An orthogonal seryl-tRNA synthetase/tRNA pair for noncanonical amino acid mutagenesis in Escherichia coli

Article abstract of DOI:10.1016/j.bmc.2020.115662

We report the development of the orthogonal amber-suppressor pair Archaeoglobus fulgidus seryl-tRNA (Af-tRNA^Ser)/Methanosarcina mazei seryl-tRNA synthetase (MmSerRS) in Escherichia coli. Furthermore, the crystal structure of MmSerRS was solved at 1.45 ? resolution, which should enable structure-guided engineering of its active site to genetically encode small, polar noncanonical amino acids (ncAAs).

Full text of DOI:10.1016/j.bmc.2020.115662

Journal Pre-proofs
“An orthogonal seryl-tRNA synthetase/tRNA pair for noncanonical amino
acid mutagenesis in Escherichia coli ”
Claudio Zambaldo, Minseob Koh, Fariborz Nasertorabi, Gye Won Han,
Abhishek Chatterjee, Raymond C. Stevens, Peter G. Schultz
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DOI:
Reference:
S0968-0896(20)30492-2
https://doi.org/10.1016/j.bmc.2020.115662
BMC 115662
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Bioorganic & Medicinal Chemistry
Received Date:
Accepted Date:
9 June 2020
18 July 2020
Please cite this article as: Zambaldo, C., Koh, M., Nasertorabi, F., Han, G.W., Chatterjee, A., Stevens, R.C.,
Schultz, P.G., “An orthogonal seryl-tRNA synthetase/tRNA pair for noncanonical amino acid mutagenesis in
Escherichia coli ”, Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115662
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“An orthogonal seryl-tRNA synthetase/tRNA pair for noncanonical amino acid
mutagenesis in Escherichia coli”
Claudio Zambaldo,a,‡ Minseob Koh,a,‡ Fariborz Nasertorabi,b,‡ Gye Won Han,b Abhishek
Chatterjee,c,* Raymond C. Stevens,b,* and Peter G. Schultz a,*
a
Department of Chemistry and Skaggs Institute for Chemical Biology, The Scripps
Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037, United States
b
Departments of Biological Sciences and Chemistry, Bridge Institute, Michelson Center
for Convergent Bioscience, University of Southern California, Los Angeles, CA 90089,
United States
c Department of Chemistry, Boston College, 2609 Beacon Street, 246B Merkert Chemistry
Center, Chestnut Hill, Massachusetts 02467, United States
Abstract
We report the development of the orthogonal amber-suppressor pair Archaeoglobus
fulgidus seryl-tRNA (Af-tRNASer)/Methanosarcina mazei seryl-tRNA synthetase
(MmSerRS) in Escherichia coli. Furthermore, the crystal structure of MmSerRS was
solved at 1.45 Å resolution, which should enable structure-guided engineering of its active
site to genetically encode small, polar noncanonical amino acids (ncAAs).
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*Corresponding Authors
*schultz@scripps.edu
*stevens@usc.edu
*abhishek.chatterjee@bc.edu

Keywords
Seryl-tRNA synthetase
E. coli orthogonality
Genetic code expansion
X-ray crystallography
Non-canonical amino acids
1. Introduction
Methods that enable the genetic incorporation of noncanonical amino acids (ncAAs)
into proteins have greatly increased our ability to manipulate protein structure and function
in vitro and in vivo.1 The site-selective incorporation of ncAAs into proteins in living cells
is reliant on laboratory-evolved tRNA/aminoacyl-tRNA synthetase (aaRS) pairs. To
ensure that the ncAA is incorporated into proteins with high fidelity and efficiency, the
evolved tRNA/aaRS pair must be orthogonal to its host counterparts (i.e., not cross-react
with the host tRNAs and aminoacyl-tRNA synthetases) and efficiently suppress a codon
that does not encode a canonical amino acid (e.g., nonsense, four-base, etc.). Several
such orthogonal tRNA/aaRS have been developed for expanding the genetic codes of
prokaryotes and eukaryotes, enabling site-specific incorporation of ncAAs encompassing
a wide spectrum of chemical functionalities in the context of amino acid side chains.2
In E. coli, three tRNA/aaRS pairs have been successfully engineered to incorporate
ncAAs: the M. jannaschii derived tyrosyl,3 archaea-derived pyrrolysyl,4 and a yeast-
derived tryptophanyl pairs.5 While these pairs have enabled the incorporation of ncAAs
with a wide variety of side chains, small polar ncAAs remain challenging to genetically
encode. The only exception is the successful genetic incorporation of phosphoserine
using an archaea-derived pair that is naturally evolved to charge this amino acid as a
biosynthetic precursor to cysteine.6,7,8,9 Additionally, recent development of heritable
unnatural base pairs10 and engineered E. coli strains with compressed genetic codes11,12
promise a further expansion of available triplet codons that can be simultaneously
reassigned to distinct ncAAs.
