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.