Sequence-selective molecular recognition between β sheets

Article abstract of DOI:10.1002/anie.200250750

Molecular recognition between peptide β sheets is sequence selective! By simply controlling the placement of two threonine (Thr) or valine (Val) residues, peptides that preferentially form either homodimers or heterodimers with about tenfold selectivity can be generated. The picture shows the structure of a Thr-Val·Val-Thr heterodimer.

Full text of DOI:10.1002/anie.200250750

Angewandte
Chemie
Sequence-Selective Recognition
Sequence-Selective Molecular Recognition
between b Sheets**
James S. Nowick* and De Michael Chung
Dedicated to Professor Larry Overman
on the occasion of his 60th birthday
Interactions between the hydrogen-bonding edges of b sheets
occur widely in protein quaternary structure, protein–protein
interactions, and protein aggregation and are involved in both
healthy biological processes and in diseases ranging from
cancer and AIDS to anthrax and Alzheimer's.[1,2] These
protein–protein interactions constitute a form of molecular
recognition that deserves more attention, both because of its
importance in biological processes and because of its funda-
mental nature. This paper asks whether interactions between
b-sheet-forming peptides can be sequence selective and finds
that patterning valine and threonine residues across the non-
hydrogen-bonded rings formed at the interface between
antiparallel b sheets can impart dramatic sequence selectiv-
ity.[3]
We had previously reported that peptides such as 1a,
which contain the amino acid Orn(iPrCO-Hao), fold into b-
sheetlike structures that dimerize through b-sheet interac-
tions in organic solvents (Scheme 1).[4] In the present study,
we chose to replace the valine and leucine residues of 1a with
threonine and valine in all possible orders: Thr–Val (1b), Val–
Thr (1c), Thr–Thr (1d), and Val–Val (1e). We selected these
residues with the expectation that Val would pair preferen-
tially with Val, and that Thr would pair preferentially with
Thr, through self-complementary noncovalent interactions
[*] Prof. J. S. Nowick, D. M. Chung
Department of Chemistry
University of California, Irvine
Irvine, CA 92697-2025 (USA)
Fax: (+1)949-824-9920
E-mail: jsnowick@uci.edu
[**] We thank the NIH for grant support (GM-49076). D.M.C. thanks the
Bill and Melinda Gates Foundation and the UCI Institute for Brain
Aging and Dementia for a fellowship and training grant support
(NIA-5T32AG000096).
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DOI: 10.1002/anie.200250750
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Communications
Figure 1. 1H NMR spectra of the hydrazide and anilide NH groups of
Thr–Val peptide 1b (a), Val–Thr peptide 1c (b), and a mixture of the
two peptides (c). The heterodimer predominates in the mixture. Spectra
were recorded at 500 MHz and 253 K in 10% [D6]DMSO in CDCl3 at a)
2.0 mm 1b, b) 2.0 mm 1c , and c) 2.1 mm 1b and 1.5 mm 1c.
Scheme 1. The b-sheet dimer of peptides 1.
and with the knowledge that these pairings frequently occur
in the non-hydrogen-bonded rings of antiparallel b sheets.[5]
To evaluate the sequence selectivity of molecular recog-
nition between threonine- and valine-containing b-sheet
peptides 1b–e, we measured the propensity of these peptides
to form heterodimers. Mixing solutions of (homodimeric)
peptides ArCO-Waa-Orn(iPrCO-Hao)-Phe-Ile-Xaa-NHMe
(Waa–Xaa) and ArCO-Yaa-Orn(iPrCO-Hao)-Phe-Ile-Zaa-
NHMe (Yaa–Zaa) gives an equilibrium mixture of homo-
dimers [(Waa–Xaa)2 and (Yaa–Zaa)2] and heterodimer
[(Waa–Xaa)·(Yaa–Zaa)] [Eq. (1)]. A heterodimeric equili-
brium constant KHet can be defined as [(Waa–Xaa)·(Yaa–
Figure 2. 1H NMR spectra of the hydrazide and anilide NH groups of
Thr–Thr peptide 1d (a), Val–Val peptide 1e (b), and a mixture of the
two peptides (c). The homodimers predominate in the mixture. Spectra
were recorded at 500 MHz and 253 K in 10% [D6]DMSO in CDCl3 at a)
2.0 mm 1d, b) 2.0 mm 1e, and c) 1.5 mm 1d and 1.7 mm 1e.
