Determination of thermal intermediate state ensemble of box 5 with restrained molecular dynamics simulations

The thermal intermediate state of the high mobility group box 5 domain in human upstream binding factor was detected at 55 °C by nuclear magnetic resonance (NMR) experiments. For insufficient data, however, the tertiary structure of the intermediate state cannot be resolved as native state with the experimental techniques. To characterize the intermediate state ensemble, here we performed ensemble-averaged molecular dynamics simulations on the box 5 protein with 421 distance restraints derived from Nuclear Overhauser Enhancement and paramagnetic relaxation enhancement, as well as 122 dihedral angle restraints obtained from the program TALOS based on atom chemical shifts. The number of replicas was 48. The 60 ns simulation was completed for each replica. The total simulation time was up to 2.88 μs. The results indicated the intermediate state ensemble of box 5 was high heterogeneity and most secondary structures were formed. The N-terminal coil and helix 1 moved toward the C-terminal region; helix 3 was more stable and native-like than the other two helices; the hydrophobic core was not formed completely in the intermediate ensemble; and the L-shaped topology of the native conformation disappeared. In addition, some experimental inconsistencies were found, which could not be resolved in one conformation. In this study, the structural characteristics of box 5 thermal intermediate state ensemble were determined, which cannot be directly achieved through wet experiments. The findings of the current work are useful for the understanding of the protein folding mechanism. In our knowledge, this is the first report on the structural and thermal characters of the intermediate state ensemble of box 5.