Development of an orthogonal suppressor seryl-tRNA (tRNASer)/SerRS13,14 pair would
potentially enable genetic incorporation of ncAAs bearing small, polar side chains.
Additionally, unlike most other pairs, SerRS does not use the anticodon of tRNASer as an
identity element, making it straightforward to reassign this pair to any other codon,
including the amber nonsense codon. Here, we report the development of an archaea-
derived tRNASer/SerRS pair that incorporates serine in response to the amber nonsense
codon in E. coli with high fidelity and efficiency. In addition, we solved the crystal structure
of MmSerRS, which will facilitate the design of synthetase libraries for use in directed
evolution experiments to genetically encode ncAAs with small side chains.
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2. Results and discussion
2.1. Identification of orthogonal tRNA/aaRS pairs
Typically, tRNA/aaRS pairs from archaea and eukaryotes serve as starting points for
developing orthogonal pairs in E. coli because of their evolutionary divergence from their
bacterial counterparts.15 Archaea-derived pairs have been particularly useful to this end,
since the corresponding aaRSs tend to be simpler and more robust scaffolds for directed
evolution.16 Consequently, we focused on archaea-derived tRNASer/SerRS pairs for
developing an orthogonal suppressor pair in E.coli.17 To this end we tested all possible
combinations of six different archaeal tRNASer and five different SerRS with divergent
sequences (Figure 1), since it has previously been shown that the combination of an aaRS
and a tRNA from two different species can provide the optimal choice for developing an
orthogonal pair.18 The SerRS genes were cloned into a pBK vector to express them using
the constitutive, low-activity glnS promoter. The TAG-suppressor mutants of the tRNASer
genes (anticodon mutated to CUA) were cloned into a pRepCM3b vector19 under a
constitutive lpp promoter. This vector also encodes a chloramphenicol-acetyl transferase
(CAT) reporter harboring a TAG mutation at a permissive site (D111TAG), which allows
assessment of TAG-suppression efficiency by monitoring the degree of chloramphenicol
resistance exhibited by cells hosting this plasmid.19
Each of the six pRepCM3b-tRNASer plasmids were separately co-transformed into
DH10b E. coli with each of the pBK-SerRS plasmids, as well as an empty pBK vector
(does not express an aaRS). The resulting 35 different strains, harboring different
combinations of tRNASer and SerRS variants, were individually plated on LB-agar medium
supplemented with antibiotics necessary for plasmid maintenance (kanamycin and
tetracycline), as well as increasing concentrations of chloramphenicol. For each distinct
tRNASer, the minimal inhibitory concentration (MIC) value observed in the presence of the
empty pBK plasmid is a measure of its cross-reactivity with host aaRSs, while the different
MIC values exhibited in the presence of different SerRS provide an estimate of the TAG-
suppression activity of the corresponding tRNASer/SerRS combination (Figure 1). We
found that two different tRNASer variants from A. fulgidus and one from P. horikoshii
demonstrated strong TAG suppression activity, while SerRS from M. mazei and P.
horikoshii were the most active. The heterologous combination of MmSerRS and CUA
mutant of Af-tRNASer(GCU) (Af2) exhibited the highest activity, enabling survival of the
resulting strain at up to 150 µg/mL of chloramphenicol.
