Research ArticleCOMPUTATIONAL CHEMISTRY

Regulation of protein-ligand binding affinity by hydrogen bond pairing

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Science Advances  25 Mar 2016:
Vol. 2, no. 3, e1501240
DOI: 10.1126/sciadv.1501240
  • Fig. 1 The s-s/w-w H-bond pairing principle and the effect of protein-ligand H-bonds on protein-ligand binding.

    (A) General schematic of the principle. Red and blue circles indicate H-bond acceptors and donors, respectively, with the symbol representing the relative H-bonding capability. (B) Competing H-bonds of two ligand atoms (LA and LB) to a protein atom P: this illustrates the effect of the H-bonding capability on the ligand binding affinity. (C) Relationship between the ΔG° of process (B) and HBHA; HA and HB are the H-bonding capability of LA and LB, respectively. The slopes of the lines are directly proportional to HWHP (the difference in H-bonding capability between water and the protein atom). (D) Relationship between ΔGHB for protein-ligand H-bonds and the H-bonding capability of ligand atom (HL). ΔGs-s° is the contribution of s-s pairing H-bonds shown in Fig. 3B to the ligand binding affinity; ΔGw-s° is associated to the polar-apolar interaction (w-s pairing H-bonds) shown in Fig. 3B. The yellow region represents H-bonds that have little effect on binding affinity.

  • Fig. 2 Validation of Eq. 6 based on the definition of H-bonding capability.

    (A) A competing H-bond pairing process shows the binding of two H-bonding acceptors A and B bound to the same nonpolar site of a protein receptor. (B and C). Because the nonpolar environment is similar to hexadecane, process (A) can be represented as two subprocesses (B and C), which are relevant to the definition of H-bonding capability. The free energy change of process (A) is HBHA (H-bonding capability) and is derived from experimental water/hexadecane partition coefficients. Because the H-bonding capability of nonpolar atoms is zero, the calculated free energy change based on Eq. 6 is similar to the experimental data, irrespective of the nature of HB and HA. Further validation is provided in text S2.

  • Fig. 3 Validation of Eq. 6 with reported experimental data.

    (A) Structures of the inhibitors used in this figure. Inhibitors 1 and 2 are scytalone dehydratase inhibitors. Inhibitors 3 and 4 are carbonic anhydrase inhibitors. (B) The competing H-bond pairing process between inhibitors 1 and 2 is used to calculate whether s-s H-bond pairings enhance ligand binding affinity. (C) The competing H-bond pairing process between inhibitors 3 and 4 demonstrates that the strong H-bond between 4 and Thr200 (s-w pairing) is less favorable to binding affinity than the weak interaction between 3 and Thr200 (w-w pairing).

  • Fig. 4 Streptavidin-biotin as a prototypical example of the contribution of s-s/w-w H-bond pairings to high binding affinity.

    (A) H-bond interactions between the ureido oxygen atom from biotin and streptavidin. The H-bonds contribute significantly to binding affinity because of the extreme H-bonding capabilities for both H-bond donors and H-bond acceptors. (B) Structure and interactions of the biotin analog 2-iminobiotin, which is highly similar in structure to biotin, yet its binding affinity to streptavidin is >3 million–fold lower (30). (C) The unfavorable positive-positive interactions between the imino group and H-bond donors in (B) are minimal because the side chains are rotatable.

  • Fig. 5 Ligand binding affinity is relatively unaffected when the H-bonding capability is close to water.

    Interactions between the H-bond acceptors of three heterocyclic aromatic sulfonamide inhibitors (5, 6, and 7) with large differences in H-bonding capabilities and the H-bond donors from the receptor Thr200. Because the H-bonding capability of the receptor Thr200 protein is close to that of water, the ligand binding affinity is relatively unaffected by the varying strengths of the H-bonds that are formed. A similar inhibitor, 1H-benzimidazole-2-sulfonamide, is excluded from our comparison because its extra polar hydrogen atom affects binding affinity.

  • Fig. 6 Favorable π–quaternary ammonium cation interactions with w-w H-bond pairings.

    (A and B) Structures of two factor Xa antagonists 8 and 9. Antagonist 8 is ~1100-fold more active than 9 because of the w-w pairing interactions between the hydrophobic aromatic rings of factor Xa and polarized CH groups [–N(Me)3+] shown in (B) (Protein Data Bank: 2JKH).

  • Fig. 7 Pathogenic s-s H-bond pairings in melamine toxicity.

