Selenocysteine in native chemical ligation and expressed protein ligation

Full text of DOI:10.1021/ja005885t

5140
J. Am. Chem. Soc. 2001, 123, 5140-5141
Scheme 1
Selenocysteine in Native Chemical Ligation and
Expressed Protein Ligation
Robert J. Hondal,† Bradley L. Nilsson,‡ and
Ronald T. Raines*,†,‡
Department of Biochemistry and
Department of Chemistry, UniVersity of Wisconsin-Madison
Madison, Wisconsin 53706
ReceiVed December 14, 2000
L-Selenocysteine (Sec or U) has been called the “21st amino
acid”.1 Like the twenty common amino acids, selenocysteine is
inserted during the translation of mRNA and has its own tRNASec
and codon, UGA. This codon also serves as the opal stop codon.
Decoding a UGA codon as one for selenocysteine requires a
special structure in the 3′ untranslated region of the mRNA called
a selenocysteine insertion sequence (SECIS) element. Because
eukaryotic and prokaryotic cells use a different SECIS element
to decode UGA as selenocysteine, the production of eukaryotic
selenocysteine-containing proteins in prokaryotes is problematic.2
Here, we describe a general semisynthetic route to proteins
containing selenocysteine.3,4
Scheme 2
In “native chemical ligation”, the thiolate of an N-terminal
cysteine residue in one peptide attacks a C-terminal thioester in
another peptide to produce, ultimately, an amide bond between
the two peptides (Scheme 1).5 “Expressed protein ligation” is an
extension in which the C-terminal thioester is produced by using
recombinant DNA (rDNA) technology.6 We reasoned that
selenocysteine, like cysteine, could effect both native chemical
ligation and expressed protein ligation, and thereby provide a
means to incorporate selenocysteine into proteins.
We used AcGlySCH2C(O)NHCH3 as a model thioester to test
the feasibility of using selenocysteine in native chemical ligation.7
Reaction with cystine ((CysOH)2) in the presence of the reducing
agent tris-(2-carboxyethyl)phosphine (TCEP) produced AcGly-
CysOH, as well as some (AcGlyCysOH)2. When selenocystine
((SecOH)2) was used in the same reaction, the product was
(AcGlySecOH)2.8
A selenolate (RSe-) is more nucleophilic than is its analogous
thiolate (RS-).9 Moreover, the pKa of a selenol (RSeH) is lower
than that of its analogous thiol (RSH).9a,10 These properties
suggested to us that native chemical ligation with selenocysteine
could be more rapid than with cysteine, especially at low pH. To
test this hypothesis, we used the chromogenic thioester AcGly-
SC6H4-p-NO2 (1; Scheme 2) to determine the rate of native
chemical ligation as a function of pH.11 The resulting pH-rate
profile is shown in Figure 1. Reaction with selenocysteine is 103-
fold faster than with cysteine at pH 5.0. Thus, native chemical
ligation with selenocysteine can be chemoselective.12
Having demonstrated the effectiveness of selenocysteine in
native chemical ligation, we next set out to explore its utility in
expressed protein ligation. As a model protein, we chose
ribonuclease A (RNase A; EC 3.1.27.5; Figure 2), which has been
the object of much seminal work in protein chemistry.14 RNase
A has 8 cysteine residues that form 4 disulfide bonds in the native
* To whom correspondence should be addressed. Telephone: (608) 262-
8588. Fax: (608) 262-3453. E-mail: raines@biochem.wisc.edu.
† Department of Biochemistry.
‡ Department of Chemistry.
(1) (a) Bock, A.; Forchhammer, J.; Heider, W.; Leinfelder, G.; Veprek,
B.; Zinoni, F. Mol. Microbiol. 1991, 5, 515-520. For other reviews, see: (b)
Odom, J. D. Structure Bonding 1983, 54, 1-26. (c) Low, S. C.; Berry, M. J.
Trends Biochem. Sci. 1996, 21, 203-208. (d) Stadtman, T. C. Annu. ReV.
