Functional Replacement of Histidine in Proteins to Generate Noncanonical Amino Acid Dependent Organisms

Article abstract of DOI:10.1021/jacs.7b13452

Simple strategies to produce organisms whose growth is strictly dependent on the presence of a noncanonical amino acid are useful for the generation of live vaccines and the biological containment of recombinant organisms. To this end, we report an approach based on genetically replacing key histidine (His) residues in essential proteins with functional His analogs. We demonstrate that 3-methyl-l-histidine (MeH) functionally substitutes for a key metal binding ligand, H264, in the zinc-containing metalloenzyme mannose-6-phosphate isomerase (ManA). An evolved variant, Opt5, harboring both N262S and H264MeH substitutions exhibited comparable activities to wild type ManA. An engineered Escherichia coli strain containing the ManA variant Opt5 was strictly dependent on MeH for growth with an extremely low reversion rate. This straightforward strategy should be applicable to other metallo- or nonmetalloproteins that contain essential His residues.

Full text of DOI:10.1021/jacs.7b13452

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Functional replacement of histidine in proteins to
generate noncanonical amino acid dependent organisms
Fei Gan, Renhe Liu, Feng Wang, and Peter G Schultz
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b13452 • Publication Date (Web): 06 Mar 2018
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Functional replacement of histidine in proteins to generate
noncanonical amino acid dependent organisms
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1, 2
Fei Gan, Renhe Liu, Feng Wang, and Peter G. Schultz*
1
California Institute for Biomedical Research (Calibr), La Jolla, California 92037, United States
2
Department of Chemistry and the Skaggs Institute for Chemical Biology, the Scripps Research Institute, La
Jolla, California 92037, United States.
Supporting Information Placeholder
ABSTRACT: Simple strategies to produce organisms whose
growth is strictly dependent on the presence of
a
Metalloproteins account for one third to one half of naturally
occurring proteins, and metal ions can play key catalytic
roles or act as essential structural elements . In particular,
noncanonical amino acid are useful for the generation of live
vaccines and the biological containment of recombinant
organisms. To this end, we report an approach based on
genetically replacing key histidine (His) residues in essential
proteins with functional His analogs. We demonstrate that 3-
methyl-L-histidine (MeH) functionally substitutes for a key
metal binding ligand, H264, in the zinc-containing
metalloenzyme mannose-6-phosphate isomerase (ManA). An
evolved variant, Opt5, harboring both N262S and H264MeH
substitutions exhibited comparable activities to wild type
ManA. An engineered Escherichia coli strain containing the
ManA variant Opt5 was strictly dependent on MeH for
growth with an extremely low reversion rate. This
straightforward strategy should be applicable to other
metallo- or non-metalloproteins that contain essential His
residues.
9,10
zinc ions are required for many hydrolytic and redox
enzymes, and histidine (His) is the most common metal
1
1-14
coordinating ligand
. It is also noted that two single
mutations are required to convert a nonsense codon
(e.g.,UAA, UAG) to either of the native codons for His (CAU
and CAC). Therefore, substitution of a metal-binding His in
an essential protein with a functional His analog, encoded by
an amber or ochre nonsense codon, should result in ncAA
conditional cell survival with a low reversion rate, as
reversion to His would be very unlikely.
Genetically encoded noncanonical amino acids (ncAAs) are a
powerful tool for probing the structure and function of
proteins, as well as creating proteins with novel or advanced
1
-3
activities . Recently, organisms strictly depending on the
presence of ncAAs have been created for biological
4
-8
containment and live attenuated vaccines . Engineered
organisms grow only when the specified ncAA is available to
translate an in-frame nonsense codon to produce a fully
functional protein. However, the incorporation of ncAAs at
catalytically active sites or at key structural elements usually
adversely affects protein activity, whereas substitution at
permissive sites suffers from high escape frequencies due to
naturally occurring mutations. Therefore, additional
strategies to create ncAA-dependent organisms with normal
growth and low escape frequencies are needed.