Zaa)]2/[(Waa–Xaa)2][(Yaa–Zaa)2]. If the equilibrium is stat-
istical, KHet = 4; if the heterodimer forms preferentially,
Analysis of spectra collected in 10% [D6]DMSO in CDCl3
(Figure 1 and 2) reveals a strong preference for Thr–Val (1b)
and Val–Thr (1c) to form a heterodimer (KHet/4 = 8.9) and for
Thr–Thr (1d) and Val–Val (1e) to remain as homodimers
(KHet/4 = 0.083).[6] In contrast with these results, controls
involving all four other possible combinations of peptides
1b–e show little or no preferences for forming either homo-
or heterodimers (KHet/4 = 0.8–1.0; Table 1). These results
confirm the hypothesis that pairing of like residues is
preferred, Thr with Thr, and Val with Val. Calculation of
free energies based on these KHet values reveals that a Thr–
Thr pair and a Val–Val pair collectively are 0.6 kcalmolÀ1
more stable than two Thr–Val pairs, under the conditions of
these experiments.
K
Het > 4; if the homodimers are preferred, KHet < 4. The
quantity KHet/4 provides a convenient gauge of the dimeriza-
tion preference, with a value of 1.0 indicating no preference.
1H NMR spectroscopic studies readily permit the direct
measurement of KHet. Chemical exchange between the homo-
and heterodimers occurs slowly on the NMR time scale at
ambient and subambient temperatures in CDCl3 or in
[D6]DMSO/CDCl3. The hydrazide and anilide NH resonance
signals of the Orn(iPrCO-Hao) group are well dispersed and
allow the various species present to be quantified accurately.
Each homodimer presents two doublets associated with the
hydrazide NH resonance signals in the d = 11–12 ppm region
of the spectrum and a singlet near d = 10 ppm associated with
the anilide NH resonance signal; the heterodimer presents
four doublets and two singlets. Integration of these peaks
allows the determination of the ratios of the species;
deconvolution of the spectra by fitting the peaks with
Lorentzian functions provides even greater accuracy. The
spectra shown in Figure 1 and 2 are representative of these
studies.
The [D6]DMSO cosolvent is essential for sequence
selectivity. When pure CDCl3 is used for these studies, no
Table 1: Propensities of peptides 1b–e to form heterodimers.
1b&1c
1d&1e
1b&1d
1b&1e
1c&1d
1c&1e
K
Het/4
8.9
0.083
0.87
0.80
1.0
1.0
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Figure 3. 1H NMR Tr-ROESY spectra of the a-proton region of Thr–Val peptide 1b (a), Val–Thr peptide 1c (b), and a mixture of the two peptides
(c), illustrating intermolecular NOEs associated with the respective homodimers and the heterodimer. Spectra were recorded at 500 MHz and
253 K with a 250 ms mixing time in 10% [D6]DMSO in CDCl3 at a) 2.0 mm 1b, b) 2.0 mm 1c, and c) 1.4 mm 1b and 2.6 mm 1c.
Keywords: b sheets · molecular recognition · peptides ·
proteins · supramolecular chemistry
significant selectivity is observed. Selectivity emerges and
increases as [D6]DMSO is added. At 3.9% [D6]DMSO, for
example, KHet/4 = 2.7 for Thr–Val (1b) and Val–Thr (1c) and
0.28 for Thr–Thr (1d) and Val–Val (1e).[7] The DMSO
.
cosolvent may effect selectivity by promoting interaction
between Val side chains (e.g., through solvophobic interac-
[1] S. Maitra, J. S. Nowick in The Amide Linkage: Structural
Significance in Chemistry, Biochemistry, and Materials Science
tions), destabilizing interactions between Val and Thr side
chains, or enhancing hydrogen-bonding interactions between
the Thr side chains. In light of the known interactions between
DMSO and water dimers, the latter explanation appears to be
most likely.[8]
1H NMR transverse-ROESY (Tr-ROESY)[9] studies
(Figure 3) corroborate that the peptides form well-defined
homo- and heterodimers. Strong NOEs between the Thr and
Val a protons in the Tr-ROESY spectra of unmixed 1b and
1c, similar to those we reported previously for peptide 1a,[4]
help establish the formation of homodimers. A strong NOE
between the two different Val a protons of the new species
formed upon mixing 1b and 1c confirms the formation of the
heterodimer.[10]
The experiments described above establish that sequence-
selective molecular recognition of b sheets may be achieved
through side chain interactions. The measurement of equili-
brium constants by these experiments is appealing, because it
provides the same information about side chain interaction
energies as a double-mutant cycle experiment, but does so
directly in a single measurement.[11,12] The peptide model
system developed here is arguably simpler and better defined
than other related model systems.[13,14] We envision modifying
this system to generate peptides that recognize protein b
sheets in a sequence-selective fashion.
(Eds.: A. Greenberg, C. M. Breneman, J. F. Liebman), Wiley,
New York, 2000, pp. 495 – 518.
[2] C. Petosa, R. J. Collier, K. R. Klimpel, S. H. Leppla, R. C.
Liddington, Nature 1997, 385, 833 – 838.
[3] For related observations in proteins associated with genetic
diseases, see: a) P. Hammarström, F. Schneider, J. W. Kelly,
Science 2001, 293, 2459 – 2462; b) E. G. Huizinga, S. Tsuji,
R. A. P. Romijn, M. E. Schiphorst, P. G. de Groot, J. J. Sixma,
P. Gros, Science 2002, 297, 1176 – 1179.
[4] J. S. Nowick, K. S. Lam, T. V. Khasanova, W. E. Kemnitzer, S.
