Proximity-enabled protein crosslinking through genetically encoding haloalkane unnatural amino acids

Article abstract of DOI:10.1002/anie.201308794

The selective generation of covalent bonds between and within proteins would provide new avenues for studying protein function and engineering proteins with new properties. New covalent bonds were genetically introduced into proteins by enabling an unnatural amino acid (Uaa) to selectively react with a proximal natural residue. This proximity-enabled bioreactivity was expanded to a series of haloalkane Uaas. Orthogonal tRNA/synthetase pairs were evolved to incorporate these Uaas, which only form a covalent thioether bond with cysteine when positioned in close proximity. By using the Uaa and cysteine, spontaneous covalent bond formation was demonstrated between an affibody and its substrate Z protein, thereby leading to irreversible binding, and within the affibody to increase its thermostability. This strategy of proximity-enabled protein crosslinking (PEPC) may be generally expanded to target different natural amino acids, thus providing diversity and flexibility in covalent bond formation for protein research and protein engineering. Copyright

Full text of DOI:10.1002/anie.201308794

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DOI: 10.1002/anie.201308794
Synthetic Biology
Proximity-Enabled Protein Crosslinking through Genetically Encoding
Haloalkane Unnatural Amino Acids**
Zheng Xiang, Vanessa K. Lacey, Haiyan Ren, Jing Xu, David J. Burban, Patricia A. Jennings,
and Lei Wang*
Abstract: The selective generation of covalent bonds between
and within proteins would provide new avenues for studying
protein function and engineering proteins with new properties.
New covalent bonds were genetically introduced into proteins
by enabling an unnatural amino acid (Uaa) to selectively react
with a proximal natural residue. This proximity-enabled
bioreactivity was expanded to a series of haloalkane Uaas.
Orthogonal tRNA/synthetase pairs were evolved to incorpo-
rate these Uaas, which only form a covalent thioether bond
with cysteine when positioned in close proximity. By using the
Uaa and cysteine, spontaneous covalent bond formation was
demonstrated between an affibody and its substrate Z protein,
thereby leading to irreversible binding, and within the affibody
to increase its thermostability. This strategy of proximity-
enabled protein crosslinking (PEPC) may be generally
expanded to target different natural amino acids, thus provid-
ing diversity and flexibility in covalent bond formation for
protein research and protein engineering.
protein manipulation. To this end, we have developed
a general strategy in which an unnatural amino acid (Uaa)
is designed to react with a natural amino acid in a protein
through proximity-enhanced reactivity.[3] The Uaa does not
react with free natural amino acids under physiological
conditions, thereby permitting in vivo genetic incorporation.
When the Uaa is placed with appropriate orientation in
proximity to its target natural amino acid in a protein, the
increased local effective concentration facilitates the reaction
of the Uaa with the target residue to build a covalent bond. By
using this strategy, we have recently shown that new inter- or
intra-protein covalent bonds could be formed between a Cys
residue and p-2’-fluoroacetylphenylalanine.[3] Herein, we
report the genetic incorporation of a series of haloalkane
Uaas and demonstrate their ability to form covalent bonds
with Cys in proximity-enabled protein crosslinking (PEPC),
thus suggesting that this overall strategy may be applicable to
a host of Uaas to generate various new covalent bonds in
proteins. To demonstrate the potential of PEPC, we show that
interprotein PEPC can enable irreversible protein binding
and that intraprotein PEPC can be used to increase a proteinꢀs
thermostability.