Biochem. 1996, 65, 83-100.
(2) Arner, E. S. Sarioglu, H.; Lottspeich, F.; Holmgren, A.; Bock, A. J.
Mol. Biol. 1999, 292, 1003-1016.
(3) For other means to incorporate selenocysteine into semisynthetic and
synthetic proteins, see: (a) Wu, Z. P.; Hilvert, D. J. Am. Chem. Soc. 1989,
111, 4513-4514. (b) Fiori, S.; Pegoraro, S.; Rudolph-Bohner, S.; Cramer, J.;
Moroder, L. Biopolymers 2000, 53, 550-564.
(9) (a) Huber, R. E.; Criddle, R. S. Arch. Biochem. Biophys. 1967, 122,
164-173. (b) Pearson, R. G.; Sobel, H.; Songstad, J. J. Am. Chem. Soc. 1968,
90, 319-326. (c) Pleasants, J. C.; Guo, W.; Rabenstein, D. L. J. Am. Chem.
Soc. 1989, 111, 6553-6558. (d) Singh, R.; Whitesides, G. M. J. Org. Chem.
1991, 56, 6931-6933. (e) Gorlatov, S. N.; Stadtman, T. C. Proc. Natl. Acad.
Sci. U.S.A. 1998, 95, 8520-8525. (f) Lee, S.-R.; Bary-Noy, S.; Kwon, J.;
Levine, R. L.; Stadtman, T. C.; Rhee, S. G. Proc. Natl. Acad. Sci. U.S.A.
2000, 97, 2521-2526. (g) Zhong, L.; Holmgren, A. J. Biol. Chem. 2000,
275, 18121-18128.
(4) For a means to produce selenopeptide libraries on the surface of M13
bacteriophage, see: Sandman, K. E.; Benner, J. S.; Noren, C. J. J. Am. Chem.
Soc. 2000, 122, 960-961.
(5) (a) Wieland, T.; Bokelmann, E.; Bauer, L.; Lang, H. U.; Lau, H. Liebigs
Ann. Chem. 1953, 583, 129-149. (b) Dawson, P. E.; Muir, T. W.; Clark-
Lewis, I.; Kent, S. B. Science 1994, 266, 776-779. For a recent review, see:
(c) Dawson, P. E.; Kent, S. B. H. Annu. ReV. Biochem. 2000, 69, 923-960.
(6) (a) Muir, T. W.; Sondhi, D.; Cole, P. A. Proc. Natl. Acad. Sci. U.S.A.
1998, 95, 6705-6710. For reviews, see: (b) Cotton, G. J.; Muir, T. W. Chem.
Biol. 1999, 6, R247-R256. (c) Evans, T. C., Jr.; Xu, M.-Q. Biopolymers 2000,
51, 333-342.
(10) Arnold, A. P.; Tan, K. S.; Rabenstein, D. L. Inorg. Chem. 1986, 25,
2433-2437.
(11) Ligation assays were performed at (23 ( 2) °C under Ar(g) in 0.10
M buffer containing thioester 1 (10 µM), TCEP (0.10 mM), and cystine (20
µM-0.50 mM) or selenocystine (1.0-20 µM). Rate ) ∂[HSC6H4-p-NO2]/∂t
(7) AcGlySCH2C(O)NHCH3 was synthesized as described by Nilsson B.
L.; Kiessling, L. L.; Raines, R. T. Org. Lett. 2000, 2, 1939-1941. We find
N-methylmercaptoacetamide to be a superb thiol for effecting both native
chemical ligation and expressed protein ligation.
(8) L-Selenocystine was synthesized from elemental selenium and â-chloro-
L-alanine as described by Tanaka, H.; Soda, K. Methods Enzymol. 1987, 143,
240-243. Ligation reactions were performed at (23 ( 2) °C under Ar(g) in
0.10 M Tris-HCl buffer (pH 8.0) containing AcGlySCH2C(O)NHCH3 (25
mM), TCEP (25 mM), and cystine or selenocystine (12.5 mM).
