Figure 6
The interactions within or between molecules are the main structural factors for stabilizing the overall or local structures, especially hydrogen bonds and hydrophobic interactions. Pace et al. reported that hydrophobic interactions and hydrogen bonds contribute approximately 60 ± 4% and 40 ± 4% to protein stability, respectively.[36] As shown in Figure 7, the interactions of mutation sites with surrounding amino acids were displayed. When Thr200 was substituted with Lys200, additional H-bonds (between site 200 and Ser160) and hydrophobic interaction (between site 200 and Met154) were increased. When Pro260 was mutated to Ser, a H-bonds was added between Ser260 and Leu263, and the distance of hydrophobic interaction between site 260 and Leu263 decreased from 5.495 Å to 5.221 Å. As for site 23, there was no increase or decrease of H-bond and hydrophobic interaction. The substrate binding pocket locates on the dimer interface of At ATA, and the substitution of local amino acids will influence the interaction between or within monomers so that the overall structure conformation may change. Via Discovery Studio 2019 Client, the number of H-bond, electrostatic, hydrophobic, and salt bridge between or within molecules of WT or M3 was counted (Table 4). The total number of interactions between or within monomers was increased from WT to M3. This might strengthen the rigidity of mutant structure for resisting the organic solvents. For dimer or multimer proteins, the subunit interface is crucial for stability, and the protein structure can be optimized by stabilizing the subunit interface. As shown in Figure 8, the hydrophobic interactions and H-bonds on the dimer interface of WT and M3 were further analyzed using the software Ligplus, and the H-bonds and hydrophobic interactions were increased from WT to M3, which could improve the rigidity of the interface, so that promote a more compact joint between two monomers. In general, the enhanced rigidity may lead to a slight decrease in catalytic efficiency.[37] In this study, the catalytic efficiency of M3 was not decreased, which might be these three sites were far from the active site K180, and they were not cause significant changes to the substrate binding pocket.