was also labeled successfully with a fluores-
cein-modified depsipeptide (see Supporting Information).
When working at low protein concentrations, prolonged
incubation with SrtA can lead to hydrolysis of the label;[10]
however, if necessary, increasing the quantity of label to 2–
3 equiv can still allow complete conversion to the product.
To confirm the generality of our depsipeptide method we
also labeled a sample of the mouse pumilio-2 Puf RNA-
binding domain[14] in which an N-terminal GT sequence had
been produced by TEV protease cleavage. The protein was
quantitatively labeled using 1.5 equivalents of labeling
reagent 14 demonstrating the generality of this approach.
We also investigated the labeling of both myoglobin and the
fly pumilio RNA-binding domain. In both cases protein
labeling was unsuccessful; however, short peptides corre-
sponding to the N-terminal sequences of myoglobin, mouse
pumilio and fly pumilio could be successfully modified (see
Supporting Information). All three peptides displayed very
similar reaction kinetics that were only slightly slower than
those displayed for diglycyl peptide 4. We attribute the
differences in protein reactivity to variation in steric bulk in
the vicinity of the labeled glycine. In the case of fly pumilio,
the glycine is only one residue removed from the globular
domain of the protein with the sequence GS (c.f. GTG for the
mouse paralog). This suggests that a minimum length of
flexible peptide is required in order to ensure that labeling
can occur.
Figure 3. Use of 14 in covalent modification of proteins. a) Labeling of
mannose-binding protein with 14. Incubation of ManBP with 0.1 equiv
SrtA and 1.5 equivalents of 14 led to quantitative labeling in 4 h: lane
1, SrtA; lane 2, 60 mm ManBP; lane 3, 60 mm ManBP + 90 mm 14 +
6 mm SrtA; lane 4, fluorescence image of lane 3. b) The same protocol
led to quantitative labeling of mouse pumilio-2 Puf domain: lane 1,
20 mm pumilio + 30 mm 14 + 2 mm SrtA; lane 2, fluorescence image of
lane 1. c) MS analysis of ManBP before (black) and after (gray)
modification demonstrates quantitative labeling of protein.
concentrations used in this study;[10,15] the relative rate of
reaction is therefore determined by the specificity constant,
kcat/Km. We suggest that this factor accounts for the success of
the depsipeptides under the conditions used in this study. The
rate of acylation by an ester substrate will be greater than that
for an amide, thus increasing kcat for both the depsipeptide
and the methyl ester. However, while the affinity of the
depsipeptide should be similar to that of a peptide substrate,
the methyl ester will presumably bind less tightly. Overall the
relative rate of reaction (as determined by kcat/Km) for the
methyl ester should be less than that for the product, and
product inhibition therefore leads to the observed low overall
The depsipeptide substrates allow rapid labeling of both
peptides and proteins. In conventional SrtA-mediated liga-
tions, the rate-determining step is the initial attack of the
enzyme to form an acyl–enzyme intermediate;[10] this is then
attacked by the nucleophile to form a product that is itself
a substrate for SrtA. The efficiency of any given substrate is
therefore controlled by the rate of its turnover relative to that
for the product. The Michaelis constants for peptide acyl
donors are typically in the same range as the substrate
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In conclusion, we have described the synthesis and
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Experimental Section
Full experimental details are in the Supporting Information.
Protocol for protein labeling. All stock solutions were prepared in
50 mm HEPES, 5 mm CaCl2, 150 mm NaCl pH 7.5 at the following
concentrations: acyl donor 14 2 mm, ManBP 120 mm, Sortase A
304 mm. In a 100 mL assay at a ratio of 1.5:1, ManBP (50 mL, 60 mm), 14
(4.5 mL, 90 mm) and Sortase A (2 mL, 6 mm) were added to HEPES
buffer (43.5 mL) and mixed well. The solution was incubated at 378C.
Reaction progress was monitored by SDS-PAGE and ESMS analysis.
Received: June 11, 2012
Published online: && &&, &&&&
Keywords: depsipeptides · enzymes · peptides · sortase ·
.
transpeptidasen
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Angew. Chem. Int. Ed. 2012, 51, 1 – 5
These are not the final page numbers!

Angewandte
Chemie
Communications
Protein Modification
“Sorting out” N-terminal labeling: The
reversibility of transpeptidase reactions
makes protein N-terminal labeling chal-
lenging. Depsipeptide substrates for sor-
tase A release alcohol by-products during
ligation which are poor nucleophiles for
the reverse reaction. Proteins with an
unhindered N-terminal glycine residue
can be labeled efficiently with only a min-
imal excess of the labeling reagent (see
scheme).
D. J. Williamson, M. A. Fascione,
M. E. Webb,*
W. B. Turnbull*
&&&& — &&&&
Efficient N-Terminal Labeling of Proteins
by Use of Sortase
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!