Traceless and site-specific ubiquitination of recombinant proteins

Article abstract of DOI:10.1021/ja202799r

Protein ubiquitination is a post-translational modification that regulates almost all aspects of eukaryotic biology. Here we discover the first routes for the efficient site-specific incorporation of δ-thiol-l-lysine (7) and δ-hydroxy-l-lysine (8) into re

Full text of DOI:10.1021/ja202799r

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pubs.acs.org/JACS
Traceless and Site-Specific Ubiquitination of Recombinant Proteins
Satpal Virdee, Prashant B. Kapadnis, Thomas Elliott, Kathrin Lang, Julia Madrzak, Duy P. Nguyen,
Lutz Riechmann, and Jason W. Chin*
Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, U.K.
S Supporting Information
b
ABSTRACT: Protein ubiquitination is a post-translational
modification that regulates almost all aspects of eukaryotic
biology. Here we discover the first routes for the efficient
site-specific incorporation of δ-thiol-L-lysine (7) and
δ-hydroxy-L-lysine (8) into recombinant proteins, via evo-
lution of a pyrrolysyl-tRNA synthetase/tRNACUA pair. We
combine the genetically directed incorporation of 7 with
native chemical ligation and desulfurization to yield an
entirely native isopeptide bond between substrate proteins
and ubiquitin. We exemplify this approach by demonstrat-
ing the synthesis of a ubiquitin dimer and the first synthesis
of ubiquitinated SUMO.
ost-translational modification of target proteins with the 76
amino acid protein ubiquitin regulates almost all aspects of
P
eukaryotic biology.1,2 Ubiquitination is the process by which the
ε-amino group of a lysine residue within the substrate protein is
linked to the C-terminal carboxylate of ubiquitin via an isopep-
tide bond. In vivo, ubiquitin is attached to its substrates by a series
of enzymes (E1s, E2s, E3s) that direct isopeptide bond forma-
tion. Studying the molecular consequences of protein ubiquiti-
nation is challenging, since there are >600 E3 ubiquitin ligases
believed to be responsible for substrate recognition,3 and the E3
ligase for specific substrates are often unknown. Moreover, even
when the ligases are known, they may not drive the reaction to
completion or at a unique site in vitro.
Figure 1. (A) 1, pyrrolysine; 2, Nε-(t-butyloxycarbonyl)-L-lysine; 3,
photocleavable auxiliary-bearing amino acid allowing native chemical
ligation (NCL) with ubiquitin 1ꢀ75 thioester; 4, Nε-protected γ-thiol-
L-lysine (R3 = carbobenzyloxy or 3,4-dimethoxy-o-nitrocarbobenzyloxy);
5, δ-thiol-Nε-allyloxycarbonyl)-L-lysine; 6, thiazolidine-protected δ-thiol
L-lysine; 7, δ-thiol-L-lysine; 8, δ-hydroxy-L-lysine; 9, δ-hydroxy-Nε-
(t-butyloxycarbonyl) lysine, 10 δ-thiol-Nε-(t-butyloxycarbonyl) lysine;
11, δ-methyldisulfanyl-Nε-(t-butyloxycarbonyl) lysine; 12, Nε-(p-nitro-
carbobenzyloxy)lysine; 13, δ-hydroxy-Nε-(p-nitrocarbobenzyloxy)lysine;
and 14, δ-thiol-Nε-(p-nitrocarbobenzyloxy)lysine. (B) Genetically direct-
ing traceless ubiquitination; i) shows a thioester of full-length ubiquitin.
While the amino acids added to cells do not have defined stereochemistry at
the R-carbon, the protein translation machinery only uses R-L-amino acids
and the chirality at the δ-carbon is destroyed upon desulfurization to
produce the final product.
Several investigators have addressed the creation of ubiquitin
conjugates that are connected via non-native linkages, including a
disulfide bond,4,5 an oxime,6 triazoles,7 and isopeptide bonds in
which the universally conserved C-terminal glycine of ubiquitin is
mutated to D-cysteine8 or alanine9 in a non-traceless native
chemical ligation. While some of these non-native linkages have
found utility,4,5,9 a clear and important challenge is to address the
creation of methods for the ubiquitination of any protein at a
user-defined site via a native isopeptide bond.
