Letters pubs.acs.org/acschemicalbiology
An Anion−π Interaction Strongly Stabilizes the β‑Sheet Protein WW Mason S. Smith, Eliza E. K. Lawrence, Wendy M. Billings, Kimberlee S. Larsen, Natalie A. Bécar, and Joshua L. Price* Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United States S Supporting Information *
ABSTRACT: Anions have long been known to engage in stabilizing interactions with electron-deficient arenes. However, the precise nature and energetic contribution of anion−π interactions to protein stability remains a subject of debate. Here, we show that placing a negatively charged Asp in close proximity to electron-rich Phe in a reverse turn within the WW domain results in a favorable interaction that increases WW conformational stability by −1.3 kcal/mol.
C
ation−π interactions are ubiquitous within proteins and contribute substantially to protein conformational stability and function.1−4 Many experimental and theoretical studies indicate that anions can engage in analogous facewise interactions with electron-deficient arenes.5−11 However, the precise nature and energetic impact of anion−π interactions is a subject of debate,12,13 especially for interactions of anions with the electron-rich protein side chains Phe, Tyr, and Trp.14 One would naturally expect the facewise interaction of an electron-rich arene with an anion to be unfavorable due to electrostatic repulsion,11 though polarization can compensate somewhat for suboptimal electrostatic interactions.6 Interestingly, structural and statistical analyses of the Protein Data Bank (PDB) suggest that facewise contacts of anions with the aryl side chains of Phe, Trp, or Tyr occur often,15−17 though others argue that they appear less frequently than expected.18−20 Moreover, theoretical studies disagree on whether such facewise anion−π interactions in proteins are substantially stabilizing14,21 or have a negligible effect.20 Alternative analyses have suggested that edgewise contact of an anion with the electron-deficient C−H edge of an electronrich arene is more prevalent than facewise contact6 and has a more favorable energetic impact.18,20 For example, Kallenbach and co-workers found that the interaction between an i-position Glu and an i + 4 Phe contributes −0.5 kcal/mol to the stability of an α-helical polyalanine model peptide; relatively weak NOEs demonstrate proximity of the Glu and Phe side chains, possibly consistent with an interaction between the aryl protons of Phe and the negatively charged oxygens of Glu.22,23 We aim to explore more fully the nature and energetic contribution of anion−π interactions in the context of a wellcharacterized β-sheet model system: the WW domain of the human protein Pin 1 (hereafter called WW).24−31 WW contains a reverse turn that brings side chains at positions 16 and 18 into close proximity (Figure 1). We wondered whether placing a negatively charged amino acid at position 16 and an aromatic © XXXX American Chemical Society
Figure 1. Close proximity of residues 16 and 18 in a reverse turn within the Pin WW domain (PDB: 2f21). We made the substitutions shown at the positions indicated.
amino acid at position 18 might facilitate a favorable anion−π interaction similar to that observed previously in the context of an α-helix.22,23 To test this hypothesis, we prepared WW variant DY, with Asp at position 16 and Tyr at position 18, along with sequencematched control compounds SY, DN, and SN, in which we replaced Asp16 with Ser and Tyr18 with Asn, respectively, in all possible combinations. We chose these replacements because Ser16 and Asn18 cannot participate in an anion−π interaction and because they closely resemble the residues that occupy these positions in the parent WW sequence from which these variants were derived.28 We used variable temperature CD experiments to assess the conformational stability of DY relative to SY, DN, and SN. The results of this analysis are shown in Table 1. Changing Ser16 to Received: September 1, 2017 Accepted: September 8, 2017 Published: September 8, 2017 A
DOI: 10.1021/acschembio.7b00768 ACS Chem. Biol. XXXX, XXX, XXX−XXX
Letters
ACS Chemical Biology Table 1. Folding Free Energies of WW Proteins SN, DN, and Their Sequence Variants at 60 °Ca protein