We designed and synthesized a series of tyrosine ana-
logues containing different halogen atoms linked with ali-
phatic chains of varying length (Figure 1a, Scheme S1 in the
Supporting Information). By exploiting specific protein
activities, small-molecule alkyl halides have been used for
activity-based protein profiling and protein tagging.[4] The
reactivity between alkyl halides and Cys has also been
employed to synthesize cyclic peptides.[5] We therefore
expect the alkyl halides, after being incorporated into
proteins, to react with the sulfhydryl group of a Cys residue
when they are in close proximity. To date, no haloalkane Uaa
has been genetically incorporated into proteins in living
cells.[6]
T
he ability to covalently crosslink proteins spontaneously
would provide a tremendous opportunity for enhancing
existing protein properties or engineering new ones because
covalent linkages are more stable and selective than the
noncovalent interactions between protein side chains. In
natural proteins, the disulfide bond formed between two Cys
residues plays a crucial role in protein folding, stability, and
activity for a vast array of proteins.[1] The inherent redox
sensitivity and reversibility of the disulfide linkage, however,
also set limitations for protein expression, engineering, and
application.[2] Adding new covalent bonds into proteins
should overcome such limitations and broaden the scope of
[*] Dr. Z. Xiang, Dr. V. K. Lacey, Dr. H. Ren, Prof. Dr. L. Wang
The Jack H. Skirball Center for Chemical Biology and Proteomics
The Salk Institute for Biological Studies
To produce an aminoacyl-tRNA synthetase specific for
haloalkane Uaas, we generated a mutant synthetase library
based on MmOmeRS,[7] which was created by directed
evolution from the Methanosarcina mazei pyrrolysyl-tRNA
synthetase (MmPylRS)for incorporating the Uaa O-methyl-
tyrosine. Based on the crystal structure of MmOmeRS in
complex with O-methyltyrosine,[7] we randomized five resi-
dues (Val346, Trp348, Ser399, Val401, Trp417) in MmOmeRS
to create a library of 3 ꢁ 107 mutants (Figure 1b).[8] Three
rounds of selection of this library with CprY yielded one clone
that showed CprY-dependent survival in chloramphenicol
(see the Supporting Information). Sequencing revealed that
the selected synthetase, named MmXYRS, contains the
following substitutions: V346A, W348A, L401V, and W417T
10010 N. Torrey Pines Road, La Jolla, CA 92037 (USA)
E-mail: lwang@salk.edu
Homepage: http://wang.salk.edu/
Dr. J. Xu, D. J. Burban, Prof. Dr. P. A. Jennings
Department of Chemistry and Biochemistry
University of California, San Diego
La Jolla, CA 92093 (USA)
[**] We thank C. Hoppman for helpful discussions, Y.-X. Guo and M.
Bowman for help with FPLC purification, J.-K. Weng for pHIS8.3-
MBP plasmid, and W. Fischer and W. Low for help with mass
spectrometry. L.W. acknowledges support from the California
Institute for Regenerative Medicine (RN1-00577-1) and US National
Institutes of Health (1DP2OD004744-01, P30CA014195).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201308794.
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Figure 1. a) Structures of the haloalkane Uaas. Uaas are abbreviated in
the following format: CprY, O-(3-Chloropropyl)-l-tyrosine. b) Active site
of MmOmeRS in complex with O-methyltyrosine (OmeY, dark) with
the residues randomized in the library shown as stick representations.
c) Amino acid sequences of the evolved MmXYRS in comparison to
WT MmPylRS and the parental MmOmeRS.
Figure 2. Incorporation of haloalkane Uaas into myoglobin in E. coli.
Pyl
SDS-PAGE analysis of myoglobin(4TAG) expressed using the tRNA
CUA
and MmXYRS in the presence of 1 mm CprY (a; tris-glycine gel); BetY,
BprY, BbtY, and BptY (c; tris-glycine gel); and IetY and IprY (d; tricine
gel). Samples were normalized for equal cell numbers for each lane. b)
High resolution ESI-FTMS of the intact myoglobin produced with
1 mm CprY (a). Monoisotopic masses of major peaks indicate that
only CprY was incorporated into myoglobin at the TAG-encoded
position. Peaks corresponding to oxidation (ox.) and deamidation
(da.) of CprY-myoglobin were also detected and labeled.
(Figure 1c). Ser399 was retained, but the codon was changed
(see the Supporting Information).
