A rationally designed pyrrolysyl-tRNA synthetase mutant with a broad substrate spectrum

Article abstract of DOI:10.1021/ja211972x

Together with tRNA_CUA^Pyl, a rationally designed pyrrolysyl-tRNA synthetase mutant N346A/C348A has been successfully used for the genetic incorporation of a variety of phenylalanine derivatives with large para substituents into superfolder green fluorescent protein at an amber mutation site in Escherichia coli. This discovery greatly expands the genetically encoded noncanonical amino acid inventory and opens the gate for the genetic incorporation of other phenylalanine derivatives using engineered pyrrolysyl-tRNA synthetase-tRNA_CUA^Pyl pairs.

Full text of DOI:10.1021/ja211972x

Communication
pubs.acs.org/JACS
A Rationally Designed Pyrrolysyl-tRNA Synthetase Mutant with a
Broad Substrate Spectrum
Yane-Shih Wang, Xinqiang Fang, Ashley L. Wallace, Bo Wu, and Wenshe R. Liu*
Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States
S
* Supporting Information
displays specific recognition toward NAAs and has a broad
substrate spectrum.
Pyl
CUA
ABSTRACT: Together with tRNA
, a rationally
designed pyrrolysyl-tRNA synthetase mutant N346A/
C348A has been successfully used for the genetic
incorporation of a variety of phenylalanine derivatives
with large para substituents into superfolder green
fluorescent protein at an amber mutation site in Escherichia
coli. This discovery greatly expands the genetically encoded
noncanonical amino acid inventory and opens the gate for
the genetic incorporation of other phenylalanine deriva-
We previously evolved two PylRS mutants that display
specific recognition of phenylalanine.21 In both mutants, N346
is mutated to alanine and C348 is mutated to a larger amino
acid, leucine or lysine. No mutation at other sites was found.
Figure 1 shows the structure of the PylRS complex with
tives using engineered pyrrolysyl-tRNA synthetase-
Pyl
CUA
tRNA
pairs.
n the past decade, we have seen genetic incorporation of
Imany noncanonical amino acids (NAAs) into proteins at
nonsense mutation sites in Escherichia coli, Saccharomyces
cerevisiae, and mammalian cells using unique aminoacyl-tRNA
synthetase (aaRS)-nonsense suppressing tRNA pairs that do
not cross-interact with endogenous aaRS-tRNA pairs.1−7
Except for the wild-type (wt) pyrrolysyl-tRNA synthetase
(PylRS) that has been directly used for genetic incorporation of
>10 NAAs,8−12 most of these unique aaRSs were evolved via
complicated positive and negative selection systems.1,2,13−16
So far, evolution is still the primary approach to identify NAA-
specific aaRSs. The prerequisites of the referred aaRS evolution
are the construction of a large mutant aaRS gene library and a
readily available selection system.1 Although several easily
accessible selection systems have been developed for E. coli and
S. cerevisiae cells, constructing a large mutant aaRS gene library
that most of time needs to cover random variations of 5−6
aaRS active-site residues is not straightforward and needs an
experienced molecular biologist to practice many times to
achieve close to the coverage of the library variations.17
Statistically, full coverage is not likely. One factor that also
significantly influences the aaRS evolution is the selection
pressure. Positive and negative selections used in the evolution
are based on either antibiotic resistance or toxic gene
expression. Varying concentrations of antibiotics and inducers
that are used to trigger toxic gene expression could lead to
dramatic different evolution results. In comparison to the
evolution approach, rational design of aaRSs is relatively more
straightforward and easier to be carried out by following
standardized site-directed mutagenesis protocols. However,
attempts to rationally design unique NAA-specific aaRSs often
led to mutant aaRSs with nonexclusive recognition of
endogenous canonical amino acids (CAAs).18,19 In this work,
we show that a rationally designed PylRS mutant N346A/N348A
Figure 1. Structure of the PylRS complex with pyrrolysyl-AMP.
