Hydrophobicity in Intrinsically Disordered Protein Force Fields: Implications for Conformational Ensembles and Protein-Protein Interactions

Abstract

Intrinsically disordered proteins (IDPs) lack a stable 3D structure under physiological conditions, making them challenging to study and simulate. In this study, we compare the hydrophobicity and water-protein interactions of amino acids in three popular all-atom molecular dynamics (MD) force fields: amber03ws (a03ws), CHARMM36m (C36m), and a99SB-disp. Using the indirect umbrella sampling (INDUS) technique, we quantify the dewetting free energies of each amino acid in the force fields. Additionally, we analyze water structuring around the amino acids using the water triplet angle distribution and measure water diffusion in the hydration shells. Our results reveal that CHARMM36m has the lowest dewetting free energies, indicating higher amino acid hydrophobicity, while a99SB-disp exhibits the highest, suggesting lower hydrophobicity. Water diffusion is significantly slower in the hydration shells of a99SB-disp due to its unique water structuring (e.g., higher frequency of tetrahedral coordination), while there is much less of a water diffusion slowdown in a03ws and CHARMM36m. We show that these differences impact the behavior of an aggregation-prone tau fragment, jR2R3 P301L, in MD simulations. We find that CHARMM36m's propensity for dimer formation is attributed to its lower dewetting free energies, whereas a99SB-disp's higher-than-expected dimerization propensity is due to favorable, entropically driven changes in water structure upon peptide association. These findings underscore the importance of accurately modeling water-protein interactions for IDPs and protein-protein interactions as well as the sensitivity of these to the underlying force field. Our study suggests that dewetting free energies and water structuring metrics, such as the water triplet angle distribution, can be valuable for future force field development and for predicting phenomena related to water-protein interactions.

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(A) Depiction of the simulation setup for the INDUS procedure. The capped amino acids are placed far apart in a water box, and then waters are iteratively pushed out of the hydration volume (B), defined by the distances from the amino acids’ heavy atoms, to ultimately determine dewetting free energies. (C) Dewetting free energies per water of each amino acid in the three force fields. Amino acids are ordered based on their hydrophilicity in a03ws. (D) Comparing water diffusivity of the hydration waters relative to bulk water in each force field; note the stretched y-axis from 0.9 to 1.0. D _bulk for a03ws, a99SBdisp, and C36m is 0.23, 0.19, and 0.58 Ų/ps, respectively.

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(A) jR2R3 P301L monomer (i) and dimer (ii) cluster and its hydration waters. (iv) Depiction of the cryo-EM structure of jR2R3 P301L, which consists of four strands, two of which form an internal side chain zipper. A dimer from the side chain zipper strand is depicted in (iii). (B) Comparing the distribution of hydration waters for monomers (left half of violins) and dimers (right half of violins) for each force field. For reference, the hydration waters of the compact cluster (A­(ii)) are indicated with a star, and the hydration waters of the cryo-EM dimer (A­(iii)) and filament (A­(iv)) are indicated with dashed lines. (C) Comparing the center-of-mass distance distribution between the two peptides in the dimer ensemble for each force field; shading denotes a 90% confidence interval. CHARMM36m has a strong propensity for the associated state (i.e., low center-of-mass distances), while a03ws has a stronger propensity for the disassociated state.

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(A) Depiction of the simulation setup with a small, capped peptide (valine-leucine-glycine with ACE and NME caps, gray sticks) diffusing within a teal box above a restrained jR2R3 P301L seed (purple). Flat-bottomed harmonic restraints prevent the chain’s center of mass from diffusing outside the teal box. (B) Free energy surface of the peptide as a function of its height above the seed at two temperatures, from umbrella sampling on the center of mass; shading denotes standard errors. (C) The docking free energy for each force field at two temperatures. a99SBdisp has a greater docking free energy at 325 K, indicating a significant entropic component of the docking process, likely due to water unstructuring upon docking. (D) Comparing the residence times (median and 90th percentile) of the peptide on the seed for each force field; a99SBdisp has longer residence times than the other force fields. (E­(i)) Comparing water tetrahedrality around the undocked and docked peptides for each force field. Relative tetrahedrality is the fraction of tetrahedral water angles (100° < θ < 120°) in the hydration shell divided by the fraction of tetrahedral water angles in bulk. (E­(ii)) Comparing water tetrahedrality in a larger box (i.e., within the dashed lines of panel A) when the peptide is undocked vs docked in each force field. Tetrahedral waters are released as the peptide’s hydration shell overlaps with the seed’s hydration shell upon binding, especially in a99SBdisp.

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(A) Comparing a cluster from the C36m dimer ensemble to a dimer from the solved cryo-EM structure (see Figure A­(iii,iv)). I297, V306, and I308 form side chain contacts in both structures, and this also strongly resembles the same motif in the LNT filament (PDB #7P6A). (B) Comparing interchain-aligned PHF6 contacts in each force field, which is a key feature in all the tau filaments. PHF6 is residues 306–311 of tau. ∼22% of the C36m ensemble has more than 12 interchain-aligned PHF6 contacts, while the other force fields have <2%. 90% confidence intervals are shown. (C) A free energy landscape of C36m dimers along two collective variables: interchain-aligned PHF6 contacts and intrachain contacts. The top cluster from five regions (i–v) of this energy landscape is shown. Panel (A­(i)) shows the top cluster from region iv, while panel (C­(v)) shows the second cluster, which forms a compact dimer that appears in Figure A­(ii). PHF6 (residues 306–311) is pink, intramolecular hydrogen bonds are blue dashes, intermolecular hydrogen bonds are purple dashes, and well-aligned PHF6 hydrogen bonds are black dashes.