Maitra, H. T. Mee, R. Liu, J. Am. Chem. Soc. 2002, 124, 4972 –
4973.
[5] a) S. Lifson, C. Sander, J. Mol. Biol. 1980, 139, 627 – 639;
b) M. A. Wouters, P. M. G. Curmi, Proteins Struct. Funct. Genet.
1995, 22, 119 – 131; c) E. G. Hutchinson, R. B. Sessions, J. M.
Thornton, D. N. Woolfson, Protein Sci. 1998, 7, 2287 – 2300; d) Y.
Mandel-Gutfreund, S. M. Zaremba, L. M. Gregoret, J. Mol.
Biol. 2001, 305, 1145 – 1159.
[6] Studies were performed at 253 K. Although the resonance
signals are resolved in the 500 MHz 1H NMR spectrum at 298 K,
they are sharper at lower temperatures. Measurement of the
relative concentrations of the homo- and heterodimers at
varying ratios of peptides verifies that KHet is invariant and
supports its use to gauge the preferences of amino acid pairings.
[7] Further addition of [D6]DMSO beyond 10% results in addi-
tional increases in selectivity, however shimming of the NMR
instrument becomes difficult at higher [D6]DMSO concentra-
tions. In pure [D6]DMSO, dimers do not form.
[8] B. Kirchner, M. Reiher, J. Am. Chem. Soc. 2002, 124, 6206 – 6215,
and references therein.
[9] a) T.-L. Hwang, A. J. Shaka, J. Am. Chem. Soc. 1992, 114, 3157 –
3159; b) T.-L. Hwang, A. J. Shaka, J. Magn. Reson. Ser. B 1993,
102, 155 – 165.
Received: December 11, 2002 [Z50750]
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[10] NOEs between the Thr a-protons of the heterodimer could not
12061 – 12068; f) T. D. Clark, J. M. Buriak, K. Kobayashi, M. P.
Isler, D. E. McRee, M. R. Ghadiri, J. Am. Chem. Soc. 1998, 120,
8949 – 8962; g) M. A. Hossain, H.-J. Schneider, J. Am. Chem.
Soc. 1998, 120, 11208 – 11209; h) B. Baumeister, S. Matile,
Chem. Eur. J. 2000, 6, 1739 – 1749; i) Y. Takahashi, A. Ueno, H.
Mihara, ChemBioChem 2001, 2, 75 – 79; j) G. Xu, W. Wang, J. T.
Groves, M. H. Hecht, Proc. Natl. Acad. Sci. USA 2001, 98, 3652 –
3657; k) A. Cox, M. M. Arroyo, K. H. Mayo, Biochem. J. 2001,
357, 739 – 747; l) Y. Hamuro, A. D. Hamilton, Bioorg. Med.
Chem. 2001, 9, 2355 – 2363; m) H. Wennemers, M. Conza, M.
Nold, P. Krattiger, Chem. Eur. J. 2001, 7, 3342 – 3347; n) S. T.
Phillips, M. Rezac, U. Abel, M. Kossenjans, P. A. Bartlett, J. Am.
Chem. Soc. 2002, 124, 58 – 66; o) H. Zeng, X. Yang, R. A.
Flowers, B. Gong, J. Am. Chem. Soc. 2002, 124, 2903 – 2910;
p) K. B. Jensen, T. M. Braxmeier, M. Demarcus, J. G. Frey, J. D.
Kilburn, Chem. Eur. J. 2002, 8, 1300 – 1309.
be observed because the resonance signals of these protons are
nearly coincident.
[11] a) C. K. Smith, L. Regan, Science 1995, 270, 980 – 982; b) C. A.
Blasie, J. M. Berg, Biochemistry 1997, 36, 6218 – 6222.
[12] A. Horovitz, Folding Des. 1996, 1, R121-R126.
[13] M. W. Peczuh, A. D. Hamilton, Chem. Rev. 2000, 100, 2479 –
2494.
[14] For some leading examples of related work involving peptide
recognition, see: a) S. R. LaBrenz, J. W. Kelly, J. Am. Chem. Soc.
1995, 117, 1655 – 1656; b) Y. Cheng, T. Suenaga, W. C. Still, J.
Am. Chem. Soc. 1996, 118, 1813 – 1814; c) X. Cha, K. Ariga, T.
Kunitake, J. Am. Chem. Soc. 1996, 118, 9545 – 9551; d) R. Zutshi,
J. Franciskovich, M. Shultz, B. Schweitzer, P. Bishop, M. Wilson,
J. Chmielewski, J. Am. Chem. Soc. 1997, 119, 4841 – 4845;
e) C. N. Kirsten, T. H. Schrader, J. Am. Chem. Soc. 1997, 119,
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