) kobs[CysOH or SecOH] - kH O was determined by using ꢀ ) 11, 230 M-1
2
cm-1 for p-nitrothiophenolate at 410 nm.
(12) The template-directed ligation of a phosphoroselenol and iodoribose
is approximately 4-fold faster than that of an analogous phosphorothiol at pH
7.0 (Xu, Y.; Kool, E. T. J. Am. Chem. Soc. 2000, 122, 9040-9041). We
observe a similar difference in Se vs S reactivity at high pH (Figure 1).
(13) Danehy, J. P.; Parameswaran, K. N. J. Chem. Eng. Data 1968, 13,
386-389.
(14) Raines, R. T. Chem. ReV. 1998, 98, 1045-1065.
10.1021/ja005885t CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/04/2001

Communications to the Editor
J. Am. Chem. Soc., Vol. 123, No. 21, 2001 5141
ribonucleolytic activities [kcat/Km ) (1.13 ( 0.06) × 107 M-1 s-1
and (1.1 ( 0.1) × 107 M-1 s-1, respectively],19 indicating that
C110U RNase A is not only intact but also folded properly (Figure
2). We conclude that the isomorphous replacement of sulfur with
selenium can be effected with expressed protein ligation.
Incorporation of selenocysteine residues into proteins could
have much utility. For example, proteins containing selenosulfide
(Se-S) or diselenide (Se-Se) bonds could have high conforma-
tional stability in a reducing environment (such as the cytosol),
as the reduction potential of selenosulfide and diselenide bonds
is less than that of the corresponding disulfide bond.19 In addition,
incorporation of selenocysteine could be used to reveal structure-
function relationships with 77Se NMR spectroscopy20 or determine
the phase of protein crystals.21 Finally, chemoselective alkylation
of the side chain of a selenocysteine residue9 or reduction of the
C-Se bond to create alanine22 would expand the scope of protein
semisynthesis.
Many natural proteins contain selenocysteine residues. For
example, mammalian thioredoxin reductase uses a selenosulfide
bond in its catalytic mechanism.9e Thiol:disulfide oxidoreductases
have a CXXC motif in their active site.23 Likewise, selenoprotein
W has a CXXU motif, and selenoprotein P has a UXXC motif
as well as 16 other cysteine and 9 other selenocysteine residues.24
The role of selenocysteine in the function of selenoprotein W or
selenoprotein P is unknown. The methods described herein make
these and other selenocysteine-containing proteins readily amen-
able to detailed structure-function analyses.
Figure 1. pH-Rate profile for the reaction in Scheme 2 with CysOH
(O) and SecOH (b).11 Data were fitted to the equation: kobs ) k[1]/(1 +
[H+]/Ka) with pKaCys ) 8.3013 and pKaSec ) 5.24,8a yielding k ) (3.7 ×
102) M-1 s-1 for CysOH and k ) (9.5 × 102) M-1 s-1 for SecOH.
Acknowledgment. We thank H. J. Reich and C. Park for advice,
and T. C. Evans, Jr. for plasmid pTXB1-RNase. R.J.H. was supported
by Postdoctoral Fellowship GM20180 (NIH). This work was supported
by Grant GM44783 (NIH).
Note Added in Proof. Subsequent to the submission of this
manuscript, van der Donk and co-workers reported the synthesis
of a selenocysteine-containing peptide by native chemical ligation.
Gieselman, M. D.; Xie, L.; van der Donk, W. A. Org. Lett. 2001,
3, 1331-1334.
Figure 2. Ribbon diagram of the three-dimensional structure of ribo-
nuclease A.14 C-Terminal residues 110-124 (white) can derive from a
synthetic peptide via expressed protein ligation.
enzyme. Of these 8 cysteine residues, Cys110 is closest to the
C-terminus.
Supporting Information Available: Procedures for all syntheses and
analyses reported herein (PDF). This material is available free of charge
via the Internet at http://pubs.acs.org.
