A glutamine-based single α-helix scaffold to target globular proteins

Abstract

The binding of intrinsically disordered proteins to globular ones can require the folding of motifs into α-helices. These interactions offer opportunities for therapeutic intervention but their modulation with small molecules is challenging because they bury large surfaces. Linear peptides that display the residues that are key for binding can be targeted to globular proteins when they form stable helices, which in most cases requires their chemical modification. Here we present rules to design peptides that fold into single α-helices by instead concatenating glutamine side chain to main chain hydrogen bonds recently discovered in polyglutamine helices. The resulting peptides are uncharged, contain only natural amino acids, and their sequences can be optimized to interact with specific targets. Our results provide design rules to obtain single α-helices for a wide range of applications in protein engineering and drug design.

Fig. 1. Design of single α-helices stabilized by Gln side chain to main chain hydrogen bonds.

a Sequences and representation as helical projections of the peptides used to investigate cooperativity, where pink arrows indicate putative Gln side chain to main chain interactions. b Selected region of the 2D ¹H¹³,C HSQC spectra of the peptides at 278 K, where the shaded area corresponds to that of peptide P3-7, which has the highest helical propensity. c Sequence and representation as helical projections of peptides (P3-7)₂ and (P3-7)₃, as in a. d Top: CD spectra of peptides (P3-7)₂, (P3-7)₃, and (P3-7)_{3 Ctrl} at the indicated temperatures. Bottom: residue-specific helical propensities as obtained from the C’, C_α, N^H, and H^N NMR chemical shifts by using CheSPI^,. e Left: CD spectra of peptide (P3-7)₃ at temperatures between 278 K (blue) and 368 K (red) in steps of 10 K; the spectrum obtained at 278 K after refolding is shown in black. Right: superimposition of a selected region of the ¹³C-detected 2D CACO NMR spectra of peptide (P3-7)₃ recorded at 278 K (blue) and 310 K (red), with indications as colored shades of amino acid-specific regions. f Thermal denaturation of peptides (P3-7)₃ and (P3-7)_{3 Ctrl} monitored by measuring the mean residue ellipticity at 222 nm.

Fig. 2. Structure of a Gln-based single α-helix.

a ¹⁵N NMR relaxation data for main chain (N^H) and Gln side chain (Nε) amide groups of peptide (P3-7)₂, measured at 14.1 and 18.8 T at 278 K in 10% D₂O (main chain) and 50% D₂O (side chain). Left: ¹⁵N R₂/R₁ ratios, error bars represent the error-propagated SD of both exponential decay fits to R₁ and R₂ data and solid horizontal lines represent the theoretical R₂/R₁ ratio of a rigid body in isotropic motion with τ_c = 4.5 ns, whose field dependency agrees well with the experimental data. Right: ¹⁵N{¹H} heteronuclear NOE, where the error bars correspond to the NOE SD and the shade corresponds to the region with the highest degree of structuration. b Spectral densities derived by reduced spectral density mapping of all ¹⁵N relaxation data: the dashed line corresponds to the behavior expected for a polypeptide. c Strips from a 3D ¹⁵N-edited TOCSY-HSQC spectrum showing the H_β and H_γ resonances of all four Gln residues in (P3-7)₂. d Residue-specific CoMAND fitting of NOE signal intensities from the 3D CNH-NOESY spectrum of (P3-7)₂. Top: example measured and back-calculated 1D ¹³C traces (Gln14 main chain N, a99sb-disp frame pool). Bottom: average R-factors obtained from 100 CoMAND iterations using either frame pool. e Density plots and mean values of the fraction of mt rotamer found in the 100 fitting iterations for all four Gln residues. The fraction of helical glutamines in mt configuration in the BBDep dataset is shown in blue f Probability density for Gln14 χ₁ and χ₂ derived using the Gaussian mixture model (GMM). g Structural ensemble of (P3-7)₂, with main chain conformations selected by CoMAND from both trajectory pools and side chain conformations generated by GMM sampling. Top: one of 20 global ensemble calculations aligned for the Leu10-Gln14 pair. Bottom: same iteration aligned for the Leu13-Gln17 pair. h Residual dipolar couplings (RDCs) for the main chain ¹H-¹⁵N moieties and relaxation-derived main chain spectral density at zero frequency, J(0). i Correlation between experimental and back-calculated RDCs.