To measure the in vivo translational efficiency and fidelity
of the evolved MmXYRS, we expressed the genes for
Pyl
CUA
tRNA
and MmXYRS in E. coli together with a sperm
whale myoglobin gene with a C-terminal His tag and a TAG
codon at position 4. After Ni2+ affinity chromatography,
a strong band running at ~ 17 kD was observed by SDS-PAGE
only from cells supplemented with 1 mm CprY, thus indicating
that full-length myoglobin was produced by the E. coli
(Figure 2a). We then analyzed the purified myoglobin with
Fourier transform ion trap mass spectrometry (ESI-FTMS). A
peak with a monoisotopic mass of 18570.75 Da was observed
(Figure 2b), which corresponds to intact myoglobin with
a single CprY residue at position 4 (expected [M + H]+ =
18570.76 Da). A second measured peak corresponds to
CprY-myoglobin lacking the initiating Met residue (expected
[M-Met + H]+ = 18439.72 Da, measured 18439.69 Da). There
were no peaks that corresponded to myoglobin variants
resulting from the incorporation of any endogenous amino
acid instead of CprY. Moreover, there were no peaks
corresponding to myoglobin with the halogen substituted by
cysteine, glutathione, 2-mercaptoethanol, or imidazole. These
results indicate that CprY was specifically incorporated into
myoglobin and that the CprY residue was stable and
unmodified during protein synthesis in E. coli and throughout
the purification process.
a high fidelity of incorporation for these Uaas (Figure S1
and Table S1 in the Supporting Information).
With MmXYRS and a set of haloalkane Uaas in hand, we
embarked on testing intermolecular PEPC between the
Z protein and the ZSPA affibody (Afb), which binds to the
Z protein with a Kd of 6 mm.[9] We chose to introduce Cys into
the Z protein at Asn6 and incorporate the Uaa into the Afb at
Asp36; these two sites are brought into close proximity when
the two proteins bind (Figure 3a).[3a,9] To clearly distinguish
the Z protein from the Afb by molecular weight by SDS-
PAGE, we fused the maltose binding protein (MBP) to the Z
protein and its mutant, Z(N6C). The purified Afb(D36Uaa)
proteins were incubated with the purified MBP–Z(N6C) in
a 2:1 ratio in PBS buffer (pH 7.4) at 378C for 1 h, and the
reaction mixture was subjected to SDS-PAGE under denatur-
ing conditions. We observed a band with a molecular weight
corresponding to MBP–Z in complex with the Afb (Fig-
ure 3b), thus indicating that the two proteins were covalently
crosslinked. By contrast, when the Afb(D36Uaa) mutants
were incubated with MBP–Z(WT), no band corresponding to
the complex was detected (Figure 3b), thus showing that the
crosslinking reaction was specific for Cys6. For the Uaas with
different halogen atoms linked by the same alkyl chain, the
order of crosslinking efficiency is I > Br> Cl, a result con-
sistent with the order of the halide leaving ability in SN2
reactions.
We next examined whether MmXYRS could incorporate
other haloalkane Uaas with structures similar to CprY.
Indeed, SDS-PAGE analyses of myoglobin expressed in the
presence of different Uaas showed that both bromo- and
iodo-containing Uaas were incorporated by the
tRNAPCyUl A/MmXYRS pair (Figure 2c,d). ESI-FTMS analyses
of the purified myoglobin samples clearly demonstrated
To explore intramolecular PEPC, we introduced both the
haloalkane Uaa and the Cys residue into the same Afb
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Figure 3. Intermolecular PEPC of the Afb to its substrate Z protein
fused to MBP (MBP–Z). a) Crystal structure of the Afb–Z-protein
complex (PDB ID 1LP1), with Asp36 in the Afb and Asn6 in the Z
protein (for substitution by the Uaa and Cys, respectively) shown as
stick representations. b) SDS-PAGE analysis of Afb–Z crosslinking. The
identities of residue 6 of the Z protein in MBP–Z and residue 36 of the
Afb are indicated. Crosslinking efficiency was calculated based on the
relative band intensities of the crosslinked complex and the MBP–Z
fusion protein.