To determine whether selenocysteine can effect expressed
protein ligation, we used rDNA technology to prepare a fragment
corresponding to residues 1-109 with a C-terminal thioester.15
We used standard solid-phase methods to synthesize a peptide
corresponding to residues 110-124 with a cysteine or a seleno-
cysteine residue16 at position 110. We then ligated the thioester
fragment and a peptide fragment, and folded and purified the
ligation product. Semisynthetic wild-type RNase A has m/z
13 819, and C110U RNase A has m/z 13 865. The ∆m/z of 46 is
in gratifying agreement with the ∆Mr ) 47 expected for the
replacement of sulfur with selenium.
JA005885T
(17) This fragment lacks His119 and the Cys58-Cys110 disulfide bond.
For their roles, see: (a) Thompson, J. E.; Raines, R. T. J. Am. Chem. Soc.
1994, 116, 5467-5468. (b) Klink, T. A.; Woycechowsky, K. J.; Taylor, K.
M.; Raines, R. T. Eur. J. Biochem. 2000, 267, 566-572.
(18) Catalysis was assessed by using the fluorogenic substrate 6-carboxy-
fluorescein∼dArU(dA)2∼6-carboxytetramethylrhodamine as described by
Kelemen, B. R.; Klink, T. A.; Behlke, M. A.; Eubanks, S. R.; Leland, P. A.;
Raines, R. T. Nucleic Acids Res. 1999, 18, 3696-3701.
(19) (a) Besse, D.; Siedler, F.; Diercks, T.; Kessler, H.; Moroder, L. Angew.
Chem., Int. Ed. Engl. 1997, 36, 883-885. (b) Pegoraro, S.; Fiori, S.; Rudolph-
Bohner, S.; Watanabe, T. X.; Moroder, L. J. Mol. Biol. 1998, 284, 779-792.
(20) For an example, see: House, K. L.; Dunlap, R. B.; Odom, J. D.; Wu,
Z. P.; Hilvert, D. J. Am. Chem. Soc. 1992, 114, 8573-8579.
(21) For examples with selenomethionine, see: (a) Hendrickson, W. A.;
Horton, J. R.; LeMaster, D. M. EMBO J. 1990, 9, 1665-1672. (b) Doublie,
S. Methods Enzymol. 1997, 276, 523-530. (c) Shao, W.; Fernandez, E.;
Wilken, J.; Thompson, D. A.; Siani, M. A.; West, J.; Lolis, E.; Schweitzer,
B. I. FEBS Lett. 1998, 441, 77-82.
The gain of function provides another indication of a successful
ligation. The fragment corresponding to residues 1-109 has no
detectable ribonucleolytic activity.17,18 In contrast, semisynthetic
wild-type RNase A and C110U RNase A have equivalent
(22) Possible routes involve photochemical, tin-hydride, or hydrogenolytic
reduction. For examples, see: (a) Pandey, G.; Soma Sekhar, B. B. V.; Bhalerao,
U. T. J. Am. Chem. Soc. 1990, 112, 5650-5651. (b) Light, J.; Breslow, R.
Org. Synth. 1995, 72, 199-208. (c) Yan L. Z.; Dawson P. E. J. Am. Chem.
Soc. 2001, 123, 526-533.
(15) This fragment was produced with plasmid pTXB1-RNase, which
directs the production in Escherichia coli of the Mxe intein fused to residues
1-109 of RNase A plus an N-terminal methionine. Evans, T. C., Jr.; Benner,
J.; Xu, M.-Q. Protein Sci. 1998, 7, 2256-2264.
(16) FmocSec(PMB)OH was synthesized essentially as described by Koide,
T.; Itoh, H.; Otaka, A.; Yasui, H.; Kuroda, M.; Esaki, N.; Soda, K.; Fujii, N.
Chem. Pharm. Bull. 1993, 41, 502-506.
(23) Chivers, P. T.; Prehoda, K. E.; Raines, R. T. Biochemistry 1997, 36,
4061-4066.
(24) Burk, R. F.; Hill, K. E. Bioessays 1999, 21, 231-237.