Allosterically Regulated Phosphatase Activity from Peptide–PNA Conjugates Folded Through Hybridization

Article abstract of DOI:10.1002/anie.201602751

The importance of spatial organization in short peptide catalysts is well recognized. We synthesized and screened a library of peptides flanked by peptide nucleic acids (PNAs) such that the peptide would be constrained in a hairpin loop upon hybridization. A screen for phosphatase activity led to the discovery of a catalyst with >25-fold rate acceleration over the linear peptide. We demonstrated that the hybridization-enforced folding of the peptide is necessary for activity, and designed a catalyst that is allosterically controlled using a complementary PNA sequence.

Full text of DOI:10.1002/anie.201602751

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International Edition: DOI: 10.1002/anie.201602751
German Edition: DOI: 10.1002/ange.201602751
Peptide Catalysts
Allosterically Regulated Phosphatase Activity from Peptide–PNA
Conjugates Folded Through Hybridization
Takuya Machida+, Som Dutt+, and Nicolas Winssinger*
Abstract: The importance of spatial organization in short
peptide catalysts is well recognized. We synthesized and
screened a library of peptides flanked by peptide nucleic
acids (PNAs) such that the peptide would be constrained in
a hairpin loop upon hybridization. A screen for phosphatase
activity led to the discovery of a catalyst with > 25-fold rate
acceleration over the linear peptide. We demonstrated that the
hybridization-enforced folding of the peptide is necessary for
activity, and designed a catalyst that is allosterically controlled
using a complementary PNA sequence.
induce a b-turn)[3a] and position the required side chains in
close proximity. Oligonucleotides adopt stable tertiary struc-
tures with relatively short sequences. However, relatively few
reactions are efficiently catalyzed by structured oligonucleo-
tides, phosphodiester cleavage by the hammerhead ribo-
zyme[4] being one example (Figure 1). Clearly, the larger
pallet of functional groups present on amino acid side chains
endows proteins with a broader scope of chemistries. We
reasoned that short peptides conjugated to flanking oligonu-
cleotides should allow for structuring through hybridization,
resulting in a productive disposition of amino acid side chains
for catalysis (Figure 1). For ease of conjugate synthesis,
peptide nucleic acids (PNA)[5] were used because the
oligomer could be synthesized by standard solid-phase
peptide synthesis (SPPS). Hybridization of PNA–peptide
conjugates has already been used to modulate the conforma-
tion of peptides involved in biologically relevant peptide-
receptor interactions;[6] however, this approach has not been
used in the context of catalysis.
The library was designed to contain a bias for residues
frequently found in hydrolytic enzymes (His, Ser, Asp, Lys) in
a hairpin loop resulting from a constant CGGC sequence
prior to the peptide and a complementary GCCG sequence at
the end of the peptide. A preliminary experiment showed that
a tetraglycine hairpin with this sequence had a Tm of 708C,
ensuring a predictable folding. A library of 1000 PNA-
peptide-PNA adducts was prepared by split-and-mix syn-
thesis[7] using 3 ꢀ 10 sets of peptide fragments with variable
length (0- to 4-mer; Figure 2). Aliquots of each synthetic pool
were kept for eventual deconvolution and the pools from the
third peptide fragment synthesis were kept as discrete entities
for screening, thus affording the library as 10 pools of 100
putative catalysts with a hairpin composed of 4–10mer
peptides. The synthesis made use of an orthogonal linker
triggered by the reduction of the para-azidobenzyloxy moiety
and 1,6-elimination upon azide reduction (Figure 2). We have
previously shown that this type of elimination proceeds with
a half-life of approximately 20 min (k = 0.035 minÀ1).[8] To
prevent acid-catalyzed cleavage of the benzyloxy moiety
during peptide deprotection, a carboxamide was introduced
ortho to the benzyloxy group. Cleavage of the present linker
with 2 mm PMe3 in THF/H2O had a comparable half-life of
30 min.
C
atalysis in living systems is generally achieved by proteins
that fold in unique three-dimensional structures. However,
only a few residues generally participate in catalysis, as
exemplified by the catalytic triad of proteases (Figure 1).[1]
The relatively large size of enzymes (> 20 kDa) is required to
achieve a stable, folded conformation. The prospect of
recapitulating a given catalytic reaction using minimal
peptides or a rigid scaffold to position the required function-
alities has attracted significant attention, and catalysis with
oligopeptides has delivered ground-breaking results.[2,3] For
catalysis with short peptides, select amino acids are frequently
included to create a conformational bias (for instance, Pro-
Aib- or Ca-tetrasubstituted amino acids have been used to
Figure 1. Representation of catalysts arising from controlled position-
ing of key functionalities in space: left, catalytic triad of trypsin (PDB
ID:2PTC); middle, hammerhead ribozyme (PDB ID:3ZD5); right,
a peptide–oligonucleotide structure folded through hybridization.
