High resolution ensemble description of metamorphic and intrinsically disordered proteins using an efficient hybrid parallel tempering scheme

Abstract

Mapping free energy landscapes of complex multi-funneled metamorphic proteins and weakly-funneled intrinsically disordered proteins (IDPs) remains challenging. While rare-event sampling molecular dynamics simulations can be useful, they often need to either impose restraints or reweigh the generated data to match experiments. Here, we present a parallel-tempering method that takes advantage of accelerated water dynamics and allows efficient and accurate conformational sampling across a wide variety of proteins. We demonstrate the improved sampling efficiency by benchmarking against standard model systems such as alanine di-peptide, TRP-cage and β-hairpin. The method successfully scales to large metamorphic proteins such as RFA-H and to highly disordered IDPs such as Histatin-5. Across the diverse proteins, the calculated ensemble averages match well with the NMR, SAXS and other biophysical experiments without the need to reweigh. By allowing accurate sampling across different landscapes, the method opens doors for sampling free energy landscape of complex uncharted proteins.

Fig. 1. List of systems studied.

a Dihedral switching of alanine dipeptide, b folding of Trp-Cage from completely unfolded structure, c folding of β-hairpin, d intrinsically disordered Histatin-5, and e metamorphic switching in bacterial RFA-H are explored using the state-of-the art REST2 and the REHT approach designed in this work.

Fig. 2. Efficiency of REHT in comparison to REST2 in capturing the folding of fast-folding protein, Trp-Cage.

a Structural overlay between the natively folded NMR structure (blue) and the REHT-generated folded structure of Trp-cage (yellow, obtained at the base replica). b Time evolution of protein backbone RMSD from the NMR structure along one of the successfully folded replicas of REHT (top) and REST2 (bottom) simulations. The RMSD evolution of other folded replicas are shown in Supplementary Figs. 4 and 5. c, d Free energy landscape of Trp-Cage, shown as the function of Radius of gyration (Rg) and RMSD against the NMR structure. The landscape is shown for the ensembles collected at the base replica of c) REHT simulation and d REST2 simulation.

Fig. 3. Ensemble description of Intrinsically disordered Histatin-5.

a Charge-hydropathy plot showing the uniqueness of Histatin-5 that is located at the disordered zone with lower mean hydrophobicity unlike other successfully studied IDPs which exist at or near the folded zone. b Comparison between the experimental (black) and theoretical ensemble-averaged SAXS profiles, represented as a Kratky plot. The theoretical prediction was made for the last 250 ns unweighted trajectories corresponding to the base replica of REHT and REST2 simulations. The distribution of Rg for the ensembles obtained from REST2 (red) and REHT (blue) simulations are shown in the inset. c Comparison of ensemble averaged chemical shifts of Hα atoms predicted from the REHT and REST2 simulations (for the same 250 ns trajectory) with reference to the experimental NMR chemical shifts. d Weakly-funneled diffusive energy landscape of Histatin-5 explored from REHT simulation is shown as a function of Rg and solvent accessible surface area (SASA).

Fig. 4. Configurational map of His-5 ensemble based on pairwise-dissimilarity of conformations shown across 2-dimensional MDS axes.

In a, b the conformations of REST2 and REHT ensembles are colored by the respective Rg values and in c, d they are colored by replica index. Maximally separated non-overlapping clusters in REST2 as marked in c indicates the trapping of conformations in independent replicas that are eventually exchanged to base replica.

Fig. 5. Conformational metamorphosis of RFA-H.

a Schematic representation of the two native structures resolved in experiments, the α-helical fold of CTD in presence of NTD and β fold in isolated CTD. b The initial and final structures of the REHT simulation. The helical state of CTD while deleting the NTD was chosen as the starting structure (left figure in b). Structural superposition of the final simulation generated β barrel structure (red) over the experimental β structure (cyan) is shown on the right side of (b). c Validation of simulation generated ensemble by comparing the predicted Cα chemical shifts with the experimental shifts. The fitted linear regression (blue line) indicates a precise match between the two sets of data. d Dual basin free energy landscape of RFA-H shown as a function of RMSDs from the experimentally found α-helix (2OUG) and β-sheet (2LCL). The conformations at each of the basins, all α-helical and all β-sheet (I and VII), early structure with the loss of helicity at both the termini (II), intermediate partially unfolded structure with residual α-helical content (III), and metastable structures with open β-sheets (V and VI) are shown along the landscape. Note that the completely unfolded structure (IV) is at the other side of the dual basin and the transition of α → β structure does not need to step through the completely unfolded structure unlike that of metamorphic lymphotactin.

Fig. 6. Extent of hydration dynamics and its tight coupling with the conformational space sampling.

In figure a, b we showed the cumulative distribution of the slow component of water orientational relaxation decay (τ₂) in REST2 (red) and REHT (blue) simulations for Trp-cage (a) and His-5 (b). In c–f we projected the conformational space of Trp cage (across RMSD to the native NMR structure and Rg) and His-5 (across SASA and Rg) with the conformations color coded by the τ₂ value as indicated in the color bar. The region with significant entropic barrier is highlighted in all the systems. Since there are a lesser number of replicas in REST2 in comparison to REHT (8 vs 12 in Trp-cage, 10 vs 15 in histatin-5), the number of points analyzed also became lesser in REST2.