Protein ubiquitination: Via dehydroalanine: Development and insights into the diastereoselective 1,4-addition step

Article abstract of DOI:10.1039/c6ob00882h

We report a strategy for site-specific protein ubiquitination using dehydroalanine (Dha) chemistry for the preparation of ubiquitin conjugates bearing a very close mimic of the native isopeptide bond. Our approach relies on the selective formation of Dha followed by conjugation with hexapeptide bearing a thiol handle derived from the C-terminal of ubiquitin. Subsequently, the resulting synthetic intermediate undergoes native chemical ligation with the complementary part of the ubiquitin polypeptide. It has been proposed that the Michael addition step could result in the formation of a diastereomeric mixture as a result of unselective protonation of the enolate intermediate. It has also been proposed that the chiral protein environment may influence such an addition step. In the protein context these questions remain open and no experimental evidence was provided as to how such a protein environment affects the diastereoselectivity of the addition step. As was previously proposed for the conjugation step on protein bearing Dha, the isopeptide bond formation step in our study resulted in the construction of two protein diastereomers. To assign the ratio of these diastereomers, trypsinization coupled with high-pressure liquid chromatography analysis were performed. Moreover, the obtained peptide diastereomers were compared with identical synthetic peptides having defined stereogenic centers, which enabled the determination of the configuration of the isopeptide mimic in each diastereomer. Our study, which offers a new method for isopeptide bond formation and protein ubiquitination, gives insights into the parameters that affect the stereoselectivity of the addition step to Dha for chemical protein modifications.

Full text of DOI:10.1039/c6ob00882h

Organic &
Biomolecular Chemistry
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PAPER
Protein ubiquitination via dehydroalanine:
development and insights into the
diastereoselective 1,4-addition step†
Cite this: Org. Biomol. Chem., 2016,
14, 4817
Roman Meledin, Sachitanand M. Mali, Sumeet K. Singh and Ashraf Brik*
We report a strategy for site-specific protein ubiquitination using dehydroalanine (Dha) chemistry for the
preparation of ubiquitin conjugates bearing a very close mimic of the native isopeptide bond. Our
approach relies on the selective formation of Dha followed by conjugation with hexapeptide bearing a
thiol handle derived from the C-terminal of ubiquitin. Subsequently, the resulting synthetic intermediate
undergoes native chemical ligation with the complementary part of the ubiquitin polypeptide. It has been
proposed that the Michael addition step could result in the formation of a diastereomeric mixture as a
result of unselective protonation of the enolate intermediate. It has also been proposed that the chiral
protein environment may influence such an addition step. In the protein context these questions remain
open and no experimental evidence was provided as to how such a protein environment affects the
diastereoselectivity of the addition step. As was previously proposed for the conjugation step on protein
bearing Dha, the isopeptide bond formation step in our study resulted in the construction of two protein
diastereomers. To assign the ratio of these diastereomers, trypsinization coupled with high-pressure liquid
chromatography analysis were performed. Moreover, the obtained peptide diastereomers were compared
with identical synthetic peptides having defined stereogenic centers, which enabled the determination of
the configuration of the isopeptide mimic in each diastereomer. Our study, which offers a new method
for isopeptide bond formation and protein ubiquitination, gives insights into the parameters that affect the
stereoselectivity of the addition step to Dha for chemical protein modifications.
Received 29th March 2016,
Accepted 25th April 2016
DOI: 10.1039/c6ob00882h
www.rsc.org/obc
In order to study ubiquitination and deubiquitination at
the molecular level one has to obtain highly pure and homo-
Introduction
Protein ubiquitination is highly important and ubiquitous geneous ubiquitin conjugates in workable quantities. Like
posttranslational modification (PTM) is utilized for controlling many other posttranslationally modified proteins, the prepa-
various cellular events.1 This process involves the enzymatic ration of such complex conjugates using the enzymatic
machinery consisting of E1, E2 and E3, which collaborate with machinery is a formidable task. Chemical approaches offer
each other to attach a ubiquitin monomer or polyubiquitin many advantages for the preparation of free ubiquitin chains
chain to a protein substrate.2 In the polyubiquitin chain, the and ubiquitinated proteins as evident by several recent
monomers are linked through isopeptide bonds, which are studies.6–11 The main privilege of the synthetic methods is the
formed between any of the seven-Lys side chains or the avoidance of using the E1–E3 enzymatic machinery and the
N-terminus in ubiquitin and the C-terminus of the subsequent opportunity to basically make any variation in these bioconju-
ubiquitin.3 This leads to a wide landscape of ubiquitin biocon- gates. These efforts include for example the preparation of ubi-
jugates, encompassing different ubiquitin chains of various quitinated proteins having ubiquitin chains with the desired
lengths and linkage types that can be linked to a substrate at length and linkage type.12,13 These conjugates can also be
different sites.4 In order to terminate/edit the signal, ubiquitin stabilized to prevent cleavage by deubiquitinases14–16 or modi-
or ubiquitin chains can be removed fully or partially by a fied by an electrophile to trap ubiquitin interacting
family of enzymes known as deubiquitinases (DUBs).5
partners.17–19 Despite these advantages, there are still several
limitations in the synthetic and semisynthetic approaches for
the preparation of ubiquitinated proteins of large size.