    (A) Structural analysis of melamine (MEL, blue)–cyanuric acid (CYA, red) interactions demonstrates an extensive H-bonding network (dashed lines) that promotes the formation of toxic insoluble crystals. (B and C) The MEL and CYA complex forms s-s H-bond pairings (fig. S7), with H-bonding capabilities of MEL and CYA and of inhibitors 2,6-dihydroxyisonicotinic acid (DHI), 3-(2,5-dioxo-4-imidazolidinyl)propanoic acid (DIPA), and tetrahydrofurandiol (THF) shown in (C). (D and E) Although DHI (D) and 2,6-diaminoisonicotinic acid (DAI) (E) are structurally similar, the hydrogen atoms of DHI have a much stronger H-bonding capability (C). (G) (left) We used this calculation of H-bonding capability to demonstrate that DHI preferentially forms a complex with CYA when compared with DAI or MEL (F). Statistically significant inhibition of MEL-CYA complex formation was also demonstrated with DIPA, which forms s-s H-bond pairings with the triol tautomer of CYA. The triol tautomer predominates in solution because of its aromatic character. DIPA also dissolved MEL-CYA crystal in solution (whereas THF did not) (G; right), illustrating how the s-s/w-w H-bond pairing principle may be applied to lead optimization in diseases caused by low solubility (error bars show SEM; P < 0.05 was considered significant using t test or Mann-Whitney U test for ranks).

  • Fig. 8 Lead optimization of the C. difficile toxin inhibitor InsP6 using the s-s/w-w H-bond pairing principle.

    (A and B) H-bond interactions between InsP6 and allosteric binding site residues on TcdB based on the crystal structure 3PA8. (C) Structures of InsP6, its derivative InsP(S)6, and the structural analog InsS6 and the relative H-bonding capability of the oxygen atoms. (D) (Top) TcdB autocleavage induced by 100 nM InsP6, InsP(S)6, or InsS6 shows intact unprocessed (270 kD) and processed toxin cleavage products (205 kD). Processed toxin is inactive as the virulent glucosyltransferase domain fails to enter the target cell. (Bottom) InsP6 binding affinity for TcdB as measured by its self-cleavage activity in vitro (half-maximum activation constant, AC50, in micromole per liter). Increasing the H-bonding capability of the oxygen atoms in InsP(S)6 enhances the AC50 by 26-fold, whereas decreasing the H-bonding capability in InsS6 leads to a 110-fold reduction of AC50 (*P < 0.05 compared with InsP6). (E) Kaplan-Meier survival plots of C57BL/6 mice inoculated intragastrically with 103 C. difficile VPI 10463 spores and with InsP6 or InsP(S)6 (1 or 10 mg kg−1 day−1; n = 5 per group; survival at day 4; P < 0.05, analysis of variance on ranks). (F) Histopathology showing that oral InsP(S)6 is protective for colonic mucosa when administered at 10 mg.kg−1 day−1 (scale bar, 50 μm).

  • Fig. 9 Estimating the contribution of H-bond interactions to the free energy barrier reduction for the isomerization of 5-androstene-3,17-dione (5-AND) in aqueous solution.

    (A) H-bond interactions in the oxyanion hole of the ketosteroid isomerase for the ground state (GS) and transition state (TS). The H-bonds in red boxes are well oriented. (B) GS and TS without H-bond interactions. (C) Difference between (A) and (B), which is a competing H-bond pairing process. (D) H-bond pairing process in which the H-bond interactions are similar to (C) but are not restricted and can adopt a broad distribution of conformations as is seen in solution. (E) Competing H-bond pairing process for estimating the free energy barrier reduction contributed by the H-bond interactions of the reference reaction. We assume that the H-bonding capabilities for the atoms in the ketosteroid isomerase are close to those in solution. On the basis of Eq. 6, we get ΔGenz = k × (0 − Henz)(HO_TSHO_GS) and ΔGref = k × (0 − HW)(HO_TSHO_GS). Gref = ΔGenz × (0 − HW)/(0 − Henz) < −6.13 × 14.04/22.0 = −3.91 (kcal/mol). Thus, the free energy barrier reduction for the isomerization of 5-AND in aqueous solution is larger than 3.91 kcal/mol.

  • Table 1 H-bonding capabilities (in kilojoules per mole) of defined atoms (red) in basic functional groups calculated from water/hexadecane partition coefficients (fig. S2): R, R1, and R2 represent alkyl groups.

    The H-bonding capability for oxygen atoms is the value of a lone pair (±SDs).