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Figure 1. ManA as a model system and ncAAs used in this
incorporated in response to an amber codon at either H99 or
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study. (A) Canonical amino acid His (left) and its selected
analogs 3-methyl-L-Histidine (MeH, middle) and 3-(3-
pyridyl)-alanine (PyA, right). (B) The reaction catalyzed by
H264 site.
We next investigated the activities of ManA variants H99X
and H264X using a growth assay with an E.coli mutant strain
lacking endogenous ManA activity. Due to the inactivation of
its native manA gene, E.coli ΔmanA mutant strain is unable
to grow on D-mannose as the sole carbon source (Figure 2A).
Heterologous expression of WT Mtb ManA in this strain
restored the capacity to use D-mannose for growth (Figure
2+
ManA. (C) The Zn -binding pocket of ManA from Candida
2+
albicans (PDB: 1PMI, Zn cofactor shown in green).
Metal-chelating ncAAs including 2,2'-bipyridin-5-yl-alanine
(Bpy-Ala) and 8-hydroxyquinolin-3-yl-alanine have been
2+
2B). To determine the activities of H99X and H264X variants,
genetically encoded to generate a Cu -dependent DNA
1
5
16
we co-transformed plasmid pH99TAG or pH264TAG
individually with pUltra-HRS into E.coli ΔmanA cells, and
cleaving protein , metal-binding biophysical probes , novel
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iron(II) binding zinc finger-like motifs , catalytic dimer
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8,19
20
grew
the
resulting
strain
(Ec.ΔmanA.H99X
or
interfaces
, and stabilized trimeric helical motifs .
Ec.ΔmanA.H264X) on D-mannose minimal medium (termed
M9M medium) supplemented with 1 mM IPTG, appropriate
antibiotics, and 1 mM MeH or PyA (Materials and Methods
in SI). No growth was observed for Ec.ΔmanA.H99TAG cells
in the presence of MeH or PyA (Figure 2C and 2E),
suggesting neither ncAA can functionally substitute for H99.
Strain Ec.ΔmanA.H264TAG also did not grow in the presence
of PyA (Figure 2D); however, MeH can functionally replace
H264 and growth was observed for this strain when
supplemented with 1 mM MeH (Figure 2F). These results
agree with previous studies proposing a dual role of H99
Engineering of Bpy-Ala dependent metalloproteins with
atom level accuracy was accomplished by Miller and
21,22
coworkers using computational design method . However,
it remains challenging to introduce these bulky bidentate
1
7,21,22
ncAAs at enzyme active sites and retain native activity
.
Structurally similar analogs to His, such as 3-methyl-L-
histidine and 3-(3-pyridyl)-L-alanine (Figure 1A), are
potential functional replacements for His with minimal
23-25
perturbation on protein structure and activity
. To
demonstrate this notion, we selected the metalloenzyme
mannose-6-phosphate isomerase (ManA) as a model. ManA
is an essential enzyme in various pathogens, such as Candida
albicans and Mycobacterium tuberculosis (Mtb), and
catalyzes the interconversion of D-mannose-6-phosphate
1
involving both imidazole nitrogens whereas the N of H264
chelates Zn
2+ 28,29
.
(
M6P) and D-fructose-6-phosphate (F6P) (Figure 1B) which
are involved in multiple metabolic pathways, cell wall
2
6,27
formation, and capsular polysaccharide biosynthesis
activity of ManA relies on a metal cofactor Zn
. The
. Two His
(H), one Glu (E), and one Gln (Q) (or Lys in certain
2+ 28
2+
organisms) commonly serve as Zn -binding ligands and are
highly conserved in ManA homologs (Figure 1C and Figure
S1). In particular Mtb ManA contains the two His residues
(
H99 and H264, Figure S1) and H99 has been proposed to act
28,29
as a general acid in the ring opening of substrate
.