Site selective modification of expressed proteins is another
powerful approach to prepare protein conjugates including
those that bear PTMs.20,21 The obvious advantage here is the
ability to use protein substrates derived by recombinant
Schulich Faculty of Chemistry, Technion-Israel Institute of Technology, Haifa,
3200008 Israel. E-mail: abrik@technion.ac.il
†Electronic supplementary information (ESI) available: All experimental pro-
cedures, analytical data for synthetic small molecules, peptides, proteins and
enzymatic hydrolysis studies. See DOI: 10.1039/c6ob00882h
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technology or isolated from natural sources. The development ence of more than one cysteine residue in the protein as long
of approaches for the site selective chemical ubiquitination of as the rest are participating in disulfide bonds.32
expressed proteins is still in its infancy. Only a few methods
Inspired by the recent approaches to prepare posttransla-
are available to non-enzymatically ubiquitinate proteins using tionally modified proteins via Dha, we envisioned a strategy
unnatural linkages such as disulfide22–24 or applying click for site selective ubiquitination based on the incorporation of
chemistry on a protein having an unnatural alkyne or azide a Cys residue at the desired position of substrate 1, followed by
incorporated via expression of proteins using the amber codon selective thiol elimination by reagent 2 to afford the Dha con-
suppression technology.15,16,25 Notably, the latter technology taining protein 3 (Scheme 1). This Dha will then serve as the
was also used to incorporate thiolysine26 which has enabled electrophilic center for the conjugation with N-terminally thia-
the preparation of native ubiquitin chains.27 In addition, the zolidine (Thz) protected hexapeptide–thiol 4, derived from the
GOPAL based strategy28 which also relies on the amber codon C-terminal of ubiquitin. In this strategy, a single point mutation
suppression approach was also developed and used to prepare of Leu to Cys is introduced in ubiquitin to enable native chemi-
ubiquitin chains.29
step forward toward facilitating ubiquitination of will undergo Thz deprotection, followed by NCL with a comp-
expressed native proteins was made recently by our group lementary part of the ubiquitin(1–70)–thioester 7.
where we succeeded in modifying the isolated α-globin with Notably, the final ubiquitinated product 8 would contain a
cal ligation (NCL).40 Later, the resulting conjugated product 6
A
natively linked polyubiquitin chains of up to four monomers close mimic of the isopeptide bond where the γ-carbon of the
in length through the disulfide linkage.24 Since the disulfide Lys side chain is replaced by sulfur. Despite the two-step
bond is sensitive to the reducing environment these conjugates approach, such a conjugation of a Dha containing substrate
cannot be used for certain biochemical studies. To overcome with a small size peptide is advantageous compared with that
this limitation, we also attempted alternative strategies linking of a bigger molecular weight protein (such as ubiquitin–thiol),
the ubiquitin chains via thioether linkages, however this is still since it allows one to use a higher ratio of nucleophile/electro-
limited mainly to mono and di-ubiquitination.24 Moreover phile and hence to push the equilibrium of the reaction
despite these successes, the obtained conjugates bear unnatural towards product formation. Remarkably, the Strieter group
linkages between the chain and the substrate, which cannot be reported an elegant method based on the use of thiol–ene
hydrolyzed by DUBs. Here we report on the development of a chemistry to prepare a thiol containing an isopeptide bond
novel strategy for site selective protein ubiquitination based on having an extra bond relative to the native isopeptide bond.41
dehydroalanine (Dha) chemistry. Subsequently, we examined
As a starting point for developing this strategy we chose ubi-
the recognition of the isopeptide bond mimic in these conju- quitin as a substrate to prepare Lys48-linked di-ubiquitin. The
gates by DUBs. For the first time, we also provide experimental N-terminally Thz protected hexapeptide–thiol 4 was prepared
information about the stereoselective addition of a nucleophile with C-terminal N-acyl-benzimidazolinone (Nbz),42 followed by
on the Dha moiety in the protein context.