    H-bond acceptorsH-bond donors
    AtomH-bonding capabilityAtomH-bonding capability
    H2O7.02 (±0.11)H2O7.02 (±0.11)
    Apolar atom0Apolar atom0
    AlcoholAlcohol6.6 (±0.21)
    RCH2OH8.7 (±0.42)RCH2OH
    RR1CHOH9.5 (±0.58)RR1CHOH6.3 (±0.20)
    SerCH2OH8.1 (±0.42)CF3CH2OH10.2 (±0.45)
    EtherAmide
    THF: (CH2)4O8.2 (±0.22)RCH2C(=O)NHR17.6 (±0.65)
    RCH2OCH2R7.2 (±0.35)C6H5C(=O)NHR17.2 (±0.86)
    AldehydePhenol
    RCH2CHO8.9 (±0.45)4-R-C6H4OH10.8 (±0.74)
    4-RC6H4CHO7.6 (±0.26)4-NO2-C6H4OH16.9 (±0.75)
    KetoneAniline
    4-RC6H4COMe10.4 (±0.27)C6H5NH22.7 (±0.51)
    RCH2COCH2R9.8 (±0.55)4-NO2-C6H4NH26.1 (±0.78)
    Ester (O.2)Amino
    4R-C6H4C(=O)OCH38.4 (±0.26)RCH2NH21.3 (±0.34)
    RCH2C(=O)OCH2R9.6 (±0.47)(RCH2)2NH1.5 (±0.61)
    AmideAcid
    RCH2C(=O)NH214.5 (±0.62)RCH2COOH11.2 (±0.86)
    RCH2C(=O)NHCH314.9 (±0.64)C6H5COOH11.5 (±1.03)
    RCH2C(=O)N(CH3)215.9 (±0.22)Cl3CCOOH20.4 (±0.77)
    C6H5C(=O)NH212.0 (±0.88)3NO2-C6H4COOH13.2 (±1.06)
    AminoOthers
    RCH2NH219.7 (±0.58)CHCl33.6 (±0.42)
    (RCH2)2NH22.9 (±0.42)CHBr32.7 (±0.31)
    (RCH2)3N22.8 (±0.31)RCH2NO20.85 (±0.16)
    NitrileRCH2CN0.6 (±0.14)
    RCH2CN18.1 (±0.36)
    C6H5CN15.4 (±0.43)
    Pyridine
    C5H5N18.2 (±0.62)
    4-CN-C4H4N12.3 (±0.64)
    Others
    C6H5O*H6.4 (±0.51)
    Furan: C4H4O*2.9 (±0.65)
    RC6H5 (pi electron)1.6 (±0.18)

    *Because the two lone pairs of electrons are not identical, H-bonding capability is the value of the oxygen atom.

    • Table 2 Enthalpy changes for the H-bond competing processes (A + B ↔ C + D) of strong ionic H-bonds (ΔH, in kilocalories per mole).

      On the basis of the H-bond energies given (25), we list H-bond competing processes, the energy of each H-bond, and the enthalpy change (ΔH) of each process. The strongest H-bond of each process is D, which is on the right side of the process. Because all processes have negative ΔH values, we conclude that the reversible H-bond competing process favors the s-s/w-w H-bond pairing in enthalpy. A, B, C, and D denote the hydrogen bonds of the H-bond competing processes. EA, EB, EC, and ED denote the hydrogen bond energy of A, B, C, and D in kilocalories per mole.

      ABCDEAEBECEDΔH
      1Cl…H-ClF…H-FCl…H-FF…H-Cl23.138.621.859.8−19.9
      2Cl…H-BrF…H-FCl…H-FF…H-Br29.038.621.865.0−19.2
      3Cl…H-IF…H-FCl…H-FF…H-I32.038.621.872.0−23.2
      4Cl…H-CNF…H-FCl…H-FF…H-CN21.038.621.839.5−1.7
      5Br…H-ClF…H-FBr…H-FF…H-Cl19.038.617.059.8−19.2
      6Br…H-BrF…H-FBr…H-FF…H-Br20.038.617.065.0−23.4
      7Br…H-CNF…H-FBr…H-FF…H-CN16.038.617.039.5−1.9
      8I…H-ClF…H-FI…H-FF…H-Cl13.038.615.059.8−23.2
      9CN…H-ClF…H-FCN…H-FF…H-Cl37.038.621.159.8−5.3
      10F…H-FCN…H-BrCN…H-FF…H-Br38.642.021.165.0−5.5
      11CN…H-CNF…H-FCN…H-FF…H-CN20.038.621.139.5−2.0
      12Cl…H-IF…H-ClCl…H-ClF…H-I32.059.823.172.0−3.3
      13Cl…H-ClF…H-CNCl…H-CNF…H-Cl23.139.521.059.8−18.2
      14Br…H-BrF…H-ClBr…H-ClF…H-Br20.059.819.065.0−4.2
      15Br…H-ClF…H-CNBr…H-CNF…H-Cl19.039.516.059.8−17.3
      16CN…H-ClF…H-CNCN…H-CNF…H-Cl37.039.520.059.8−3.3
      17Cl…H-IF…H-BrCl…H-BrF…H-I32.065.029.072.0−4.0
      18Cl…H-BrF…H-CNCl…H-CNF…H-Br29.039.521.065.0−17.5
      19Br…H-BrF…H-CNBr…H-CNF…H-Br20.039.516.065.0−21.5
      20F…H-CNCN…H-BrCN…H-CNF…H-Br39.542.020.065.0−3.5
      21Cl…H-IF…H-CNCl…H-CNF…H-I32.039.521.072.0−21.5
      22Br…H-BrCl…H-FBr…H-FCl…H-Br20.021.817.029.0−4.2
      23I…H-ClCl…H-FI…H-FCl…H-Cl13.021.815.023.1−3.3
      24CN…H-FCl…H-ClCl…H-FCN…H-Cl21.123.121.837.0−14.6
      25CN…H-FCl…H-BrCl…H-FCN…H-Br21.12921.842.0−13.7
      26Br…H-BrCl…H-ClBr…H-ClCl…H-Br20.023.119.029.0−4.9
      27CN…H-CNCl…H-ClCl…H-CNCN…H-Cl20.023.121.037.0−14.9
      28Br…H-BrCl…H-CNBr…H-CNCl…H-Br20.021.016.029.0−4.0
      29CN…H-CNCl…H-BrCl…H-CNCN…H-Br20.029.021.042.0−14.0
      30I…H-ClBr…H-FI…H-FBr…H-Cl13.017.015.019.0−4.0
      31Br…H-ClCN…H-FBr…H-FCN…H-Cl19.021.117.037.0−13.9
      32Br…H-BrCN…H-FBr…H-FCN…H-Br20.021.117.042.0−17.9
      33Br…H-BrCN…H-ClBr…H-ClCN…H-Br20.037.019.042.0−4.0
      34Br…H-ClCN…H-CNBr…H-CNCN…H-Cl19.020.016.037.0−14.0
      35CN…H-CNBr…H-BrBr…H-CNCN…H-Br20.020.016.042.0−18.0
      36I…H-ClCN…H-FI…H-FCN…H-Cl13.021.115.037.0−17.9