To incorporate His analogs in response to an amber codon at
site H99 or H264 of Mtb ManA, we first constructed a
recombinant plasmid pWT for expression of wild type (WT)
ManA under the T5 promoter. The codons at site 99 and 264
were then independently mutated to TAG to afford plasmids
pH99TAG and pH264TAG for expression of ManA variants
H99X and H264X (X: MeH or PyA), respectively. Each of the
constructs was individually co-transformed into Escherichia
coli (E. coli) DH10B cells with plasmid pUltra-HRS, which
harbors the genes encoding the cognate aaRS and tRNA for
Figure 2. A ManA deficient E.coli strain expressing Mtb WT
ManA and ncAA-containing variants. (A) and (B) Growth of
the E.coli ΔmanA mutant and strain expressing Mtb wild type
ManA (ManA WT) respectively. (C) and (E) Growth of
Ec.ΔmanA.H99TAG strain in the presence of 1 mM PyA
(H99PyA) and 1 mM MeH (H99MeH) on M9M selection
medium
Ec.ΔmanA.H264TAG strain in the presence of 1 mM PyA
H264PyA) and 1 mM MeH (H264MeH) on M9M selection
respectively.
(D)
and
(F)
Growth
of
3
0
specific incorporation of the His analogues . The aminoacyl-
tRNA synthase HRS is polyspecific and uses both MeH and
PyA as substrates, but does not recognize His. Protein
(
medium respectively.
expression was induced with
1 mM isopropyl β-D-1-
thiogalactopyranoside (IPTG) in the presence of 1 mM MeH
or PyA. The yields for ncAA-containing variants after
purification were ~ 10-30 µg/L, while a higher yield was
obtained for WT ManA (250 µg/L). Mass spectrometry
analysis showed that the observed masses for WT, H99MeH,
H99PyA, H264MeH, and H264PyA were 44194 Da, 44208 Da,
Considering several canonical amino acid residues (e.g., Glu,
Cys) can function as metal binding ligands, we next
determined if H264 can be replaced by any of the common
amino acids. We therefore mutated H264 to each of the
other 19 canonical amino acids. None of the canonical ManA
variants restored growth of the E.coli ΔmanA mutant on D-
mannose (Figure S3). These data demonstrate that any
canonical substitution at 264 site, which could occur through
a single mutation of an amber codon, leads to inactivated
4
4205 Da, 44208 Da, and 44205 Da respectively (Figure S2),
which were consistent with the expected masses. These data
indicate that both MeH and PyA can be specifically
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ManA. A low reversion rate is thus expected for the
H264MeH (Figure S5), we reasoned that the faster cell
growth could be due to an improvement in activity. The
catalytic activities of purified WT, Opt5 and H264MeH
ManA proteins were then measured using a coupled reaction
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engineered strain containing the His264MeH ManA variant.
To test this notion, we grew Ec.ΔmanA.H264MeH cells
continuously in the presence of 1 mM MeH, and screened
1
1
+
1
x10 cells for survival on non-permissive medium (in the
monitoring absorbance at 340 nm due to reduction of NADP
absence of MeH) for a 2-week incubation at 30°C. The
(Figure 4, Figures S7 and S8). The kcat and K values of variant
m
-
1
-1
reversion rate determined for Ec.ΔmanA.H264MeH was less
Opt5 (kcat = 29.5 ± 1.0 s ; K
s ) are comparable to those of WT enzyme (kcat = 29.3 ± 1.2 s ;
m
= 1.2 ± 0.1 mM; kcat/K
m
= 24.6 mM
-
11
-1
-1
than 10 escapees per colony forming unit. Importantly,
-1 -1
K = 0.7 ± 0.1 mM; k /K = 41.9 mM s ). We could not obtain
these values are below the acceptable reversion frequency
m
cat
m
−8
kcat and Km values for H264MeH which exhibited a low
(
10 ) for biocontainment requirement in the guidelines of
31
activity. To explain the increased activity of the N262S
the National Institutes of Health (NIH) .