We recently described a new approach, in protein chemistry
termed GOPAL, for creating native isopeptide bonds between
ubiquitin and a specific lysine in a target protein.10 Using the
M. barkeri (Mb) pyrrolysyl-tRNA synthetase (PylRS)/tRNACUA
pair, which naturally introduces pyrrolysine (1, Figure 1) into
proteins in certain methanogens, we site-specifically inserted
Nε-(t-butyloxycarbonyl)-L-lysine (2) into ubiquitin and devel-
oped a series of selective protection and deprotection steps that
allowed us to direct site-selective isopeptide bond formation.
Using this approach, we were able to generate important
ubiquitin dimers linked through specific isopeptide bonds, solve
the crystal structure of Lys6-linked diubiquitin, and reveal new
deubiquitinase specificity. Since this method relies on cellular
protein synthesis to generate the component proteins, it is in
principle scalable for the creation of traceless isopeptide bonds
between proteins of any size. However, this method does require
multiple protection and deprotection steps and generates highly
protected hydrophobic intermediates that can be poorly soluble.
Moreover, the method may be challenging to apply to proteins
that cannot be refolded.
Received: March 28, 2011
Published: June 28, 2011
r
2011 American Chemical Society
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1,2-Aminothiols can react reversibly with thioesters to form a
new thioester-linked intermediate. Intramolecular nucleophilic
attack on the thioester intermediate, in a favorable S-to-N acyl
shift, leads to formation of an amide bond. This reaction has been
used to assemble polypeptides from fragments bearing C-term-
inal thioesters and fragments bearing N-terminal cysteines and
forms a basis for native chemical ligation.11 Recently, four distinct
lysine derivatives (3ꢀ6), bearing 1,2-aminothiols, which can be
incorporated into synthetic peptides via solid-phase peptide
synthesis methods have been described.12ꢀ18 3 can be ligated
with C-terminal thioesters of ubiquitin, and subsequent auxiliary
removal allows the generation of a native isopeptide bond.12,13
The deprotection of 4ꢀ6 allows their ligation with C-terminal
thioesters, and subsequent desulfurization yields native isopep-
tide bonds.14ꢀ17 Unfortunately, while these amino acids can be
incorporated into longer peptides via rounds of native chemical
ligation with thioesters, these steps require further thiol protec-
tion, decrease the yield of protein conjugates, and ultimately limit
the length of proteins and/or the positions within proteins to
which these approaches might be applied.
Here we demonstrate the site-specific incorporation of
δ-thiol-L-lysine (7), which has recently been used in peptide
ligation at several sites,17,18 into proteins that are overexpressed
using the cell’s translational machinery (Figure 1). Native
chemical ligation of proteins containing 7 with a ubiquitin
thioester and subsequent desulfurization yields ubiquitin con-
jugates linked via a native isopeptide bond. This work provides a
simple, scalable, and broadly accessible route to ubiquitinated
proteins. In the process of this work, we also incorporated another
important post-translational modification, δ-hydroxy-L-lysine (8).
Since 7 differs from lysine only by the insertion of a sulfur
atom, it may be thermodynamically challenging to create a
synthetase that will recognize 7 but exclude lysine by a factor
of 103ꢀ104, as required to maintain the fidelity of natural protein
translation. t-Butyloxycarbonyl (Boc)-protected lysine (2) is a
good substrate for PylRS,19,20 and we have previously demon-
strated that, while the PylRS/tRNACUA pair does not direct the
incorporation of Nε-methyl-L-lysine, it does direct the incorpora-
tion of an Nε-methyl derivative of lysine bearing an Nε-Boc
group.20 Since the Boc group can be removed after incorporation
of the amino acid into the protein, this provides a paradigm for
installing modifications on the ε-amino group that cannot be
installed directly. Therefore, we investigated whether the addi-
tion of an Nε-Boc group will also facilitate the incorporation of
δ-substituted lysine derivatives (9, 10).
that confer chloramphenicol resistance on cells bearing a chloram-
phenicol acetyl-transferase gene with an amber codon at a permissive
site (D112TAG) in the presence of δ-hydroxy-Nε-(t-butyloxycar-
bony)lysine (9). We performed the initial selections in the presence
of 9 since it is valence isoelectronic with its thiol analogue (10) but
can be prepared in gram quantities in a single step from commercial
starting materials (SI Scheme 1), and since we were concerned that
a fraction of the δ-thiol compound might undergo oxidation that
could potentially lead to the selection of synthetases that recognize
oxidized forms of the amino acid. These selections yielded a single
mutant, Y349W, in PylRS. Subsequent selections using the δ-thiol
compound 10 directly yielded the same mutation. Selections using a
number of more complex libraries did not yield alternative or
improved mutants, nor did any of the libraries tested allow the
incorporation of the disulfide-protected compound 11 (data not
shown).