protein and determined whether covalent crosslinking could
spontaneously occur. Based on the structure of the Afb,[9] two
proximal sites, F30 and E47, were chosen for incorporating
the Uaa and Cys, respectively (Figure 4a). The Uaa was
incorporated at the Phe site to minimize potential perturba-
tion of the Afb structure. E. coli cells harboring the expression
plasmids were grown in rich medium 2xYT supplemented
with 1 mm of BetY or BprY. After induction with 0.2% l-
arabinose for 5 h at 308C, the proteins were purified by Ni2+
affinity chromatography. For each mutant protein, only one
band, running at the same position as the WTaffibody in SDS-
PAGE (Figure S2), was detected. As another control, we
incorporated BetY at position 30 while maintaining the WT
E47 (Afb-30BetY). ESI-FTMS analyses of Afb-30BetY
indicate that BetY was selectively incorporated at the TAG
position with high fidelity and the Br group was not
substituted by any other molecule or amino acid side chain
of the Afb (Figure S3). By contrast, when Cys was introduced
at position 47, the mutant proteins Afb-30BetY-47C and Afb-
30BprY-47C clearly showed strong peaks with monoisotopic
masses corresponding to the Afb with a covalent Cys–Uaa
bond at the introduced positions (Figure 4b,c), thus indicat-
ing successful intramolecular crosslinking. For the Afb-
30BetY-47C mutant, a peak with a monoisotopic mass
corresponding to non-crosslinked Afb ([M-Met + H]) was
also detected, albeit with very weak intensity (less than 2% of
the crosslinked Afb). Afb-30BprY-47C showed no peak for
non-crosslinked Afb, thus indicating complete intramolecular
crosslinking.
Figure 4. Intramolecular PEPC within the Afb. a) Structure of the Afb
(PDB ID 1LP1) showing Phe30 and Glu47, which were used for
introducing the Uaa and Cys, respectively. b) High resolution ESI-
FTMS analysis the Afb with BetY incorporated at residue 30 and Cys at
residue 47. The crosslinking reaction of the Uaa with Cys results in the
loss of HBr. Crosslinked products: [M-HBr+H], expected 7737.90 Da,
measured 7737.89 Da; [M-Met-HBr+H], expected 7606.86 Da, mea-
sured 7606.86 Da. Non-crosslinked products: [M+H], expected
7817.82 Da, not detected; [M-Met+H], expected 7686.78 Da, mea-
sured 7686.83. c) High resolution ESI-FTMS analysis of the Afb with
BprY incorporated at residue 30 and Cys at residue 47. Crosslinked
products: [M-HBr+H], expected 7751.91 Da, measured 7751.92 Da;
[M-Met-HBr+H], expected 7620.87 Da, measured 7620.87 Da. Non-
crosslinked products: [M+H], expected 7831.84 Da, not detected; [M-
Met+H], expected 7700.80 Da, not detected.
To assess the effects of intramolecular PEPC on protein
properties, we measured the thermal stability of the FPLC-
purified WT Afb, Afb-30BetY-47C, and Afb-30BprY-47C by
using circular dichroism (CD) spectroscopy. All CD spectra
showed negative minima at 222 nm and a signal sign change
from negative to positive at 200 nm (Figure S4a), results
consistent with the helical structure of the Afb. The melting
curves were analyzed based on a two-state unfolding model of
a monomeric protein (Figure S4b–d).[10] WT Afb and Afb-
30BprY-47C showed similar Tm values of (46.7 Æ 0.2)8C and
(46.5 Æ 0.5)8C, respectively. By contrast, Afb-30BetY-47C
showed a markedly higher Tm value of (60.4 Æ 0.7)8C. These
results suggest that an intramolecular covalent linkage of an
appropriate length improves the thermal stability of Afb.
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In summary, we genetically encoded a series of haloalkane
Uaas that possess proximity-enabled bioreactivity toward
Cys. In contrast to the Uaas incorporated to date, which have
been either chemically inert or bioorthogonal, these uniquely
bioreactive Uaas enable both inter- and intra-molecular
PEPC in a controlled and specific manner. In contrast to
disulfide bonds, the covalent bond formed between a haloal-
kane Uaa and Cys is irreversible and stable under reducing
conditions. The series of haloalkane Uaas used also increases
the diversity and flexibility in side chain length, type, and
reactivity compared to our previously reported p-2’-fluoro-
acetylphenylalanine approach.[3a] We showed that interpro-
tein PEPC enables irreversible protein binding and that
cotranslational PEPC within an Afb can increase its thermo-
stability. We are developing new Uaas to expand proximity-
enabled bioreactivity to other natural amino acid residues and
are applying PEPC to study protein interactions and develop
protein therapeutics.[3b] We expect that this spontaneous
PEPC will provide new avenues to novel protein properties
and functions for a wide range of applications in basic and
synthetic biology.
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Received: October 9, 2013
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Keywords: protein crosslinking · protein engineering ·
proximity-enabled reactivity · tRNA · unnatural amino acids
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