Fig. 3. Gln side chain to main chain hydrogen bonds can be accepted by different residues.

a Top: Sequence, numbering, and representation, as helical projection, of the L₃XQ₁₆ variants studied in this work. Bottom: residue-specific helical propensity of the L₃XQ₁₆ variants. The type of residue X (position 10) is indicated by the colored circles. Left: helical profile of the 7 most helical single variants. Center: helical profile of the least helical single variants and L₄Q₁₆. Right: Helical profile of the outlier variants (X = T, S) and L₄Q₁₆ (spectra in Supplementary Fig. 4b). b Effect of the rotameric state of the acceptor on the interaction of atom H_ε21 with H₂O. Left: two frames of the L₄Q₈ Charmm36m trajectory showing Gln4 involved in a bifurcated hydrogen bond with Leu 4 in either the mt (top) or tp (bottom) rotamer. Right: radial distribution function for Gln4 H_ε21 in the frames where Leu 4 populates the mt (red) or tp (gold) rotamer (shades show the 95% CI obtained from 10 block bootstrapping). c The frequency of the side chain to main chain hydrogen bond is strongly correlated with the solvent accessibility surface area (SASA) of H_ε21, which depends on the type of residue X. d Multiple regression correlating intrinsic helicity (x1) (Pace and Scholtz scale) and SASA (x2) with the average helicity (y): the data were standardized to estimate the relative weight of each variable in defining the model. e Measured versus predicted average L₃XQ₁₆ helicity. f Squared r correlation scores (r²) for the multiple regression shown in e, using different reported scales for intrinsic amino acid helicity and H_ε21 SASA values derived from two sets of 1 μs MD trajectories independently generated with different force fields. The results are shown for all and apolar (L, I, V, F, Y, M, W, A) residues in position X. g Number of ScanProsite-identified protein sequences in UniprotKB (including the Swiss-Prot and TrEMBL databases) containing the (P3-7)_n motif with an increasing number of Gln_i+4 → Ω_i pairs (Ω = W, L, Y, F, I, M; X = any amino acid). h Representative (P3-7)_n-like natural sequences with UniprotKB annotation score = 5.

Fig. 4. Introduction of a pH-sensitive conformational switch.

a The side chains of Gln and Glu at pH 2.8, but not that of Glu at pH 7.4 can donate a hydrogen to the main chain CO. b QxE variants of L₄Q₁₆ with pH-sensitive Glu_i+4 → X_i interactions shown in red. c Helical propensities at pH 7.4 (black) and 2.8 (dark red) compared to those of L₄Q₁₆ at pH 7.4 (orange). d Residue-specific differences in helical propensity due to substitution of Gln by Glu at pH 7.4 (black) and pH 2.8 (dark red). e–g Gln side chain N_ε2-H_ε21 regions of the ¹H-¹⁵N HSQC spectra of QxE variants at pH 7.4 (left) and 2.8 (right) with the spectrum of L₄Q₁₆ overlaid as an orange shade. The spectra of variants Q1E and Q4E with ¹⁵N labeling of only the Gln in position i+4 to the mutated residue are superimposed in green. h QM/MM-derived hydrogen bond electron densities for the Glu_i+4 → Leu_i interaction. Mean values for the Gln_i+4 → Leu_i interaction are shown as an orange line. First and second panels: normalized histograms showing the distribution of the electron density ρ(r) of the main chain to main chain interaction in the absence (white background) and in the presence (gray) of the side chain to main chain hydrogen bond. Third panel: electron densities for the side chain to main chain hydrogen bond. Fourth panel: electron densities for the bifurcated hydrogen bond. i Natural population analysis (NPA) charges on the donor hydrogen atom in Glu and Gln, showing that its charge depends on whether it participates in the hydrogen bond (lower values) or not. j, k pH-dependent helicity and thermal stability for peptide E(P3-7)_n. Top: Helical projections. Center: CD spectra (278 K) of peptides E(P3-7)_n and (P3-7)_n at the indicated conditions. Bottom: thermal denaturation of peptides E(P3-7)_n and (P3-7)_n monitored by measuring the mean residue ellipticity at 222 nm. l 2D CACO NMR spectra of E(P3-7)₃. m Residue-specific helical propensities for peptide E(P3-7)₃ at pH 2.8.