The selected synthetase (δSHKRS)/tRNACUA pair conferred
chloramphenicol resistance on cells containing a chlorampheni-
col acetyltransferase gene with an amber codon at position 112 of
200 μg mLꢀ1 in the presence of 10 and <50 μg mLꢀ1 in the
absence of 10. We produced C-terminally His-tagged ubiquitin
with an amber codon at position 6 from UbTAG6-His6 in the
presence of δSHKRS/tRNACUA and 9 or 10, in reasonable yield
(0.5 mg Lꢀ1 (SI Figure 2)). No protein was produced in the
absence of the unnatural amino acid. The incorporation of each
amino acid was conclusively demonstrated by ESI-MS analysis
(SI Figure 3), and ubiquitin bearing the δ-thiol-L-lysine (7) at
position 6 was prepared by the quantitative removal of the Boc
group from ubiquitin bearing δ-thiol-Nε-(t-butyloxycarbony)-L-
lysine at position 6 by the addition of 60% TFA for 1 h at 22 °C
and characterized by mass spectrometry (SI Figure 3).
Taken together, the phenotypic experiments, protein expres-
sion experiments, and mass spectrometry data conclusively
demonstrate that δSHKRS/tRNACUA, in the presence of 9 or
10, directs the incorporation of δ-hydroxy-Nε-(t-butyloxycar-
bonyl)-L-lysine or δ-thiol-Nε-(t-butyloxycarbonyl)-L-lysine into
recombinant proteins in response to the amber codon and allows
the preparation of proteins containing a site-specifically incorpo-
rated δ-thiol-L-lysine (7). However, we were interested in
improving two aspects of this approach. First, the yield of
recombinant protein produced when using this synthetase was
∼10 times lower than that obtained with 2 and the PylRS/
tRNACUA pair, a combination that we and others have shown is
very efficient.10,19,20 Second, the deprotection conditions are
denaturing, making this approach to installing 7 incompatible
with proteins that cannot be reversibly refolded. To improve the
method, we combined our progress up to this point with some
observations we had made while investigating the evolvability of
the PylRS/tRNACUA pair. This allowed us to very efficiently
install δ-substituted derivatives of lysine (7 and 8) into proteins
under native conditions.
In the process of investigating the scope of amino acids that
can be incorporated using the PylRS/tRNACUA pair, we dis-
covered a variant synthetase (nitroCbzKRS) that incorporates
Nε-(p-nitrocarbobenzyloxy)-L-lysine (12). This synthetase was
selected by five rounds of positive and negative selection,22 in the
presence and absence of 12, on a 109-member synthetase library,
which contains all combinations of mutations at residues M241,
A267, Y271, L274, and C313. The evolved synthetase contains
the mutations Y271M, L274G, and C313A. The selected ni-
troCbzKRS/tRNACUA pair conferred chloramphenicol resis-
tance on cells containing a chloramphenicol acetyltransferase
We first synthesized Nε-Boc-protected versions of amino
acids bearing δ-substituents (9ꢀ11) (Supporting Information
(SI) Schemes 1 and 2 and Methods). We demonstrated that
none of these amino acids are incorporated into proteins in
response to the amber codon using the wild-type PylRS/tRNACUA
pair. Next, we aimed to discover an evolved PylRS/tRNACUA pair
for the incorporation of amino acids 9ꢀ11. The crystal structure of
PylRS in complex with pyrrolysine21 reveals two prominent residues
(N311 and Y349) that are within 5 Å of the δ-carbon of pyrrolysine
(SI Figure 1). N311 in PylRS binds to the carbonyl group in the
bound pyrrolysine, and mutation of this amino acid destroys the
ability of the enzyme to discriminate this substrate from natural
amino acids (data not shown). Since this carbonyl group is
conserved in our designed substrates (9ꢀ14), we decided to
maintain N311 as a potential positive specificity determinant.
