Molecular dynamics simulations suggest stabilizing mutations in a de novo designed α/β protein

Abstract

Designing functional proteins that can withstand extreme heat is beneficial for industrial and protein therapeutic applications. Thus, elucidating the atomic-level determinants of thermostability is a major interest for rational protein design. To that end, we compared the structure and dynamics of a set of previously designed, thermostable proteins based on the activation domain of human procarboxypeptidase A2 (AYEwt). The mutations in these designed proteins were intended to increase hydrophobic core packing and inter-secondary-structure interactions. To evaluate whether these design strategies were successfully deployed, we performed all-atom, explicit-solvent molecular dynamics (MD) simulations of AYEwt and three designed variants at both 25 and 100°C. Our MD simulations agreed with the relative experimental stabilities of the designs based on their secondary structure content, Cα root-mean-square deviation/fluctuation, and buried-residue solvent accessible surface area. Using a contact analysis, we found that the designs stabilize inter-secondary structure interactions and buried hydrophobic surface area, as intended. Based on our analysis, we designed three additional variants to test the role of helix stabilization, core packing, and a Phe → Met mutation on thermostability. We performed the additional MD simulations and analysis on these variants, and these data supported our predictions.

Keywords: molecular dynamics; protein design; protein thermostability.

Fig. 1

Protein structures and sequences. (a) Initial and final structures from simulation shown for all seven proteins. Representative final structures were chosen as the frame with the median Core Cα RMSD among the five replicate simulations. On the initial structures, mutated residues relative to the template structure were shown in spheres. For simplicity, hydrogen atoms were not displayed throughout, and only Cα atoms of mutated residues were shown for AYEdes. (b) Sequences for all seven proteins with mutations relative to AYEwt shown in bold and residue numbers of buried residues underlined. All sequences have been aligned and numbered relative to the AYEdes structure. (c) Secondary structure elements shown for β1 (residues 4–9), α1 (13–24), β2 (31–36), β3 (43–47), α2 (52–62), and β4 (65–68).

Fig. 2

Backbone dynamics at low and high temperature. Cα RMSF is reported for all proteins at both temperatures. Secondary structure elements are shown between the two plots.

Fig. 3

Core Cα RMSD and side chain SASA of buried residues. Average (a) core (residues 4–68) Cα RMSD and (b) SASA of buried residue side chains (residues 6, 8, 10, 15, 16, 19, 22, 36, 43, 45, 47, 55 and 59) for all seven proteins at 25 (dark) and 100°C (light). Error bars represent SEM, n = 5.

Fig. 4

Persistence of secondary structure elements. Average percent of simulation time that all residues in each secondary structure element spent in either α-helix or β-sheet as defined by DSSP for all seven proteins at 25 (dark) and 100°C (light). See Fig. 1c for residue secondary structure assignments. Error bars represent SEM, n = 5.

Fig. 5

Contacts between secondary structure elements and buried residues. Average number of hydrogen bonds, hydrophobic interactions and other interactions between (a) α1 and the β-sheet, (b) α2 and the β-sheet, (c) α1 and α2 and (d) average number of hydrophobic interactions among buried residues (6, 8, 10, 15, 16, 19, 22, 36, 43, 45, 47, 55 and 59) for all seven proteins at 25 (dark) and 100°C (light). Error bars represent SEM, n = 5.

Fig. 6

Inter-secondary-structure contacts. Representative final structures from simulations at 25°C showing Phe residues and their rescued contacts in sticks for (a) AYEwt and (b) AYEdes. Representative final structures from simulations at 100°C showing residues 18 and 23 and all residues they contacted for any amount of simulation time in the last 50 ns at 25°C in sticks for (c) AYEwt and (d) AYEwt-3mut. Representative final structures from simulations at 25°C showing residue 58 and all residues it contacted for any amount of simulation time in the last 50 ns in sticks for (e) AYEdes and (f) AYEdes-M58F.