Systematically Scrutinizing the Impact of Substitution Sites on Thermostability and Detergent Tolerance for Bacillus subtilis Lipase A

Abstract

Improving an enzyme's (thermo-)stability or tolerance against solvents and detergents is highly relevant in protein engineering and biotechnology. Recent developments have tended toward data-driven approaches, where available knowledge about the protein is used to identify substitution sites with high potential to yield protein variants with improved stability, and subsequently, substitutions are engineered by site-directed or site-saturation (SSM) mutagenesis. However, the development and validation of algorithms for data-driven approaches have been hampered by the lack of availability of large-scale data measured in a uniform way and being unbiased with respect to substitution types and locations. Here, we extend our knowledge on guidelines for protein engineering following a datadriven approach by scrutinizing the impact of substitution sites on thermostability or/and detergent tolerance for Bacillus subtilis lipase A (BsLipA) at very large scale. We systematically analyze a complete experimental SSM library of BsLipA containing all 3439 possible single variants, which was evaluated as to thermostability and tolerances against four detergents under respectively uniform conditions. Our results provide systematic and unbiased reference data at unprecedented scale for a biotechnologically important protein, identify consistently defined hot spot types for evaluating the performance of data-driven protein-engineering approaches, and show that the rigidity theory and ensemble-based approach Constraint Network Analysis yields hot spot predictions with an up to ninefold gain in precision over random classification.

Aqueous ionic liquids redistribute local enzyme stability via long-range perturbation pathways

“…For alkylimidazolium-based IL, simulation results revealed a complex, enthalpically driven binding mechanism [76] . Finally, the CNA approach to probe structural stability has been applied before retro- [77] , [78] , [79] , [80] , [54] and prospectively [67] , [81] in the context of improving protein thermostability on pairs [79] , [80] and series [77] , [78] , [81] of proteins. It was also benchmarked against the complete SSM library of Bs LipA [53] for systematically scrutinizing the impact of substitution sites on thermostability and detergent tolerance [54] .…”

“…Finally, the CNA approach to probe structural stability has been applied before retro- [77] , [78] , [79] , [80] , [54] and prospectively [67] , [81] in the context of improving protein thermostability on pairs [79] , [80] and series [77] , [78] , [81] of proteins. It was also benchmarked against the complete SSM library of Bs LipA [53] for systematically scrutinizing the impact of substitution sites on thermostability and detergent tolerance [54] . The breadth of these CNA applications is rooted in the rigorous analysis of structural rigidity [82] and the use of structural ensembles to improve robustness [83] .…”

“…For the identification, structural ensembles of the target enzyme in aIL generated via long MD simulations are screened towards aIL-induced PP; this screening can be supported by a prior analysis of changes in local structural stability. The number of substitution sites in PP can be further reduced by considering that predominantly solvent-exposed residue side chains interact with IL ions and that substitutions at buried positions are more likely to yield inactive variants [26] , [54] , [98] . Finally, by determining specific IL ion-residue interactions guided by Δ G 0 computations, beneficial substitutions can be suggested, e.g., to R/K for cation-based PP and to D/E for anion-based PP.…”

“…CNA was performed as described in ref. [54] : The Constraint Network Analysis (CNA) aims at linking structural rigidity and flexibility to the biomolecule’s structure, (thermo)stability, and function [66] , [70] , [122] . The CNA software acts as front- and back-end to the graph theory-based rigidity analysis software Floppy Inclusions and Rigid Substructure Topography (FIRST) [82] .…”

“…Bs LipA is a small lipase, and the fold does not contain a lid domain [46] . It has often been used as a model system in similar experimental and computational studies [26] , [36] , [39] , [41] , [44] , [47] , [48] , [49] , [50] , [51] , [52] , [53] , [54] , [55] . Furthermore, high-resolution X-ray structures (PDB-IDs 1I6W [46] & 1ISP [56] ) are available.…”