Insights into the role of electrostatics in temperature adaptation: a comparative study of psychrophilic, mesophilic, and thermophilic subtilisin-like serine proteases

Abstract

To investigate the role of electrostatics in different temperature adaptations, we performed a comparative study on subtilisin-like serine proteases from psychrophilic Vibrio sp. PA-44 (VPR), mesophilic Engyodontium album (Tritirachium album) (PRK), and thermophilic Thermus aquaticus (AQN) using multiple-replica molecular dynamics (MD) simulations combined with continuum electrostatics calculations. The results reveal that although salt bridges are not a crucial factor in determining the overall thermostability of these three proteases, they on average provide the greatest, moderate, and least electrostatic stabilization to AQN, PRK, and VPR, respectively, at the respective organism growth temperatures. Most salt bridges in AQN are effectively stabilizing and thus contribute to maintaining the overall structural stability at 343 K, while nearly half of the salt bridges in VPR interconvert between being stabilizing and being destabilizing, likely aiding in enhancing the local conformational flexibility at 283 K. The individual salt bridges, salt-bridge networks, and calcium ions contribute differentially to local stability and flexibility of these three enzyme structures, depending on their spatial distributions and electrostatic strengths. The shared negatively charged surface potential at the active center of the three enzymes may provide the active-center flexibility necessary for nucleophilic attack and proton transfer. The differences in distributions of the electro-negative, electro-positive, and electro-neutral potentials, particularly over the back surfaces of the three proteases, may modulate/affect not only protein solubility and thermostability but also structural stability and flexibility/rigidity. These results demonstrate that electrostatics contributes to both heat and cold adaptation of subtilisin-like serine proteases through fine-tuning, either globally or locally, the structural stability and conformational flexibility/rigidity, thus providing a foundation for further engineering and mutagenesis studies.

Fig. 1. Cartoon representations of the crystal structures of the three serine proteases and their backbone superposition. (A) Psychrophilic VPR. (B) Mesophilic PRK. (C) Thermophilic AQN. (D) Backbone superposition of the three structures. In (A), (B), and (C), α-helices, β-strands, loops, substrate-binding segments (residues 100–104 and 132–136) are colored red, yellow, green, and purple, respectively; catalytic triads (D37-H70-S220, D39-H69-S224, and D39-H70-S222 in VPR, PRK, and AQN, respectively) and oxyanion holes (N157, N161, and N157 in VPR, PRK, and AQN, respectively) are represented as stick models, with carbon, oxygen, and nitrogen atoms colored cyan, red, and blue, respectively; disulfide bridges (S–S) are shown as stick models in orange; calcium ions are represented as blue spheres and numbered Ca1, Ca2, or Ca3 according to their numbering order in the crystal structures. In (D), the backbones of VPR, PRK, and AQN, as well as their contained calcium ions, are colored red, cyan, and blue, respectively.

Fig. 2. Salt bridges with P-degree greater than 20% in the concatenated equilibrium MD trajectories of the three proteases at the respective habitat temperatures of the source organisms. (A) Psychrophilic VPR at 283 K. (B) Mesophilic PRK at 300 K. (C) Thermophilic AQN at 343 K. The representative 3D structures are shown as backbone traces (yellow). The salt-bridging residues are shown as stick models, with the negatively and positively charged residues colored red and blue, respectively. Salt bridges are shown as green dashes. The boxed regions (i.e., I–IV) are those where salt bridges are relatively enriched (for details, see text in Discussion section).

Fig. 3. Molecular surface representations showing the electrostatic surface potentials of the three proteases. (A), (B), and (C) are the front surfaces of VPR (at 283 K), PRK (at 300 K), and AQN (at 343 K), respectively; (D), (E), and (F) are their respective back surfaces. Front surface is the surface containing the catalytic active center/catalytic triad; back surface is opposite to the front surface. The positively and negatively charged surfaces are colored blue and red, respectively, and the electro-neutral (or nonpolar/hydrophobic) surface is colored white. The catalytic triad residues and approximate locations of the substrate-binding sites/pockets, i.e., S2′, S1, S2, S3, and S4a and S4b (which are sub-sites of S4), are labeled on the front surface.