Fig. 5. The polyQ tract of TBP forms a helix stabilized by Gln side chain to main chain hydrogen bonds and an electrostatic interaction.

a Sequence, numbering and helical projection of peptide TBP-Q₁₆. The electrostatic interaction is color-coded in purple. b CD spectra of peptides TBP-Q₁₆ (black) and TBP-Q₂₅ (gold). c Scheme of the electrostatic interaction between Arg and Glu at physiological pH and its absence at acidic pH. d ¹³C-detected CACO spectra of the TBP polyQ tract at pH 7.4 (top, black) and 2.8 (bottom, dark red). e Residue-specific helicity of TBP at pH 7.4 (black) and 2.8 (dark red). Shades color-coded as in a are shown to guide the eye. f Region of the ¹H-¹⁵N HSQC spectra of TBP at pH 7.4 (left, black) and 2.8 (right, dark red) showing the N_ε2-H_ε21 correlations. g Strips from the 3D H(CC)(CO)NH spectra of TBP at pH 7.4 (left, black) and 2.8 (right, dark red) displaying the side chain aliphatic ¹H resonances of the first three Q residues in the polyQ tract. Strips were chosen from either N^H or N_ε2 for clarity. h Frames from the MD trajectory obtained for TBP. An orthogonal view of the helix is shown. In the left panel, a frame is shown where Arg13 (i) establishes an electrostatic interaction with E10 (i-3), represented by the purple dashed line. Simultaneously, both Gln11 and Gln12 establish bifurcated hydrogen bonds with Ile7 and Leu8, respectively (pink dashed lines). Right: a frame where Arg13 (i) establishes an electrostatic interaction with Glu9 (i-4), while the two bifurcated hydrogen bonds previously described are also present.

Fig. 6. Design of Gln-based single α-helical peptides that interact with RAP74-CTD.

a Top: design of peptide δ, blending the consensus RAP74-CTD binding motifs of FCP1 with peptide (P3-7)₃. Peptide δ_Stpl features a chemical staple covalently linking the side chains at positions 13 and 17, whereas all Leu residues outside the binding motif were substituted by Ala. Peptide δ_ctrl is devoid of helix-stabilizing interactions. Bottom: CD spectra of peptides δ (gold), δ_Stpl (purple), and δ_ctrl (dark red) at 298 K (solid) and 278 K (dashed). b Averaged chemical shift perturbations observed in the ¹H-¹⁵N resonances of the RAP74-CTD upon the addition of 15 molar equivalents of the (P3-7)₃ (teal), δ (gold), δ_Stpl (purple), and δ_ctrl (dark red) peptides. NMR-derived K_D values (Supplementary Figure 11a) are shown in the legend. The dashed line sets the threshold for the top 10% peaks with the most intense averaged CSPs upon the addition of δ. c Left: structure of the RAP74-CTD domain of TFIIF (PDB entry 1NHA) colored in a gradient representing the averaged CSPs observed upon the addition of 15 molar equivalents of the δ peptide (white, minimum CSP_av; gold, maximum CSP_av). Right: region of the overlaid ¹H-¹⁵N BEST-TROSY spectra of RAP74-CTD in the absence (black) and in the presence of 15 molar equivalents of the (P3-7)₃ (teal), δ (gold), δ_Stpl (purple), and δ_ctrl (dark red) peptides. d Top: design of peptide γ blending the RAP74-CTD binding motif AR-WHTLF with peptide (P3-7)₂. Peptide γ_Stpl features a chemical staple covalently linking the side chains at positions 10 and 14, whereas all Leu residues outside the binding motif were substituted by Ala. Peptide γ_Ctrl is devoid of helix-stabilizing interactions. Bottom: CD spectra of peptides AR-WHTLF (green), γ (blue), γ_Stpl (purple), and γ_Ctrl (dark red) at 298 K (solid) and 278 K (dashed). e as in b for (P3-7)₂ (dark gray), AR-WHTLF (green), γ (blue), γ_Stpl (purple), and γ_Ctrl (dark red). f Left: as in c, left, for peptide γ. Right: as in c, right, for the (P3-7)₂ (purple), AR-WHTLF (green), γ (blue), γ_Stpl (purple), and γ_Ctrl (dark red) peptides.