We created a library in which Y349 of MbPylRS is mutated to
all natural amino acids and selected MbPylRS/tRNACUA variants
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Figure 2. Incorporation of 7 into recombinant proteins. (A) SDS-PAGE
reveals that incorporation into position 6 of ubiquitin by the ni-
troCbzKRS*/tRNACUA is dependent on amino acid 14. (B) Deconvo-
luted mass spectrum ofubiquitin-containingpyruvate-derivedthiazolidine
adducts is shown in blue. Thiazolidine adduct expected mass = 9490 Da,
found = 9490 Da; decarboxylated thiazolidine adduct expected mass =
9446 Da, found 9446 Da; unmodified expected mass = 9420 Da, found =
9420 Da. The spectrum of protein treated with 200 mM methoxyamine
for 24 h is shown in green. Expected mass = 9420 Da, found = 9420 Da.
Figure 3. Genetically encoded 7 directs site-specific traceless isopeptide
bond formation via native chemical ligation and desulfurization. (A)
SDS-PAGE analysis of ligation. UbSR is ubiquitin thioester. UbSHK6 is
ubiquitin His6 with 7 at position 6. Ub2SH is the ligation product. Time
zero is quenched within minutes of mixing. (B) Deconvoluted MS
spectrum and SDS-PAGE of K6-linked diubiquitin resulting from
desulfurization DiUbSHK6-His6 and purification. Full spectra are pre-
sented in SI Figure 9. Detailed experimental procedures for the ligation
and purification are in the SI Methods.
gene with an amber codon at position 112 of >300 μg mLꢀ1 in
the presence of 12 and <50 μg mLꢀ1 in the absence of 12.
Cells containing the nitroCbzKRS/tRNACUA pair directed
expression of UbTAG6-His6 in the presence 12 to produce good
yields of ubiquitin (10 mg Lꢀ1). Ubiquitin expression was clearly
amino acid dependent (SI Figure 4).
The amino acid dependence observed in the phenotypic and
protein expression experiments demonstrated that 12 is incor-
porated during protein translation and that there is little transla-
tional incorporation of natural amino acids in response to the
amber codon. However, mass spectrometry of the purified
protein revealed that the protein contained lysine in place of
12 (SI Figure 4). We therefore postulated that 12 was incorpo-
rated into the protein during cellular translation, but the p-
nitrocarbobenzyloxy group was subsequently removed from the
ε-amino group of lysine.
We next synthesized δ-substituted derivatives of Nε-
(p-nitrocarbobenzyloxy)lysine (13 and 14; SI Scheme 3 and
Methods) and aimed to site-specifically incorporate these into
proteins. The incorporation of these amino acids and subsequent
removal of p-nitrocarbobenzyloxy group, as described above,
should lead to a clear mass shift in the protein, corresponding to
the mass added by the δ-substituent, and provide a direct route to
the incorporation of δ-substituted lysine derivatives.
As expected, the nitroCbzKRS/tRNACUA pair did not direct
the efficient incorporation of the δ-substituted amino acids
(Figure 2). To discover a synthetase/tRNA pair that uses 13
and 14 to yield 8 and 7 in recombinant proteins, we combined
the mutation in δSHKRS that allows the incorporation of
δ-hydroxy-Nε-(t-butyloxycarbonyl)-L-lysine & δ-thiol-Nε-(t-butyl-
oxycarbonyl)-L-lysine with those in nitroCbzKRS for introducing
Nε-(p-nitrocarbobenzyloxy)-L-lysine to create a new synthetase
(nitroCbzKRS*).
(10 mg Lꢀ1) was comparable to that for the incorporation of 2
with the PylRS/tRNACUA pair.10 Mass spectrometry revealed a
mass of 9490 Da, corresponding to removal of the p-nitrocarbo-
benzyloxy group from the ε-amine of lysine and the formation of
a thiazolidine adduct between the resulting 1,2-aminothiol and
pyruvate23 (Figure 2, SI Figure 6). A second minor peak corres-
ponds to the decarboxylation of the thiazolidine adduct. These
adducts are well precedented for free 1,2-aminothiols resulting
from N-terminal cysteines. Treatment of the protein with
200 mM methoxyamine23 in phosphate-buffered saline (PBS)
led to quantitative removal of the pyruvate adducts to reveal
δ-thiol-L-lysine (7) at the genetically directed site in the protein,
as characterized by mass spectrometry. We have repeated this
experiment at several different, biologically relevant sites in
ubiquitin and in SUMO to demonstrate the generality of
this approach (SI Figures 7 and 11); it nonetheless remains a
possibility that some other proteins will behave differently.