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mutation, a model of Mtb ManA with I-TASSER was
constructed (Figure S9). N262 is located in a loop that
connects the β-sheet containing H264, and is in close
Despite growing continuously in the presence of MeH, the
Ec.ΔmanA.H264MeH strain grew slower than the
Ec.ΔmanA.WT strain (Figure S4). Western blot analysis
showed a lower amount of variant H264MeH relative to WT
ManA was expressed in cells (Figure S5) possibly due to the
efficiency of MeH incorporation by the cognate HRS/tRNA
pair at this site. Considering ManA plays critical roles in
cellular metabolism, a lower ManA content could lead to a
decrease in growth rate. Another possibility is that the
3
proximity (~4 Å) to the N atom of H264. It is likely that this
mutation helps sterically to accommodate the H264MeH
substitution, and potentially improves the geometry of the
2+
Zn coordination site. Although one might like to further
increase the growth rate of the Opt5 strain to that of the WT
strain by improving protein expression levels, a conditional
vaccine will be produced ex vivo and therefore the observed
growth rate should be sufficient.
3
methyl group at N of MeH might slightly alter the geometry
2+
of the Zn site, leading to reduced activity. We therefore
performed random mutagenesis on ManA coding sequence
by error prone PCR in an effort to evolve an H264MeH
variant with increased activity. The amount of template
pH264TAG was adjusted to achieve a high mutation rate (9-
Perhaps the most critical experiment for a conditional
vaccine is to evaluate whether the N262S mutation affects
11
the low escape frequency. We therefore screened 1x10
Ec.ΔmanA. Opt5 cells for survival on non-permissive
medium (in the absence of MeH) for a 2-weeks incubation at
16 mutations per kb) during error prone PCR. The resulting
-
11
3
0°C. The reversion rate was less than 10 escapees per
library (pH264TAG-RM) was introduced into E.coli ΔmanA
mutant cells harboring the pUltra-HRS plasmid, followed by
screening on MeH-containing M9M selection medium.
Sequencing of colonies exhibiting faster growth revealed a
single mutation N262S along with the amber codon at H264
site (Figure S6A) (denoted Opt5). The observed mass of
purified Opt5 protein was 44181 Da, which was consistent
with expected mass of a ManA variant with both N262S and
H264MeH substitutions (Figure S6B).
colony forming unit, comparable to that of strain
Ec.ΔmanA.H264MeH.
Figure 4. Catalytic activity of WT ManA and variants.
Activity was measured by monitoring A3
from reduction
40nm
+
of NADP in a coupled reaction. Error bars represent
standard deviation of triplicates. M6P: D-mannose-6-
phosphate.
In summary, we describe a strategy for creating ncAA
dependent organisms by genetically substituting key His
residues in essential proteins with functional His analogs.
This strategy uses an orthogonal tRNA/aaRS pair to insert
MeH at in-frame amber codons, and thus requires minimal
genetic modification of the host organisms. Notably, this
strategy results in stringent ncAA dependence and extremely
low reversion rates. Although we chose a His residue that
Figure 3. Growth of E.coli ΔmanA cells expressing WT and
variants Opt5 and H264MeH on solid M9M selective
medium in the presence or absence of 1 mM MeH. Two
single colonies for each were assayed. Equal number of cells
(
2
after 10-fold serial dilution) were spotted and cultivated for
4 hours.
2+
We further characterized the ManA variant Opt5 in
comparison with WT ManA and variant H264MeH. The
Ec.ΔmanA.Opt5 strain grew only in the presence of 1 mM
MeH on selective medium with improved rate compared
with Ec.ΔmanA.H264MeH strain (Figure 3 and Figure S4).