[*] T. Machida,[+] Dr. S. Dutt,[+] Prof. N. Winssinger
Department of Organic Chemistry, NCCR Chemical Biology
University of Geneva
Quai Ernest Ansermet 30, 1211 Geneva (Switzerland)
E-mail: nicolas.winssinger@unige.ch
[+] These authors contributed equally to this work.
Several methods have been reported to screen libraries of
catalysts on beads.[3b,d,9,10] We opted to screen for the
hydrolysis of a phosphate ester based on the fact that the
background hydrolysis is very slow and cleavage can be
monitored using a fluorogenic precipitating dye (quinazoli-
none precipitating dye: QPD; Figure 3)[11] which is routinely
used to image alkaline phosphatase activity.[12,13]
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under
http://dx.doi.org/10.1002/anie.201602751.
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discernable amongst the 10 pools, with the pool correspond-
ing to the fragment C8 showing some of the brightest beads.
The brightest beads were removed with a pipette tip,
distributed on a MALDI plate and treated with 2 mL of
a 2 mm PMe3 solution followed by matrix for direct MALDI
analysis. Three of the beads had the mass corresponding to
the sequence Lys-His-Ile-Ser-Glu-Ser (A5-B2-C8), with
a mass redundancy between pool B2, B5, and B6. Based on
this redundancy, the three pools corresponding to redundant
masses (A5-Bx-C8) were remade from the aliquot of resin
and rescreened according to the same procedure (Figure S3).
On the basis of this comparison, the sequence Lys-His-Ile-Ser-
Glu-Ser was selected for further studies.
To validate this result, the sequence was resynthesized on
a Rink amide resin to obtain the product in solution following
TFA cleavage. To ascertain the contribution of PNA hybrid-
ization, control sequences lacking the PNA (1; Figure 4), with
Figure 2. General structure of the library composed of 4- to 10-mer
peptides constrained by a hairpin structure of self-complementary
PNAs (left). The library was prepared by three rounds of split-and-mix
combinatorial synthesis. The peptide fragments used in each consec-
utive round are shown in green, black, and blue, respectively. The
sequence used for each pool is shown on the right (A1–10: green; B1–10
black; C1-10: blue).
:
Figure 4. Catalytic activity of identified catalyst and control sequences
measured in PBS (25 mm, pH 8.0) at 378C. The fluorescence was
measured at lex =345 nm and lem =530 nm (Y-axis shows arbitrary
fluorescent units). Each data point is the average of 3 experiments.
only one side of the PNA (at the N- and C-terminus, 2 and 3
respectively; Figure 4) or
a different hairpin forming
sequence (CGGC-peptide-GCCG and AAAAA-peptide-
TTTTT: 4 and 5; Figure 4) were prepared. As a further
controls, a sequence containing the linear peptide Lys-His-Ile-
Ser-Glu-Ser emanating from a hexaglycine hairpin duplex (6)
and a conjugate that cannot fold (7) were also prepared.
Analysis of the phosphate hydrolysis using the fluorogenic
substrate (100 mm catalyst, 2 mm substrate, 25 mm PBS buffer,
pH 8) confirmed the activity of the selected peptide (4;
Figure 4), and showed that the hybridization-enforced hairpin
was necessary for catalysis. The peptide alone (1), the peptide
with only one side of the PNA (2,3), the PNA-hairpin
conjugate to the linear peptide (6), and unfolded PNA–
peptide adduct (7) showed dramatically less activity
(Figure 4). Furthermore, specific nucleobases are unlikely to
be involved in the catalysis since comparable phosphate
hydrolysis activity was obtained for the GC-based hairpin as
with the AT-based hairpin (4 vs. 5). Comparing the slope of
the reaction for compound 4 vs. 6 indicates a rate acceleration
of > 25-fold upon hairpin formation. Performing the reaction
with 2 mm EDTA did not alter the rate of reaction, suggesting
metal chelation is not involved.
Figure 3. Screening of the library for phosphatase activity using
a fluorogenic substrate (QPD-OPO3-2) yielding a green quinazolinone-
based precipitating fluorophore.
The library was screened by exposing 400–700 beads of
each pool (i.e. 4–7 copies of each library member) to the
fluorogenic precipitating dye substrate (2 mm) in PBS buffer
(pH 8.0). The development of fluorescence was monitored as
a function of time by fluorescence microscopy using a high-
content screening microscope at 378C over a period of 5 h
(Figure 3; see Figure S1 for images of all pools as a function of
time and Figure S2 for quantitative analysis). A clear
discrepancy in the number of highly fluorescent beads was
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note that at 378C compound 13 had 50% of the activity of
compound 4. However, at 258C, both compounds showed
comparable activity. This is consistent with the measured Tm
of the hairpin (758C for 4 and 458C for 13; Figure S4); at
258C, both 4 and 13 are predominantly in the hairpin
configuration, whereas at 378C a significantly larger portion
of compound 13 is in the non-productive open form than
compound 4. As shown in Figure 6, the catalytic activity of
compound 13 was indeed dramatically attenuated in the
presence of the complementary PNA (ATAGCG) that opens
the hairpin, demonstrating that a programmable allosteric
control is possible. Concurringly, the activity of 13 could also
be attenuated during the course of the reaction by the
addition of the complementary PNA (ATAGCG).