While we do not yet know the exact mechanism by which the
nitro-substituted Cbz group is removed, a likely mechanism
would include the reduction of the aromatic nitro group, pre
or post cell lysis, and the subsequent fragmentation to reveal a
free ε-amino group (SI Figure 6).
To demonstrate the utility of this system for genetically
directing chemoselective protein ubiquitination, we synthesized
K6-linked diubiquitin—an important ubiquitin linkage that may
be involved in DNA repair-related signaling processes in mam-
malian cells.24,25 Ubiquitin bearing 7 at position 6 was ligated
with an equivalent of ubiquitin thioester, prepared by intein
fusion thiolysis.10 After 4 h, the yield of the ubiquitin conjugate
linked via an amide bond between δ-thiol lysine (7) at position 6
in one ubiquitin and the C-terminus of a second ubiquitin
(DiUbδSHK6-His6) was ∼70%, as judged by SDS-PAGE
(Figure 3). Ubiquitin species were folded by dialysis against
folding buffer (PBS þ 1 mM DTT), and the K6-linked diubi-
quitin conjugate was purified from residual monoubiquitin by ion
exchange chromatography (SI Figure 8). The purified ubiquitin
chain (DiUbδSHK6-His6) was a single band by SDS-PAGE, a
single peak by HPLC, and the expected mass, confirming the
formation of the amide bond (SI Figures 8 and 9). To reveal
the entirely native isopeptide bond, DiUbδSHK6-His6 was
Mass spectrometry of ubiquitin purified from cells containing
the nitroCbzKRS*/tRNACUA pair, 13 and a ubiquitin gene
bearing in an amber codon at position 6 demonstrates the
incorporation of 8 (Supplementary Figure 5). This is consistent
with the translational incorporation of δ-hydroxy-Nε-(t-butyloxy-
carbonyl)-L-lysine into ubiquitin and the subsequent removal of the
p-nitrocarbobenzyloxy group from the protein.
Finally, we incorporated δ-thiol-Nε-(p-nitrocarbobenzyloxy)-L-
lysine into ubiquitin at position 6 using the nitroCbzKRS*/
tRNACUA pair and 14. Again, protein expression was amino
acid-dependent (Figure 2). The yield of recombinant protein
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desulfurized via a free radical method.26 Desulfurization was
complete after 2 h, as determined by LC-MS, yielding the first
genetically directed native isopeptide linkage to ubiquitin
(DiUbK6-His6; Figure 3, SI Figure 9). Desulfurization reagents
were removed, and concomitant folding was achieved by dialysis
of the reaction mixture against 10 mM Tris, pH 7.6 buffer,
conditions that are known to yield folded ubiquitin chains.27 We
confirmed that the K6-linked diubiquitin we have synthesized is
folded by circular dichroism spectroscopy, using a folded K6-
linked diubiquitin of which we have previously solved the X-ray
structure10 as an authentic standard (SI Figure 10). To further
confirm the biological integrity of the ubiquitin dimers synthe-
sized by our method, we demonstrated that they are efficient
substrates for members of the ubiquitin-specific protease (USP)
family of deubiquitinases (USP2 and USP5) that we have
previously shown are able to readily hydrolyze K6-linked
diubiquitin10 (SI Figure 10). To further demonstrate the general-
ity of our approach, we incorporated δ-thiol-Nε-(p-nitrocarbo-
benzyloxy)-L-lysine into position 11 of recombinant SUMO and
prepared ubiquitinated SUMO, a modified protein implicated in
promyelocytic leukemia,28 by procedures analogous to those
used to prepare K6-linked ubiquitin (SI Figure 11).
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In conclusion, we have discovered the first route for the
efficient site-specific incorporation of δ-thiol-L-lysine (7) and
δ-hydroxy-L-lysine (8) into recombinant proteins. We have
combined the genetically directed incorporation of 7 with
native chemical ligation and desulfurization to yield an entirely
native isopeptide bond between substrate proteins and ubiquitin.
Moreover, we have discovered that the p-nitrocarbobenzyloxy
group can be used to site-specifically install lysine and its close
analogues into proteins, and this may facilitate the preparation of
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’ ASSOCIATED CONTENT
S
Supporting Information. Supplementary figures and ex-
b
perimental procedures. This material is available free of charge
via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
chin@mrc-lmb.cam.ac.uk
’ ACKNOWLEDGMENT
We are grateful to the ERC and MRC for funding.
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