position 16
position 18
SN DN SY DY SH DH SF DF SZ DZ SX DX
Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp
Asn Asn Tyr Tyr His His Phe Phe F5Phe F5Phe Cha Cha
ΔGf (kcal/mol)
Tm (°C) 65.7 68.7 58.3 64.2 60.8 65.4 52.8 65.9 49.7 62.6 58.4 61.3
± ± ± ± ± ± ± ± ± ± ± ±
−0.53 −0.80 0.15 −0.37 −0.06 −0.45 1.05 −0.53 1.33 −0.26 0.22 −0.13
0.1 0.1 0.3 0.2 0.1 0.1 0.2 0.1 0.1 0.1 0.1 0.1
± ± ± ± ± ± ± ± ± ± ± ±
ΔΔΔGf (kcal/mol) Xxx16−Yyy18 interaction
0.01 0.01 0.03 0.02 0.01 0.01 0.04 0.01 0.03 0.01 0.01 0.01
−0.26 ± 0.04 −0.12 ± 0.02 −1.31 ± 0.05 −1.33 ± 0.03 −0.08 ± 0.02
Folding free energies are given ± standard error in kcal/mol at 60 °C (333.15 K) in 20 mM sodium phosphate buffer (pH 7). Z = pentafluorophenylalanine; X = cyclohexylalanine. a
Asp increases WW conformational stability by −0.26 ± 0.04 kcal/mol when Asn occupies position 18 (which would preclude any contribution from an anion−π interaction). In contrast, when Tyr occupies position 18, the S16D mutation stabilizes WW by a moderately larger amount (ΔΔGf = −0.52 ± 0.03 kcal/mol), suggesting that the Asp16−Tyr18 interaction contributes slightly to the stability of DY relative to DN (ΔΔΔGf = −0.26 ± 0.04 kcal/mol; see Table 1). We wondered whether other proteinogenic arenes might more readily engage in a stabilizing interaction with Asp16. To test this hypothesis, we prepared WW variants SW, DW, SH, DH, SF, and DF, in which Trp, His, or Phe have replaced Tyr18. Variable temperature CD data for SW displayed a hightemperature transition that was not consistent with two-state folding, preventing us from assessing the strength of the hypothesized Asp16−Trp18 interaction. Comparison of SH and DH vs SN and DN indicates that the Asp16−His18 interaction is only marginally stabilizing (ΔΔΔGf = −0.12 ± 0.02 kcal/mol, Table 1). In contrast, the Asp16−Phe18 interaction contributes −1.31 ± 0.05 kcal/mol to WW conformational stability (compare ΔGf values for SF and DF vs SN and DN in Table 1), an increment nearly 5 times larger than observed for the Asp16−Tyr18 interaction. Although these results unambiguously demonstrate that the Asp16−Phe18 interaction can stabilize WW, the precise orientation of Asp16 relative to Phe18 remains unclear. If this interaction involves the facewise orientation of Asp16 relative to Phe18, then replacing Phe with electron-poor pentafluorophenylalanine (F5Phe; one-letter abbreviation is Z) should result in a much more favorable interaction. In contrast, an edgewise interaction of Asp16 with F5Phe should be much less favorable, because the negatively charged Asp carboxylate should be repelled by the electron-rich fluorine-modified edge of the ring. We explored this possibility by preparing WW variants DZ and SZ, in which we replaced Phe18 with F5Phe. Contrary to our expectations, the Asp16−F5Phe18 interaction contributes −1.33 ± 0.03 kcal/mol to the stability of DZ relative to SZ, an amount indistinguishable from the contribution of the Asp16− Phe18 interaction observed above. This result is consistent both with edgewise orientation of Asp16 relative to Phe18 in DF and with facewise orientation of Asp16 relative to F5Phe18 in DZ and could indicate that anion−π interactions are dynamic enough to permit local conformational rearrangements that optimize interaction energetics.
Finally, we explored the role of aromaticity in the Asp16− Phe18 interaction by preparing variants DX and SX, in which we incorporated the nonaromatic residue cyclohexylalanine (Cha; one-letter abbreviation is X) at position 18. The interaction of Asp16 with Cha18 contributes a negligible amount to WW stability (0.05 ± 0.02 kcal/mol), indicating that aromaticity is a crucial determinant of the favorable Asp16− Phe18 interaction observed above. Here, we have shown that anionic Asp can interact favorably with Phe to increase the stability of the WW domain. Our results complement previous computational predictions5−11 along with earlier studies of an α-helical model system,22,23 suggesting that the anion−π interaction should be considered as an important addition to the tool box of noncovalent interactions used to understand protein folding, to design new proteins, and to develop small-molecule effectors of protein function.