Phenylalanyl-AMP that potentially binds the N346A mutant is shown
as an overlay with pyrrolysyl-AMP. The structure is based on the PDB
entry 2Q7H.20
pyrrolysyl-AMP. Phenylalanyl-AMP was also modeled into the
active site of the N346A mutant of PylRS and is shown as an
overlay with pyrrolysyl-AMP in Figure 1. The structure in
Figure 1 suggests how two mutations N346A and C348L (or
C348K) induce the substrate specificity change from
pyrrolysine (Pyl) to phenylalanine. In the wt PylRS, the side-
chain amide nitrogen of N346 forms a hydrogen bond with the
side-chain amide oxygen of Pyl, an interaction that anchors Pyl
at the active site. The amide of N346 also shows a steric clash
with the modeled phenylalanine in the active site, excluding its
binding to the wt PylRS. The N346A mutation will not only
significantly decrease the binding of PylRS to Pyl and also
relieve the steric hindrance that prevents the binding of
phenylalanine. Since both the aromatic side chain of Y384 and
two backbone amides of residues 419−421 could form π−π
stacking interactions with the phenyl group of a bound
phenylalanine, we think that dismissing the steric hindrance
Received: December 22, 2011
Published: January 30, 2012
© 2012 American Chemical Society
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Journal of the American Chemical Society
Communication
between a bound phenylalanine and N346 is the major
contributing factor for the direct binding of two PylRS mutants
to phenylalanine. The mutation of C348 to an amino acid with
a larger side chain that apparently occupies the space of
pyrrolidine of Pyl in the active site is believed to bring more
interactions with the bound phenylalanine and therefore
increase its binding potential. Since the N346A mutation
relieves the steric hindrance that prevents the binding of
phenylalanine and there is left a large empty space at the active
site around the para position of phenylalanine after it binds to
the N346A mutant of PylRS, we speculate that the N346A
mutant could bind a phenylalanine derivate with a large para
substituent better than phenylalanine itself. Given that
mutations at C348 were prevalent in almost all evolved NAA-
specific PylRS mutants and its mutation to alanine was
observed in PylRS mutants specific for NAAs with large side
chains,22−25 we think an extra C348A mutation to the N346A
mutant will generate a larger active site pocket that could
provide a para-substituted phenylalanine with more structural
flexibility to bind to the active site. To test this idea, we
constructed a plasmid pBK-N346A/C348A that carries the
gene coding the PylRS mutant N346A/C348A and used it
together with pET-pylT-sfGFP2TAG to transform BL21 cells.
phenylalanine. To see whether N346A/C348A can recognize
a phenylalanine derivative with a large para substituent, the
same BL21 cells were grown in GMML supplemented with
5 mM p-propargyloxy phenylalanine (1, Figure 3). In comparison
Figure 3. Structures of 1−7 and their specific incorporation into
sfGFP at the S2 position.
to the condition with 5 mM phenylalanine, the expressed sfGFP at
this condition displayed a 5-fold increase in the fluorescence
The plasmid pET-pylT-sfGFP2TAG carries genes coding
Pyl
CUA
tRNA
and an IPTG-inducible superfolder green fluorescent
protein (sfGFP) with an amber mutation at the S2 position.
The transformed BL21 cells were then used to examine the
recognition of N346A/C348A toward 20 CAAs by expressing
sfGFP in liquid glycerol minimal media (GMML) supple-
mented with 5 mM of a designated CAA. The sfGFP
expression was induced by the addition of 1 mM IPTG.
Since the sfGFP expression levels at all twenty conditions were
low, we chose to detect the fluorescent emission of the
expressed sfGFP at these conditions and show their relative
intensities to represent the corresponding sfGFP expression
levels in Figure 2. The condition that contained 5 mM
emission intensity.
With our initial success with 1, we then either purchased or
synthesized several other para-substituted phenylalanine
derivatives shown as 2−7 in Figure 3 and tested their
recognition by N46A/C348A. To better quantify the expression
levels of sfGFP incorporated with these NAAs, another plasmid
pEVOL-pylT-N346A/C348A was constructed. This plasmid
Pyl
CUA
carries genes coding both tRNA
and N346A/C348A. Its
Pyl
CUA
tRNA
is under control of a strong proK promoter that can
in E. coli.26 Together
Pyl
boost up the expression level of tRNA
CUA
with pET-pylT-sfGFP2TAG, this plasmid was used to trans-
form BL21 cells. The transformed cells were grown in GMML
supplemented with 2 mM of a designated NAA (7 can only be
provided as 1 mM because of its low solubility). As shown in
Figure 3, all seven NAAs promoted high levels of sfGFP
expression 10 h after induction with 1 mM IPTG. The
molecular weights of the expressed sfGFP variants determined
by electrospray ionization spectrometry (ESI-MS) analysis
agree well with their theoretical molecular weights (SI Table 1
and Figures 2−8). Without a NAA in the medium, no sfGFP
was expressed. With the addition of 2 mM phenylalanine in the
medium, an sfGFP expression level (1.5 mg/mL) close to that
for 5 was obtained. Since 1 contains a terminal alkyne that
undergoes the Cu(I)-catalyzed azide−alkyne cyclization re-
action,27 the expressed sfGFP incorporated with 1 (sfGFP-1)
was also used separately to label with a fluorescein azide (8 in
Figure 4) in an optimized labeling condition that contained 0.1
mM Cu(I):tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine
(TBTA) complex, 0.4 mM additional TBTA, 5 mM ascorbate,
and 1 mM NiCl2.28 A 3-h incubation led to specific labeling of
sfGFP-1. A parallel labeling reaction of the wt sfGFP in the
same condition gave nondetectable labeling with 8.
