AMBER and CHARMM Force Fields Inconsistently Portray the Microscopic Details of Phosphorylation

Abstract

Phosphorylation of serine, threonine, and tyrosine is one of the most frequently occurring and crucial post-translational modifications of proteins often associated with important structural and functional changes. We investigated the direct effect of phosphorylation on the intrinsic conformational preferences of amino acids as a potential trigger of larger structural events. We conducted a comparative study of force fields on terminally capped amino acids (dipeptides) as the simplest model for phosphorylation. Our bias-exchange metadynamics simulations revealed that all model dipeptides sampled a great heterogeneity of ensembles affected by introduction of mono- and dianionic phosphate groups. However, the detected changes in populations of backbone conformers and side-chain rotamers did not reveal a strong discriminatory shift in preferences, as could be anticipated for the bulky, charged phosphate group. Furthermore, the AMBER and CHARMM force fields provided inconsistent populations of individual conformers as well as net structural trends upon phosphorylation. Detailed analysis of ensembles revealed competition between hydration and formation of internal hydrogen bonds involving amide hydrogens and the phosphate group. The observed difference in hydration free energy and potential for hydrogen bonding in individual force fields could be attributed to the different partial atomic charges used in each force field and, hence, the different parametrization strategies. Nevertheless, conformational propensities and net structural changes upon phosphorylation are difficult to extract from experimental measurements, and existing experimental data provide limited guidance for force field assessment and further development.

An Inherent Structural Difference Between Serine and Threonine Phosphorylation: Phosphothreonine Prefers an Ordered, Compact, Cyclic Conformation

“…Given the critical roles of protein phosphorylation in intracellular regulation, the development of appropriate force fields for the accurate modeling of the structural effects of post-translational modifications has attracted considerable interest, as well as the recognition that current force fields are insufficient for accurate modeling of the structural effects of phosphorylation. [65][66][67][68][69][70][71][72] The work herein provides important structural constraints to allow the improved modeling of the effects of protein Ser and Thr phosphorylation.…”

A predictive coarse-grained model for position-specific effects of post-translational modifications

“…All-atom explicit solvent simulations readily incorporate post-translationally modified amino acids (32)(33)(34)(35)(36) and have been used to probe the contacts leading to phase separation (37,38). However, the ability of atomistic force fields to accurately capture the properties of IDRs depends strongly on parameterization (39,40). Additionally, simulations of sufficiently long length and timescales to predict phase-separation behavior (e.g., saturation concentration) are beyond current computational capability for fully atomistic simulations with explicit water.…”

Development and validation of AMBER-FB15-compatible force field parameters for phosphorylated amino acids

“…[47][48][49][50][51] The validation of phosphorylated amino acid parameters by comparing simulation predictions to experiment is lacking in the literature, in contrast to the case of the canonical amino acids. A recent article by Vymětal et al 52 has pointed out inconsistencies in calculated quantities by the AMBER and CHARMM force fields for the phosphorylated amino acids over a wide range of properties related to their conformational ensembles, such as NMR 3 J couplings, intramolecular hydrogen bonding propensities, conformational preferences, and more; inconsistencies in calculated conformational preferences by AMBER and CHARMM for larger IDPs have also been noted by Rieloff and Skepö 53 . This raises questions about the suitability of current force field parameter sets for simulation of these systems.…”

“…ForceBalance is used to optimize bonded parameters starting from those created by Homeyer et al 43 for the phosphorylated side chains in order to keep consistent parameters for the backbone atoms, with the nonbonded parameters unmodified from those implemented by Homeyer et al 43 and Steinbrecher et al 45 Detailed potential energy surface comparisons are made between parameters from AMBER ff99SB, 63 which we use as a starting point for the force field optimization, the "FB18" optimized parameters presented in this work and the QM reference data for validation of the energies predicted by the new parameters. Additionally, we compare the new force field to experimental data for blocked dipeptide forms of each of the amino acids parameterized here, similarly to the procedure performed by Vymětal et al 52 These are essentially the simplest phosphorylated systems possible, for which a multitude of data exists, such as 3 J NMR couplings and chemical shifts, intramolecular hydrogen bond propensities, and backbone conformational preferences. [64][65][66] The simple nature of these systems allows for relatively straightforward comparison between MD simulation results and experiment.…”