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Figure 1. Identification of an orthogonal seryl pair. The suppression efficiency of six synthetases
expressed with five orthogonal amber suppressor tRNA was measured using a chloramphenicol
resistance assay. The MIC value is presented as a heatmap. Af: Archaeoglobus fulgidus, Mj:
Methanocaldococcus jannaschii, Ss: Sulfolobus solfataricus, Hb: Halobacterium sp. NRC-1, Mm:
Methanosarcina mazei, Ph: Pyrococcus horikoshii, Af1: Af-tRNASer(CGA), Af2: Af-tRNASer(GCU)
and Mock: No synthetase expressed (empty pBK). Anticodons of tRNASers were mutated to CUA
Even though this seryl-tRNA/aaRS pair shows high suppression activity, the relatively
high MIC exhibited by Af2 in the absence of a cognate SerRS suggests that it cross-reacts
significantly with a host aaRS, compromising its fidelity. The cross-reactivity of various
suppressor tRNAs can be reduced – without substantially compromising their suppression
efficiency in the presence of the cognate aaRS – through the use of directed evolution.5,18-
20
More specifically, mutations in the acceptor and the T-stem regions have led to such
improvements. To this end, we used site-saturation mutagenesis to fully randomize eleven
bases in the acceptor stem of Af-tRNASer(GCU). Additionally, the adenine immediately
following the anticodon (position 36) was randomized to both A and G (Figure 2A). The
resulting library was cloned into a pBK vector under the lpp promoter and co-transformed
into DH10b E. coli harboring a pREP plasmid that expresses the MmSerRS gene from a
constitutive glnS promoter, as well as a CAT gene with a TAG codon at a permissive site
(D111TAG). Growing the resulting pool of cells in the presence of 50 µg/mL
chloramphenicol (on LB-agar plates) led to the enrichment of active Af-tRNASer(GCU)
mutants (positive selection). The surviving tRNA mutants were isolated and co-
transformed into a DH10b strain containing a pNeg plasmid that expresses an arabinose-
inducible toxic barnase gene harboring two TAG nonsense codons at permissive sites
(Q3TAG and D45TAG). Upon arabinose induction, any cross-reactive tRNA mutant would
lead to cell death by enabling the expression of full-length barnase protein. After
subjecting the library to two rounds each of alternating positive and negative selections,
individual tRNA clones were tested using the chloramphenicol resistance assay described
above in the presence or absence of MmSerRS. Two clones exhibited significantly
attenuated MICs (<20 µg/mL) in the absence of MmSerRS, while retaining strong activity
(>100 µg/mL) in its presence (Figure 2B). One of these, hit 49, had a substantially altered
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acceptor stem (Figure 2C), while the other (AfS.G; Figure 2D) had a wild-type acceptor
stem but had an A to G mutation following the anticodon.
Clone 49 exhibits very low cross-reactivity, which is ideal for developing a selection
system for engineering the substrate specificity of MmSerRS. To confirm this hypothesis,
the MmSerRS gene was cloned into pBK3 (Figure S1) and tRNA clone 49 was inserted
into pREP (Figure S2),21 the plasmid system typically used for the directed evolution of
aaRSs. These vectors were then co-transformed into DH10b E. coli and serial dilutions of
an overnight culture were plated onto GMML-agar containing increasing concentrations
of chloramphenicol. A pBK plasmid devoid of the synthetase (pBKempty) was used as a
negative control. As expected, cells harboring tRNA clone 49/MmSerRS were able to
survive high concentrations of CM, but tRNA clone 49 alone did not support cell growth in
the presence of chloramphenicol (Figure S4), demonstrating the suitability of this system
to enrich active MmSerRS mutants from a synthetic library.
In contrast, AfS.G shows significantly higher suppression efficiency, which makes it a
better choice for developing an efficient expression system where this pair would be used
for ncAA incorporation into target proteins. Previous studies suggest that the low cross-
reactivity of AfS.G is unlikely to cause a loss of fidelity when the cognate MmSerRS is
coexpressed.22 To demonstrate this, we cloned the tRNA-AfS.G/MmSerRS pair into the
pUltra suppressor plasmid, which has been previously optimized to provide highly efficient
ncAA incorporation into target proteins.23 This plasmid was co-transformed into DH10b
with a pET22b plasmid expressing the sfGFP-151-TAG reporter from a strong T5-lac
promoter.24 Upon induction with isopropyl 1-thio-β-D-galactopyranoside, this culture
showed robust fluorescence, indicative of efficient suppression of the TAG codon in the
GFP gene. The full-length reporter protein was purified by immobilized metal-ion
chromatography (IMAC) with excellent yield (~250 mg/L, ~50% relative to the parent
protein) using a C-terminal hexahistidine tag. The resulting protein was analyzed by SDS-
PAGE and ESI-MS, which revealed a single specie with a molecular weight consistent
with the incorporation of serine at position 151. These observations confirm that this
orthogonal serine pair incorporates serine in response to the TAG nonsense codon with
high fidelity and efficiency (Figure S6 A and B).
.
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Figure 2. Directed evolution of an Af-tRNASer library. (A) Af-tRNASer(GCU) sequence is shown.
