PEGylation Increases the Strength of a Nearby NH-π Hydrogen Bond in the WW Domain

Abstract

Here we show that an NH-π interaction between a highly conserved Asn and a nearby Trp stabilizes the WW domain of the human protein Pin1. The strength of this NH-π interaction depends on the structure of the arene, with NH-π interactions involving Trp or naphthylalanine being substantially more stabilizing than those involving Tyr or Phe. Calculations suggest arene size and polarizability are key structural determinants of NH-π interaction strength. Methylation or PEGylation of the Asn side-chain amide nitrogen each strengthens the associated NH-π interaction, though likely for different reasons. We hypothesize that methylation introduces steric clashes that destabilize conformations in which the NH-π interaction is not possible, whereas PEGylation strengthens the NH-π interaction via localized desolvation of the protein surface.

Figure 1.

(A) Space-filling and (B) stick structures of the WW domain of the human protein Pin1 (PDB: 1PIN) with Asn26 highlighted in orange and Trp11, Pro9, Ile28, and Thr29 highlighted in black. Hydrogen bonds between Asn26 side-chain HD21 and Pro9 backbone CO; between Asn26 side-chain CO and Ile28 backbone NH; between Asn26 side-chain CO and Thr29 backbone NH; and NH-π interaction between Asn side-chain HD22 and Trp11 indole are shown as dashed orange lines. (C) Structures of residue 26 within WW variants WN, WQ, and WNm, along with folding free energies (and their enthalpic vs. entropic contributions) of variants WQ and WNm relative to unmodified parent variant WN, obtained from variable temperature CD data at 50 μM protein concentration in 20 mM sodium phosphate buffer (pH 7), and calculated at the melting temperature of variant WN. (D) Calculated interaction energies for complexes of formamide with indole, benzene, naphthalene, and phenol, calculated via the MP2 method in Gaussian 16 using the aug-cc-pVDZ basis set and corrected for basis set superposition error (see supporting information for details). (E) Relationship between melting temperatures (T_m) of WW variants FN, YN, WN, and ZN and the calculated interaction energies for the corresponding formamide/arene complexes described above.

Figure 2.

(A) Structure of PEGylated Asn26 in variant WNp along with PEG-based stabilization of WNp relative to its non-PEGylated counterpart WN. Also shown is the simulated radial distribution function of water around Trp11 in WNp (red line) vs. WN (black line) along with an inset histogram of the number of water molecules within 3 Å of Trp11 in each variant. Adapted with permission from reference . Copyright © 2014 American Chemical Society. Also shown is the structure of methylated Asn26 in variant WNm along with its conformational stability relative to non-PEGylated WN. (B) Relationship between the T_m of WW variants FN, YN, WN, and ZN or the calculated NH-π interaction energies for complexes of formamide and benzene, phenol, indole, and naphthalene and the stabilization (ΔΔG) of Asn-PEGylated FNp, YNp, WNp, and ZNp (red circles) or of Asn-methylated FNm, YNm, WNm, and ZNm (gray circles) relative to their unmodified counterparts FN, YN, WN, and ZN. Solid red and black lines represent fits of these relationships via linear least-squares regression for PEGylation and methylation, respectively, with the R², F statistic, and p values shown. Dotted vertical lines represent the difference in ΔΔG values for PEGylation vs. methylation for a given WW sequence.

Figure 3.

Relationship between calculated NH-π interaction energies for complexes of formamide with indole, benzene, phenol, and naphthalene (bottom row) or experimental melting temperatures (T_m; top row) of WW variants WN, FN, YN, and ZN with calculated properties of benzene, phenol, indole, and naphthalene, including molar volume (yellow circles); molecular polarizability (blue circles); the maximum electrostatic potential above the benzene centroid within each arene (orange circles); and c log P (purple circles); Solid yellow, blue, orange, and purple lines represent fits of these relationships via linear least-squares regression, with the R², F statistic, and p values shown. Vertical dotted yellow, blue, yellow, orange, and purple lines connect NH-π interaction energy, T_m, and ΔΔG for each variant. c log P values are from reference ; all other properties were calculated using Gaussian16 (see supporting information for details).