    Supplementary Materials

    • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/3/e1501240/DC1

      Text S1. Theoretical proof of the H-bond pairing principle.

      Text S2. Relationship between the free energy change for a reversible protein-ligand H-bond competing process and the H-bonding capability of the H-bond–forming atoms.

      Text S3. The H-bonding capability of the protein atoms with which a ligand atom interacts and the effect of H-bond geometry on the H-bond interaction.

      Fig. S1. Schematic illustration of the free energy change for the H-bond competing process.

      Fig. S2. Calculation of H-bonding capability based on water/hexadecane partition coefficients.

      Fig. S3. Contributions of the H-bonds between CN and the Tyr-OH from scytalone dehydratase to protein-ligand interactions.

      Fig. S4. Proof for the strong H-bond interactions between the CN group of inhibitor 2 and tyrosine hydroxyls from scytalone dehydratase.

      Fig. S5. Binding affinities of 1H-imidazole-2-sulfonamide and thiophene-2-sulfonamide.

      Fig. S6. Quaternary ammonium cation [–N(Me)3+]-π interactions are more favorable than ammonium ion (–NH3+)-π interactions.

      Fig. S7. A pathogenic role for the s-s/w-w H-bonding principle in melanin toxicity.

      Fig. S8. The thermodynamic cycle that demonstrates the contribution of H-bonds to enzymatic catalytic power equates to their contribution to protein-ligand binding.

      References (4652)

    • Supplementary Materials

      This PDF file includes:

      • Text S1. Theoretical proof of the H-bond pairing principle.
      • Text S2. Relationship between the free energy change for a reversible protein-ligand H-bond competing process and the H-bonding capability of the H-bond–forming atoms.
      • Text S3. The H-bonding capability of the protein atoms with which a ligand atom interacts and the effect of H-bond geometry on the H-bond interaction.
      • Fig. S1. Schematic illustration of the free energy change for the H-bond competing process.
      • Fig. S2. Calculation of H-bonding capability based on water/hexadecane partition coefficients.
      • Fig. S3. Contributions of the H-bonds between CN and the Tyr-OH from scytalone dehydratase to protein-ligand interactions.
      • Fig. S4. Proof for the strong H-bond interactions between the CN group of inhibitor 2 and tyrosine hydroxyls from scytalone dehydratase.
      • Fig. S5. Binding affinities of 1H-imidazole-2-sulfonamide and thiophene-2-sulfonamide.
      • Fig. S6. Quaternary ammonium cation –N(Me)3+-π interactions are more favorable than ammonium ion (–NH3+)-π interactions.
      • Fig. S7. A pathogenic role for the s-s/w-w H-bonding principle in melanin toxicity.
      • Fig. S8. The thermodynamic cycle that demonstrates the contribution of H-bonds to enzymatic catalytic power equates to their contribution to protein-ligand binding.
      • References (46–52)

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