The library sites are highlighted as red circle; N = A, G, C, and U (B) Orthogonality of synthetic
tRNAs in E. coli. The chloramphenicol resistance assay was used to measure the chloramphenicol
MIC values for E. coli expressing the indicated tRNA. (C) Clone 49 sequence. Modified bases are
in red; the non-canonical base pair is highlighted with a red bar; the anticodon is modified to CUA.
(D) AfS.G sequence. Modified base is in red; anticodon is modified to CUA.
2.2. Protein production and crystallization of MmSerRS mutants
With an efficient and orthogonal tRNAser/SerRS pair in hand, we next determined the
three-dimensional crystal structure of the MmSerRS in order to facilitate the design of
active site libraries for future studies aimed at evolving synthetases with altered
specificities. To express the protein, the cDNA of MmSerRS was cloned into a pET22B
vector (Figure S5) under the control of an inducible T7-lac promoter. Various hexahistidine
(6His)-tagged constructs were tested, including N- or C-terminal tagged proteins together
with a TEV protease-cleavable N-term-6His construct (Figure S3). After protein
overexpression and purification by IMAC and ion-exchange chromatography, yielding 150
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mg/L of homogenous material, the different construct permutations were screened in
crystallization experiments under a variety of conditions (JCSG 1-4 core suite, 24 and 4
°C). Only a handful of conditions gave rise to crystals, with the best set of conditions
(LiOAc/PEG3500) yielding crystals diffracting at an unsatisfactory 6-10 Å resolution.
A systematic approach investigating the crystal packing of closely related structures of
similar synthetases (PDB ID: 2DQ3, 3LSQ and 1SES) was undertaken, looking for
residues or short regions that could engage in intramolecular contacts with the
neighboring molecules. This search pointed to some charged residues in our target
sequence that could potentially disrupt a common antiparallel β-sheet otherwise
observed, therefore causing suboptimal diffraction patterns. Our analysis highlighted a
noticeable sequence variety among known structures in the C-terminal portion of the
enzyme, specifically, from residues 400 to 422. We therefore introduced mutations
reverting two glutamic acids to valine (E402V, E416V) in order to keep the antiparallel β-
sheet intact, and also shorten the peptide chain from residues 417-422 to diminish
flexibility and avoid possible clashes. We then expressed and purified a set of MmSerRS
C-terminal mutant constructs (Figure S7): in a preliminary crystallization screen, two
mutants (Figure S7A and S7B, SerRSM1 and SerRSM2 throughout) gave rise to crystals
diffracting at 3.4 Å resolution.
We then turned our attention to the synthesis and co-crystallization of both SerRSM1
and SerRSM2 with a non-hydrolysable serine-AMP analogue (SerAMS, 1) to map the key
active site interactions and potentially enhance crystal formation. 25,26,27,28,29
Scheme 1. Synthesis of SerAMS (1): 2’-3’-O-isopropylideneadenosine (1.0 equiv.), bis(tributyltin)
oxide (2.0 equiv.), benzene 80 °C; sulfamoyl chloride (5.0 equiv.) 0.3 M in dioxane 0 °C to RT.
Intermediate 3 (1.0 equiv.) 0.4 M in DMF, intermediate 4 (2.0 equiv.), DBU (2.5 equiv.), RT, 12
hours. Intermediate 5, in TFA/H2O 5:2, lyophilize.
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2.3. Ligand synthesis and SerM2 co-crystal
The synthesis of SerAMS 1 (5'-O-[N-(L-seryl)-sulfamoyl]adenosine) was carried out
following a previously reported scheme,30 with slight variations to improve yields and avoid
palladium impurities.26 Briefly (Scheme 1), 2’,3’-O-isopropylideneadenosine was heated
to reflux with a molar excess of bis(tributyltin) oxide giving its 5’-O-tributyltin ether (2);31
the mixture was then treated with an excess of sulfamoyl chloride yielding the crystalline
intermediate 3 after column chromatography. Compound 3 was reacted with the NHS
ester of methyl (S)-(−)-3-Boc-2,2-dimethyl-4-oxazolidinecarboxylate (4) in the presence of
DBU to afford compound 5, which was subjected to global acidic deprotection in aqueous
TFA yielding SerAMS (1).
Binary complex formation was carried out by mixing protein and ligand at a 1:10 molar
ratio to a final concentration of 0.45 mM for the protein and 5 mM for the ligand. Single
crystals appeared after 3 days of hanging drop crystallization and grew to their maximum
size within a week. Homogenous crystals grew in 18% PEG3350 and 200 mM thiocyanate
at 22 °C. Single crystals were flash-frozen in liquid nitrogen before they were mounted for
data collection. The structure of the binary complex MmSerRS:SerAMS was then
determined and refined at 1.45 Å resolution (Figure 3A).