In conclusion, we have identified a peptide sequence that
undergoes a dramatic gain of activity (> 25-fold) for phos-
phate hydrolysis when folded into a hairpin loop through
hybridization. While this peptide was identified from a rela-
tively small library (103), its discovery supports the notion that
hybridization can be used to induce and interrogate different
conformations within a peptide library. The fact that the
activity can be modulated by oligonucleotides that compete
for hybridization should make this design applicable in
responsive systems for nucleotide sensing.[16] The present
demonstration that the hybridization of a peptide–PNA
conjugate confers a protein-like function with allosteric
regulation should also find application in the design of
chemical networks that respond to environmental cues.
Finally, the methodology presented herein complements
alternative library design or screening methods based on
phage display[17] and catalytic dendrimers.[18] The advent of
DNA-templated synthesis[19] or the use of hybrid structures
with aptamers would also potentially benefit from iterative
cycles of selection/diversification.
Figure 5. Alanine-scan of the catalyst measured in PBS (25 mm,
pH 8.0) at 378C. The fluorescence was measured at lex =345 nm and
lem =530 nm (Y-axis shows arbitrary fluorescent units). Each data
point is the average of 3 experiments.
We next investigated the relative contribution of each
residue with a heteroatom using an Ala scan of the sequence.
As shown in Figure 5, Lys or His substitution (compounds 8
and 9) led to loss of activity, whereas the other positions
(compounds 10–12) yielded products with comparable activ-
ity. It should be noted that compound 4 failed to cleave other
aryl phosphates (phosphorylated form of 4-nitrophenol, 2-
nitrophenol, 7-hydroxycoumarin, and salicylamide), suggest-
ing some substrate specificity (for prior example of substrates
specificity with peptide catalysts[14]). Taken together, these
results support the hypothesis that the observed activity
results from catalysis mediated by a constrained conformation
of the Lys-His dipeptide within a hairpin loop that provides
a favorable interaction with the substrate.
Based on the impact of the hairpin on catalysis, we next
asked whether allosteric control of activity could be engi-
neered by toehold strand displacement switching the hairpin
configuration to an open configuration.[15] To this end,
compound 13 (Figure 6) with a hairpin made up of a 3-mer
PNA with a 3-mer overhang was prepared. It is interesting to
Acknowledgements
The authors thank the University of Geneva, the Swiss
National Science Foundation, and the NCCR Chemical
Biology for financial support.
Keywords: catalysis · peptides · PNA · system chemistry
[1] B. W. Matthews, P. B. Sigler, R. Henderso, D. M. Blow, Nature
1967, 214, 652 – 656.
[2] K. Johnsson, R. K. Allemann, H. Widmer, S. A. Benner, Nature
1993, 365, 530 – 532.
[3] For reviews, see: a) E. A. C. Davie, S. M. Mennen, Y. J. Xu, S. J.
Miller, Chem. Rev. 2007, 107, 5759 – 5812; b) J. D. Revell, H.
Wennemers, Curr. Opin. Chem. Biol. 2007, 11, 269 – 278; c) B.
Lewandowski, H. Wennemers, Curr. Opin. Chem. Biol. 2014, 22,
40 – 46; For recent examples, see: d) P. A. Lichtor, S. J. Miller,
Nat. Chem. 2012, 4, 990 – 995; e) K. W. Fiori, A. L. A. Puchlo-
pek, S. J. Miller, Nat. Chem. 2009, 1, 630 – 634; f) J. L. Gustafson,
D. Lim, S. J. Miller, Science 2010, 328, 1251 – 1255; g) K. T.
Barrett, S. J. Miller, J. Am. Chem. Soc. 2013, 135, 2963 – 2966;
Figure 6. Allosteric switch of catalytic activity of 13 measured in PBS
(25 mm, pH 8.0) at 258C. The fluorescence was measured at
lex =345 nm and lem =530 nm (Y-axis shows arbitrary fluorescent
units). Each data point is the average of 3 experiments.
Angew. Chem. Int. Ed. 2016, 55, 1 – 5
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Angewandte
Communications
Chemie
h) D. K. Romney, S. M. Colvin, S. J. Miller, J. Am. Chem. Soc.
2014, 136, 14019 – 14022.
Chem. Int. Ed. 2015, 54, 1822 – 1826; Angew. Chem. 2015, 127,
1842 – 1846.