switching with cysteamine. The ubiquitin bearing Cys at posi-
tion 48 was subjected to the elimination reaction by using
reagent 2 to afford 9, followed by conjugation with 4 and
methoxylamine treatment to afford 10 in 57% isolated yield.
Ligation with ubiquitin (1–70)–thioester afforded di-ubiquitin
Results and discussion
Dha based chemistry is a promising strategy for the prepa-
ration of protein conjugates and proteins bearing PTM
mimics.30–32 Being the Michael acceptor for the 1,4-addition
reaction with various thiols, Dha has become a useful syn-
thetic precursor to obtain a variety of modified proteins. For
example, mimics of acetylated histone H3 were prepared and
shown to behave as substrates for histone deacetylases.30,32,33
One of the key aspect of this method is the ability of site selec-
tive incorporation of the Dha moiety into a desired protein.
During the last decade, several research groups reported
different strategies of Dha formation in such a way that it
would be applicable for peptides34–36 and proteins.33,37–39
Davis and co-workers developed a highly efficient approach for
site selective bis-alkylation–elimination of Cys to Dha.32 The
bisamide of 1,4-dibromobutane was found to be the most
efficient reagent for such a transformation. Importantly, this
strategy is compatible for reactions in aqueous buffers with
mild pH and temperature, making it very suitable for proteins.
In addition, Dha formation can be selective even in the pres- Scheme 1 General strategy of Dha based protein ubiquitination.
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Scheme 2 Synthesis of Lys48- and Lys29-linked di-ubiquitin analogues
via the Dha strategy, (K* = lysine analogue).
Fig. 1 HPLC and MS analyses: (A) peak a corresponds to ubiquitin–
Dha48, 9, with the observed mass 8485.9
0.5 Da, calcd 8487.8; (B)
11 in 39% isolated yield (Scheme 2 and Fig. 1). At this stage,
Cys71 was either kept intact to give 11, converted to Ala71 via
desulfurization reaction43 to give 12, or alkylated with iodoace-
tamide to generate 13 (Scheme 2).
To examine the influence of the changes on the isopeptide
bond as well as the mutations of Leu71, the obtained ana-
logues, 11, 12 and 13, were subjected to cleavage with USP2—a
DUB that is known to cleave all Lys-linked di-ubiquitin
chains.44 Interestingly, we observed similar cleavage patterns,
peak b corresponds to 10, which is the result of conjugation of hexapep-
tide–thiol 4 to 9, with the observed mass 9206.3 0.6 Da, calcd 9206.6
Da; (C) NCL of ubiquitin(1–70)-thioester 7 with 10, where peak c corres-
ponds to the ligated product 11 with the observed mass 17082.1 2 Da,
calcd 17082.6 Da, peak b corresponds to unreacted 10, peak d corres-
ponds to unreacted ubiquitin(1–70)-thioester 7 with the observed mass
8043.4 0.7 Da, calcd 8045.0 Da.
compared with the WT di-ubiquitin in which within 30 min all observed on a model system involving the addition of metha-
analogues were hydrolyzed to near completion to the mono- nethiol to proline–dehydroalanine dipeptides in ethanol,
ubiquitin (Fig. S17†). These results indicate that subtle which afforded the D- and L-isomers in a 85 : 15 ratio, respect-
changes in this site apparently do not harm USP2 activity ively.47 Darses and coworkers have achieved even higher
although we cannot exclude a more noticeable effect on other diastereoselectivity of up to 95% when conjugating potassium
DUBs. Nevertheless, one could overcome the Leu71Cys point trifluoroorganoborates to Dha derivatives, catalyzed by a chiral
mutation by introducing mercaptoleucine for NCL instead of rhodium catalyst, Binap as a chiral ligand and guaiacol as the
N-terminal Cys in 4 followed by selective desulfurization to proton source in toluene.48 In contrast, the group of van der
generate the native Leu at this site.45,46
Donk did not observe diastereoselectivity in the conjugation of
Despite the wide usage of Dha based chemistry for protein farnesylthiolate and thioglycosides to dehydropeptides in DMF
modifications, it has been proposed that the Michael addition or methanol.35 Despite this, the two diastereomers were separ-
step could result in the formation of a diastereomeric mixture able by reverse phase HPLC, which could be useful in the
as a result of unselective protonation of the enolate intermedi- preparation of conjugates with defined diastereomeric forms.