Figure 3. Co-crystal structure of MmSerRS:SerAMS (1) (PDB ID: NNNN). (A) Overall structure.
Catalytic site (sky blue) and tRNA recognition site (pink) are highlighted with circles. (B) Amino
acid binding site. SerAMS (1) forms a strong hydrogen bonding (orange dashed lines) network
with surrounding residues (Thr229, Glu231, Glu281, and Ser379) at the amino acid binding site.
Electron density as a 2F0Fc contour map (3s) is shown in meshed surface around SerAMS and
amino acid binding residues. Other residues are omitted for clarity.
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2.4. Structural analysis
Crystals belong to space group C 2 2 21 and contain one molecule per asymmetric
unit. However, data from size exclusion chromatography (Figure S9) confirmed the protein
is dimeric in solution; the extensive intermolecular contacts across a crystallographic two
fold axis also indicates its dimeric nature, similar to other available structures from human,
bacteria and archaea.32,33,34 As depicted in figure 3A, MmSerRS shows the distinctive
features of a typical class II synthetase: the nucleotide binding module is composed of
antiparallel β sheets and within this module lie three conserved motifs (motif 1,2 and 3)
that are characteristic of class II synthetases. 14,35,36,37,38
The overall structural fold of this protein is highly similar to other known structures of
SerRS family members. A protein sequence comparison identified bacterial Aquifex
aeolicus VF5 (PDB ID: 2DQ3) as the protein with the highest sequence similarity (57%
sequence identity and 74% sequence similarity). The superimposition of Cα atoms of the
full-length structure shows a low root mean square deviation (rmsd) of 0.52, underscoring
the high degree of similarity between the two macromolecules. As shown in the crystal
structure, each monomeric unit is composed of a solvent exposed N-terminal antiparallel
coiled-coil arm; this 57 Å long helical arm has tRNA-binding function, specifically for the
variable arm recognition.38 The catalytic site is located within the C-terminal portion of the
monomer and is structured around seven-stranded antiparallel β sheets (Figure 3A).
There are 13 residues contributing 16 hydrogen bonds to the ligand in both MmSerRS
and its homologue from Aquifex aeolicus, where only one residue is not identical,
suggestive of a highly conserved Ser-AMP binding pocket. The serine recognition site
comprises Thr229, Glu231, Glu281, and Ser379 which form an extensive hydrogen
bonding network with serine. With respect to Ser hydroxyl side chain recognition,
hydrogen bonding with Glu281 and Ser379 appears to be essential, and the gamma-
carboxyl side chains of Glu231 and Glu281 form salt bridges with the amino group of the
serine. In addition to the charged residues, Thr229 plays a key role in accommodating
both the amino group as well as the carbonyl group of SerAMS (1) (Figure 3B). In fact,
two residues that appear to be the main determinants in accommodating Ser side chain,
Glu281 and Ser379, were sensitive to Ala mutation as assessed by the chloramphenicol
resistance assay (Figure S11). These results suggest a possible rationale for library
design to create a pocket to accommodate ncAAs by mutating residues surrounding the
Ser side chain including Thr229, Cys256, Lys279, Glu281, Ser348, Ser350, Asn377, and
Ser379.
3. Conclusion
We have generated an orthogonal amber suppressor pair Af-tRNASer/MmSerRS of
archaeal origin that can be used for ncAA mutagenesis in E. coli. Directed evolution
experiments showed that the acceptor stem and/or the anticodon loop of suppressor tRNA
can be modified to enhance orthogonality of the tRNA. In addition, we have solved the X-
ray co-crystal structure of MmSerRS:SerAMS at high resolution. This structural
information will be useful for generating active site mutants of SerRS that accommodate
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small and/or polar ncAAs into proteins in E. coli. These latter experiments are currently
underway.
Acknowledgments
Kristen Williams for assistance in manuscript preparation, Dr. Sean A. Reed, Dr. Angad
P. Mehta and Dr. Michael J. Bollong for helpful discussions and manuscript revision. We
would like to acknowledge Dr. Marc-André Elsliger from the Wilson lab at Scripps
Research for initial crystallization screening efforts, together with Dr. Bernhard Kuhle from
the Schimmel lab for invaluable assistance throughout the project. This work is supported
by NIH R01 GM062159 (Schultz).
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Declaration of competing interest
The authors declare no competing financial interest
Author Contributions
‡C. Z., M.K., and F.N. contributed equally.
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