The interplay between effector binding and allostery in an engineered protein switch

Abstract

The protein design rules for engineering allosteric regulation are not well understood. A fundamental understanding of the determinants of ligand binding in an allosteric context could facilitate the design and construction of versatile protein switches and biosensors. Here, we conducted extensive in vitro and in vivo characterization of the effects of 285 unique point mutations at 15 residues in the maltose-binding pocket of the maltose-activated b-lactamase MBP317-347. MBP317-347 is an allosteric enzyme formed by the insertion of TEM-1 b-lactamase into the E. coli maltose binding protein (MBP). We find that the maltose-dependent resistance to ampicillin conferred to the cells by the MBP317-347 switch gene (the switch phenotype) is very robust to mutations, with most mutations slightly improving the switch phenotype. We identified 15 mutations that improved switch performance from twofold to 22-fold, primarily by decreasing the catalytic activity in the absence of maltose, perhaps by disrupting interactions that cause a small fraction of MBP in solution to exist in a partially closed state in the absence of maltose. Other notable mutations include K15D and K15H that increased maltose affinity 30-fold and Y155K and Y155R that compromised switching by diminishing the ability of maltose to increase catalytic activity. The data also provided insights into normal MBP physiology, as select mutations at D14, W62, and F156 retained high maltose affinity but abolished the switch's ability to substitute for MBP in the transport of maltose into the cell. The results reveal the complex relationship between ligand binding and allostery in this engineered switch.

Full and Partial Agonism of a Designed Enzyme Switch

“…However, engineering these switches is still far from rational; it entails extensive trial-and-error and/or directed evolution, particularly with regards to the linkers used for tethering together the functional modules 12 . Individual point mutants can also tune the behavior of such protein switches, through effects on either the input domain (effector binding) and/or the output domain (catalysis) 13 . While combinatorial libraries of randomized gene insertions and their point mutants can be tested to find combinations with the desired effect, it will nonetheless be advantageous to instead design these types of switches in a more rational way: this will enable the switch’s output(s) to not only be predicted, but also tuned for use in different environments and timescales.…”

Insertion of a Calcium-Responsive β-Roll Domain into a Thermostable Alcohol Dehydrogenase Enables Tunable Control over Cofactor Selectivity

“…In these systems, protein structures change in response to input signals (ligands, pH, etc. ), leading to outputs such as altered ligand affinities and enzymatic activities. − Examples of protein switches include a barnase/ubiquitin system and a maltose binding protein/β-lactamase system where the catalytic activity is modulated by maltose. ,− These switches can be used in applications including biosensors, therapeutic agents, and smart biomaterials.…”

Converting a Periplasmic Binding Protein into a Synthetic Biosensing Switch through Domain Insertion

“…In order to explore the structural basis of local amino acid residues important to the allosterism generated by domain insertion, Choi and coworkers [61] used the engineered protein MBP317-347 for the construction of 285 mutants. MBP317-347 binding pocket was the target for all possible amino acid replacements at 15 positions, which were selected due to their known involvement with maltose binding.…”