ate in 5 (Scheme 1).30,32,33 It has also been proposed that the Proteins, however, are much more challenging to study when
chiral protein environment may influence such an addition compared to these model peptides, yet they provide a unique
step.33 However, in the protein context this question remains chiral environment, which might affect the addition step.33
open and no experimental evidence was provided as to how Moreover, since proteins prefer aqueous media for solubility,
such a protein environment affects the diastereoselectivity of water is likely to act as a proton donor, which may limit the
the addition step. Asymmetric induction was previously stereoselectivity.49 How these parameters as well as the protein
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sequence, folding and the nearby residues may affect the Since we did not observe two separable peaks in the RP-HPLC
diastereoselectivity of the addition product remains unclear. analysis having the same mass which correspond to the two
To examine this we turned our attention towards the isomers, assuming that these diastereomers indeed formed,
characterization of the diastereomeric mixture formed as a we trypsinized the product(s) hoping that shorter peptides will
result of the conjugation of hexapeptide–thiol 4 to ubiquitin– likely separate in the HPLC. In the case of formation of two
Dha 9 at position 48 under folding and denaturing conditions. protein diastereomers, the trypsinization step will lead to the
two peptides of interest, each containing the isopeptide bond
mimic with L- or D-configuration in a ratio that likely reflects
the diastereoselectivity of the addition step.
Indeed, when the peptide mixture of the digestion was ana-
lyzed by RP-HPLC, two peptides with the same mass were
detected (Fig. 2C). These could correspond to the expected two
peptide diastereomers bearing also the Gly–Gly from the
C-terminal of ubiquitin linked to the peptide via the isopep-
tide mimic (Fig. 2A and Scheme 3). A similar procedure was
repeated for the conjugation step, which was carried out under
denaturing conditions followed by applying trypsin digestion
and HPLC analysis (Fig. 2B). Under both conditions, we
observed similar patterns in which the two peaks that corres-
ponded to the peptides with the same mass (1477 Da) were
obtained in a ratio of ∼60 : 40 based on the HPLC analysis.
Next, we aimed to confirm the structure of the two peptides
and assign the HPLC peaks to the specific diastereomer. To
achieve this, we prepared chemically two peptides bearing the
defined D- and L-forms of an isopeptide bond mimic
Fig. 2 (A) Sequence of full ubiquitin, highlighting the products of tryp-
sinization in D- and L-forms. Asterisk indicates Cys bearing an isopeptide
bond mimic with the GG from ubiquitin (shown in bold). HPLC and mass
analysis for: (B) trypsinization of the hexapeptide-conjugated product
under denaturing conditions with the observed mass 1477.8 0.2 Da,
calcd 1479.5 Da. (C) Trypsinization of the hexapeptide conjugated
product under folding conditions with the observed mass 1477.7 0.2
Da, calcd 1479.5. (D & E) Synthetically prepared peptides corresponded
Scheme 3 General scheme of trypsinization of conjugated products
(10a = hexapeptide–thiol conjugated to either ubiquitin48, ubiquitin29 or
α-globin). Synthesis of L- and D-Lys mimics from L or D-Cys, followed by
their incorporation into the expected peptide sequence for the compari-
son study with the actual peptide mixture obtained from trypsinization.
to the
D and L peptide diastereomers obtained from trypsinization,
respectively.