Given variant Opt5 was expressed at a similar level as
coordinates a catalytic Zn ion, this approach can likely be
2+
applied to structural Zn sites, and His residues that play
other critical roles in catalysis. We are currently exploring
the feasibility of using this system to create ncAA conditional
live vaccines.
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(18) Bos, J.; Browne, W. R.; Driessen, A. J. M.; Roelfes, G. J. Am.
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Chem. Soc. 2015, 137, 9796.
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ASSOCIATED CONTENT
Chem., Int. Ed. 2012, 51, 7472.
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Chem. Biol. 2016, 23, 1098.
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
(21) Mills, J. H.; Khare, S. D.; Bolduc, J. M.; Forouhar, F.;
Mulligan, V. K.; Lew, S.; Seetharaman, J.; Tong, L.; Stoddard, B.
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Supplementary Materials and Methods, Figures s1-s9, and
Table S1.
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AUTHOR INFORMATION
1
5012.
Corresponding Author
(23) Pott, M.; Hayashi, T.; Mori, T.; Mittl, P. R. E.; Green, A. P.;
Hilvert, D. J. Am. Chem. Soc. 2018.
(24) Sharma, V.; Wang, Y. S.; Liu, W. R. ACS Chem. Biol. 2016,
11, 3305.
*schultz@scripps.edu.
Notes
(
25) Green, A. P.; Hayashi, T.; Mittl, P. R.; Hilvert, D. J. Am.
Chem. Soc. 2016, 138, 11344.
26) Griffin, J. E.; Gawronski, J. D.; Dejesus, M. A.; Ioerger, T.
The authors declare no competing financial interests.
(
ACKNOWLEDGMENT
R.; Akerley, B. J.; Sassetti, C. M. PLoS Pathog. 2011, 7,
e1002251.
(27) Cleasby, A.; Wonacott, A.; Skarzynski, T.; Hubbard, R. E.;
Davies, G. J.; Proudfoot, A. E.; Bernard, A. R.; Payton, M. A.;
Wells, T. N. Nat. Struct. Biol. 1996, 3, 470.
The authors thank Dr. Jeremy Mills for initial computional
analysis. The authors also acknowledge Dr. Weimin Xuan
and Dr. Minseob Koh for discussions and Kristen Williams
for assistance in manuscript preparation. This work was
supported, in part, by Bill and Melinda Gates Foundation.
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(
Schultz, P. G. ACS Chem. Biol. 2014, 9, 1092.
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014, 15, 822.
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Journal of the American Chemical Society
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Journal of the American Chemical Society
Page 6 of 7
SYNOPSIS TOC (Word Style “SN_Synopsis_TOC”). If you are submitting your paper to a journal that requires a synopsis graphic and/or
synopsis paragraph, see the Instructions for Authors on the journal’s homepage for a description of what needs to be provided and for the
size requirements of the artwork.
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To format double-column figures, schemes, charts, and tables, use the following instructions:
Place the insertion point where you want to change the number of columns
From the Insert menu, choose Break
Under Sections, choose Continuous
Make sure the insertion point is in the new section. From the Format menu, choose Columns
In the Number of Columns box, type 1
Choose the OK button
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Now your page is set up so that figures, schemes, charts, and tables can span two columns. These must appear at
the top of the page. Be sure to add another section break after the table and change it back to two columns with a
spacing of 0.33 in.
Table 1. Example of a Double-Column Table
Column 1
Column 2
Column 3
Column 4
Column 5
Column 6
Column 7
Column 8
Authors are required to submit a graphic entry for the Table of Contents (TOC) that, in conjunction with the
manuscript title, should give the reader a representative idea of one of the following: A key structure, reaction,
equation, concept, or theorem, etc., that is discussed in the manuscript. Consult the journal’s Instructions for
Authors for TOC graphic specifications.
Insert Table of Contents artwork here
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Journal of the American Chemical Society
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TOC Image
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6x21mm (300 x 300 DPI)
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