Figure 2. Relative fluorescence emission intensities of sfGFP expressed
in BL21 cells transformed with pBK-N346A/C348A and pET-pylT-
sfGFP2TAG and grown in GMML supplemented with different amino
acids. Cell lysates were excited at 450 nm and fluorescence emission
intensities were detected at 510 nm. Background emission from cell
lysate of same cells grown in GMML and induced with the addition of
1 mM IPTG was subtracted from each data set. Twenty CAAs are
shown as one-letter abbreviations in the x-axis labels.
One intriguing question is whether N346A/C348A could
also recognize a phenylalanine derivative with a small para
substituent. Given that 5 has a relatively small para substituent
and providing 5 in the growth medium led to a reasonable
sfGFP expression level, one would expect N346A/C348A also
recognizes a phenylalanine derivative with a small para
phenylalanine displayed the highest sfGFP expression level,
confirming our initial speculation that the N346A mutation can
change the substrate specificity of PylRS from Pyl to
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Journal of the American Chemical Society
Communication
Tyr
jannaschii tyrosyl-tRNA synthetase (MjTyrRS)-tRNA
CUA
pairs,32−34 specifically evolved MjTyrRS variants for individual
phenylalanine derivatives are usually required and the
Tyr
MjTyrRS-tRNA
pair cannot be used in eukaryotic cells
CUA
Tyr
because of the recognition of tRNA
by endogenous
CUA
eukaryotic aaRSs (Liu and Schultz, unpublished data). Using
Pyl
CUA
the N346A/C348A-tRNA
pair will resolve both issues. In
addition, phenylalanine derivatives 3, 4, 6, and 7 that are taken
by N346A/C348A are also genetically encoded in E. coli for the
first time. Given that N346A/C348A has a relatively deep and
big binding pocket, the current study also opens a gate to test
the recognition of this mutant toward other large phenylalanine
Figure 4. Site-selective labeling of sfGFP-1 with 8. (A) SDS-PAGE
analysis of sfGFP-1 and wt sfGFP after their reactions with 8. The gel
was stained with Coomassie blue. (B) Fluorescent imaging of the same
gel under 365 nm UV irradiation.
derivatives. Another potential application of N346A/C348A is
Pyl
to couple its pair with tRNA
together with evolved
UUA
Tyr
MjTyrRS-tRNA
pairs for the genetic incorporation of two
CUA
different phenylalanine derivatives into one protein.35 This
may find applications in enzyme mechanistic studies, protein
FRET labeling, and phage-displayed unnatural peptide library
construction.
substituent such as Cl, Br, I, CN, etc. To test this possibility, we
examined the genetic incorporation of 9−15 shown in Figure 5
ASSOCIATED CONTENT
■
S
* Supporting Information
Plasmid constructions, NAA synthesis, and protein expression.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Figure 5. Structures of 9−14 and their genetic incorporation into
Corresponding Author
wliu@chem.tamu.edu
sfGFP at S2. The protein expression yields are <1 mg/L for all NAAs.
Pyl
into sfGFP at S2 using the N346A/C348A- tRNA
pair. As
CUA
Notes
shown in Figure 5, providing 2 mM of any of these NAAs in
GMML led to sfGFP expression levels that are significantly
lower than those for NAAs shown in Figure 3 and
phenylalanine but still higher than the background expression
level in GMML in which no NAA was provided. Therefore,
N346A/C348A recognizes 9−15. Since the expression levels of
sfGFP incorporated with 9−15 are very low, we did not
attempt to characterize these proteins by the ESI-MS analysis.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported in part by the National Institutes of
Health (grant 1R01CA161158 to W.R.L.) and the Welch
Foundation (grant A-1715 to W.R.L.). We thank Dr. Yohannes
H. Rezenom from Laboratory for Biological Mass Spectrometry
at Texas A&M University for characterizing our proteins using
ESI-MS.
Although the current analysis indicates that it is not applicable
Pyl
CUA
to use the N346A/C348A-tRNA
pair to express proteins
incorporated with 9−15, it suggests that PylRS can be
engineered to recognize phenylalanine derivatives with small
para substituents. Since a PylRS mutant specific for 5 evolved
by Wang and co-workers contains mutations at A302 and
V401,29 We are now introducing additional mutations at these
two sites of N346A/N348A to search for PylRS mutants that
allow efficient incorporation of phenylalanine derivatives with
small para substituents.
In summary, we have rationally designed a PylRS mutant
N346A/C348A that shows low recognition toward CAAs in
minimal media but, together with tRNACPyUlA, mediates efficient
incorporation of NAAs 1−7 into proteins at amber mutation
sites in E. coli. These NAAs contain functional groups such as
alkyne and alkene and can be applied to install different
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