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METHODS
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ASSOCIATED CONTENT
Protein Synthesis. Proteins were prepared by solid-phase peptide synthesis, purified by reverse-phase HPLC, and identified by electrospray ionization time-of-flight mass spectrometry, with purity evaluated by analytical HPLC, as described in the Supporting Information. CD Measurements. CD data were obtained using an Aviv 420 spectropolarimeter as described in the Supporting Information. Data from variable temperature CD experiments for each variant were fit to equations derived from a two-state folding model to obtain the folding free energy values shown in Table 1.
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.7b00768. Experimental methods, compound characterization, and variable temperature CD data (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Joshua L. Price: 0000-0002-0116-0968 Notes
The authors declare no competing financial interest. B
DOI: 10.1021/acschembio.7b00768 ACS Chem. Biol. XXXX, XXX, XXX−XXX
Letters
ACS Chemical Biology
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Protein Data Bank: Searching for Anion-pi Pairs. Biochemistry 50, 2939−2950. (21) Marsili, S., Chelli, R., Schettino, V., and Procacci, P. (2008) Thermodynamics of stacking interactions in proteins. Phys. Chem. Chem. Phys. 10, 2673−2685. (22) Olson, C. A., Shi, Z. S., and Kallenbach, N. R. (2001) Polar interactions with aromatic side chains in alpha-helical peptides: Ch center dot center dot center dot O H-bonding and cation-pi interactions. J. Am. Chem. Soc. 123, 6451−6452. (23) Shi, Z. S., Olson, C. A., Bell, A. J., and Kallenbach, N. R. (2001) Stabilization of alpha-helix structure by polar side-chain interactions: Complex salt bridges, cation-pi interactions, and C-H center dot center dot center dot O H-bonds. Biopolymers 60, 366−380. (24) Jäger, M., Nguyen, H., Crane, J. C., Kelly, J. W., and Gruebele, M. (2001) The Folding Mechanism of a β-sheet: The WW Domain. J. Mol. Biol. 311, 373−393. (25) Kowalski, J. A., Liu, K., and Kelly, J. W. (2002) NMR Solution Structure of the Isolted Apo Pin1 WW Domain: Comparison to the XRay Crystal Structures of Pin1. Biopolymers 63, 111−121. (26) Nguyen, H., Jäger, M., Moretto, A., Gruebele, M., and Kelly, J. W. (2003) Tuning the free-energy landscape of a WW domain by temperature, mutation, and truncation. Proc. Natl. Acad. Sci. U. S. A. 100, 3948−3953. (27) Deechongkit, S., Nguyen, H., Powers, E. T., Dawson, P. E., Gruebele, M., and Kelly, J. W. (2004) Context-dependent contributions of backbone hydrogen bonding to β-sheet folding energetics. Nature 430, 101−105. (28) Jäger, M., Zhang, Y., Bieschke, J., Nguyen, H., Dendle, M., Bowman, M. E., Noel, J. P., Gruebele, M., and Kelly, J. W. (2006) Structure-function-folding relationship in a WW domain. Proc. Natl. Acad. Sci. U. S. A. 103, 10648−10653. (29) Fuller, A. A., Du, D., Liu, F., Davoren, J. E., Bhabha, G., Kroon, G., Case, D. A., Dyson, H. J., Powers, E. T., Wipf, P., Gruebele, M., and Kelly, J. W. (2009) Evaluating β-turn mimics as β-sheet folding nucleators. Proc. Natl. Acad. Sci. U. S. A. 106, 11067−11072. (30) Gao, J., Bosco, D. A., Powers, E. T., and Kelly, J. W. (2009) Localized thermodynamic coupling between hydrogen bonding and microenvironment polarity substantially stabilizes proteins. Nat. Struct. Mol. Biol. 16, 684−690. (31) Ranganathan, R., Lu, K. P., Hunter, T., and Noel, J. P. (1997) Structural and functional analysis of the mitotic rotamase Pin1 suggests substrate recognition is phosphorylation dependent. Cell 89, 875−886.
ACKNOWLEDGMENTS This work was supported by start-up funds from the Department of Chemistry and Biochemistry at Brigham Young University.
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REFERENCES
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DOI: 10.1021/acschembio.7b00768 ACS Chem. Biol. XXXX, XXX, XXX−XXX