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(Scheme 3). Briefly, the syntheses started with methyl esterifi-
Next, we were motivated to investigate if the nearby residues
cation of L- or D-Cys(Trt)-OH (16, 20) followed by trityl group and the location in the same protein could have an influence
removal using 50% TFA in DCM. The obtained free thiol 17 on the diastereoselectivity of the conjugation step. Therefore,
was subjected to substitution with ethylamine bromide in the we chose to locate the Dha at position 29 in ubiquitin to
presence of DIEA to give the S-substituted ethylamine Cys. The prepare Lys29-linked di-ubiquitin. In this case, the conjugation
side chain amine of this analogue was further protected with products were separable and the two diastereomeric products,
alloc to give 18 and subsequent hydrolysis of the ester gave the under folding conditions, were obtained in an ∼60 : 40 ratio.
Lys mimic 19 or 21.
Interestingly, this ratio under denaturing conditions was
Having these analogues in hand, we incorporated each one reversed, highlighting the effect of folding on the stereo-
in the expected peptide sequence 14 or 15 from the digestion selectivity of the conjugation step (Fig. 3).
mixture. After purification, these peptides were injected into
Similar to the previous case with the ubiquitin–Dha at
RP-HPLC under similar conditions that we have used for ana- Lys48, we applied trypsinization on the whole conjugation
lyzing the trypsinization mixture. Comparison of the retention mixture as well as on each individual peak and for their
times of these peptides confirmed the identity of each diaster-
eomer, where the D-form was eluted first and found to be the
slightly preferred isomer in the conjugation reaction under
both conditions (Fig. 2).
Fig. 4 HPLC and mass analysis for (A) pure α-globin with the observed
Fig. 3 HPLC and mass analysis for (A) Ubiquitin–Dha29, 9 with the
observed mass 8485.9 0.6 Da, calcd 8487.8 Da. (B) and (C) Conju-
gation of hexapeptide–thiol 4 to ubiquitin–Dha29 9 under folding and
mass 15123.5
1.4 Da, (calcd 15125.3 Da). (B) α-globin–Dha with the
observed mass of 15090.5 1.6 Da, (calcd 15093 Da). (C) Conjugation of
hexapeptide–thiol to α-globin–Dha under denaturing conditions. Peak c
and d correspond to conjugation products with the observed mass
15811.9 2.0 Da and 15812.2 2.1 Da, (calcd 15813.1 Da). (D) Ligation
product of hexapeptide conjugated α-globin and ubiquitin (1–70)–thio-
ester with the observed mass of 23686.3 2.2 Da, (calcd 23689.1 Da).
denaturing conditions, respectively. Peak b and c correspond to the
conjugation products with the observed mass 9206.1
0.5 Da, calcd
9206.6. Peak a corresponds to unreacted ubiquitin–Dha29 9. Asterisk
corresponds to an unidentified compound.
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assignment we compared the retention times of the two pep- the exact behavior of the different isomers could be character-
tides having the same mass with their respective synthetic pep- ized. For example, although in our study the USP2 seems to
tides with the L- or D-isopeptide bond. Our analyses indicated cleave both isomers rapidly one could obtain more detailed
that the diastereomer that eluted first is the one that has the information about the enzymatic activity of the obtained con-
L-configuration while the second has the D-configuration of the jugates with different DUBs. These and other directions are
isopeptide mimic (Fig. S18†).
currently under investigation in our group.
Finally, we examined the stereoselectivity of conjugation to
α-globin–Dha as a different protein system with a single natu-
rally occurring Cys at position 104. Here, also we observed sep-
aration of the two diastereomer proteins in which the two
Acknowledgements
products were obtained in a ∼70 : 30 ratio under denaturing We thank the Israel Science Foundation for financial
conditions. Notably, the conjugation of hexapeptide–thiol, 4, support. A. Brik is a Neubauer Professor and a Taub Fellow-
to folded α-globin–Dha did not proceed at all under different Supported by the Taub Foundations. SMM is thankful to the
reaction conditions (Fig. 4), apparently due to this cysteine Schulich foundation for the postdoctoral fellowship.
being in a less exposed region. The trypsinization-based analy-
sis as described in the case of ubiquitin was performed and
the retention time of each peak was compared with synthetic
peptides having the D- and L-form of the isopeptide bond
Notes and references
mimic. Importantly, the obtained ratio of the D- and L-peptides
(∼70 : 30), derived from trypsin digestion was very similar to
the observed ratio of the conjugation products in the full
protein, highlighting the high efficiency of the trypsinization.
In this system we found a preference for the product with the
D-configuration, which eluted first in the HPLC (Fig. S19†).
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