Enhancing protein robustness is crucial for fundamental research as well as for
technological applications. Our previous computational protein design effort
yielded an 11-point mutant haloalkane dehalogenase DhaA115 with outstanding
thermostability (Tm = 73.5 °C, DTm = 24 °C). Precise understanding of molecular
basis for this thermostability remained sparse. Here we report 1.55 Å and
1.6 Å resolution structures of DhaA115 obtained by X-ray crystallography.
We show that the placement of bulky aromatic amino acids on the protein surface
triggered novel long-distance backbone changes, establishing a new double-lock
system that: (i) closed access gates, (ii) reduced volumes of both main and
slot access tunnels, and (iii) made the active site occluded. Despite of these extensive
structural changes, experimental tracking of entry pathways by high-pressure
krypton derivatization of DhaA115 crystals revealed transport of small ligands through
enzyme’s tunnels. Experimental observations are in full agreement with the
results from computer simulations. Our findings unravel a novel structural
basis of enzyme thermostabilisation, which will pave the way for designing highly
thermostable biocatalysts and therapeutics.
This work was supported by MSCA Marie Sklodowska-Curie Actions (792772) and GAMU (MUNI/H/1561/2018).