[4] M. Martick, W. G. Scott, Cell 2006, 126, 309 – 320.
[5] M. Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M.
Freier, D. A. Driver, R. H. Berg, S. K. Kim, B. Norden, P. E.
Nielsen, Nature 1993, 365, 566 – 568.
[11] J. J. Naleway, C. M. J. Fox, D. Robinhold, E. Terpetschnig, N. A.
Olson, R. P. Haugland, Tetrahedron Lett. 1994, 35, 8569 – 8572.
[12] W. G. Cox, V. L. Singer, J. Histochem. Cytochem. 1999, 47, 1443 –
1455.
[6] a) K. J. Oh, K. J. Cash, A. A. Lubin, K. W. Plaxco, Chem.
Commun. 2007, 4869 – 4871; b) L. Rçglin, M. R. Ahmadian, O.
Seitz, Angew. Chem. Int. Ed. 2007, 46, 2704 – 2707; Angew.
Chem. 2007, 119, 2759 – 2763; c) S. Thurley, L. Roglin, O. Seitz, J.
Am. Chem. Soc. 2007, 129, 12693 – 12695; d) S. Rapireddy, L.
Nhon, R. E. Meehan, J. Franks, D. B. Stolz, D. Tran, M. E.
Selsted, D. H. Ly, J. Am. Chem. Soc. 2012, 134, 4041 – 4044.
[7] A. Furka, F. Sebestyen, M. Asgedom, G. Dibo, Int. J. Pept. Prot.
Res. 1991, 37, 487 – 493.
[8] a) K. Gorska, A. Manicardi, S. Barluenga, N. Winssinger, Chem.
Commun. 2011, 47, 4364 – 4366; b) K. K. Sadhu, T. Eierhoff, W.
Romer, N. Winssinger, J. Am. Chem. Soc. 2012, 134, 20013 –
20016.
[9] a) S. J. Taylor, J. P. Morken, Science 1998, 280, 267 – 270; b) A.
Berkessel, Curr. Opin. Chem. Biol. 2003, 7, 409 – 419.
[10] a) R. Sambasivan, Z. T. Ball, Angew. Chem. Int. Ed. 2012, 51,
8568 – 8572; Angew. Chem. 2012, 124, 8696 – 8700; b) R. Samba-
sivan, W. W. Zheng, S. J. Burya, B. V. Popp, C. Turro, C.
Clementi, Z. T. Ball, Chem. Sci. 2014, 5, 1401 – 1407; c) N.
Maillard, R. Biswas, T. Darbre, J. L. Reymond, ACS Comb. Sci.
2011, 13, 310 – 320; d) K. Akagawa, N. Sakai, K. Kudo, Angew.
[13] a) A. Berkessel, D. A. Herault, Angew. Chem. Int. Ed. 1999, 38,
102 – 105; Angew. Chem. 1999, 111, 99 – 102; b) C. Schmuck, J.
Dudaczek, Org. Lett. 2007, 9, 5389 – 5392.
[14] For prior examples of substrate specificity with peptide catalysts,
see: S. Han, S. J. Miller, J. Am. Chem. Soc. 2013, 135, 12414 –
12421.
[15] For a conceptually related approach to sensing, see: A. A. Lubin,
K. W. Plaxco, Acc. Chem. Res. 2010, 43, 496 – 505.
[16] K. Gorska, N. Winssinger, Angew. Chem. Int. Ed. 2013, 52, 6820 –
6843; Angew. Chem. 2013, 125, 6956 – 6980.
[17] Y. Maeda, N. Javid, K. Duncan, L. Birchall, K. F. Gibson, D.
Cannon, Y. Kanetsuki, C. Knapp, T. Tuttle, R. V. Ulijn, H.
Matsui, J. Am. Chem. Soc. 2014, 136, 15893 – 15896.
[18] T. Darbre, J. L. Reymond, Acc. Chem. Res. 2006, 39, 925 – 934.
[19] Y. Brudno, M. E. Birnbaum, R. E. Kleiner, D. R. Liu, Nat.
Chem. Biol. 2010, 6, 148 – 155.
Received: March 18, 2016
Revised: May 10, 2016
Published online: && &&, &&&&
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Communications
Peptide Catalysts
T. Machida, S. Dutt,
N. Winssinger*
&&&& — &&&&
Allosterically Regulated Phosphatase
Activity from Peptide–PNA Conjugates
Folded Through Hybridization
A library of peptides that are constrained
in a hairpin loop by flanking peptide
nucleic acids upon hybridization were
synthesized. A screen for phosphatase
activity led to the discovery of a catalyst
with >25-fold rate acceleration over the
linear peptide. The hybridization-
enforced folding of the peptide is decisive
for the activity.
Angew. Chem. Int. Ed. 2016, 55, 1 – 5
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!