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 β-lactamase MBP317-347. MBP317-347 is an allosteric enzyme formed by the insertion of TEM-1 β-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.

Keywords: allostery; ligand binding; maltose binding protein; maltose transport; protein engineering; protein switch.

Figure 1

MBP317‐347 fusion protein as a platform for the study of ligand binding and switching activity. (A) Schematic diagram of the MBP317‐347 fusion protein and its activation mechanism upon maltose binding. The MBP domain is shown in red and BLA domain is shown in blue. (B) A structural model was constructed by MODELLER software24 based on the crystal structures of RG13 (PDB ID: 4DXC25), MBP (PDB ID: 1OMP26), and BLA (PDB ID: 1M4027). In a graphical illustration of the MBP317‐347 sequence, MBP domain sequences are in red, BLA domain sequences are in blue, and the linker sequence is in gray. (C) A cross‐section view of maltose bound in the binding pocket of the MBP domain. The 15 residues selected for mutation are labeled.

Figure 2

MalE complementation assay for estimating maltose affinity. (A) Schematic diagrams of in vivo of the malE complementation assay. (B) A heat map representation of minimum growth concentration of maltose (MGC_Mal) for the malE knockout E. coli strain PM9F' as a function of the mutation in the MBP domain. (C) Average MGC_Mal for types of residues as categorized in (B) for each target residue. All MGC_Mal values can be found in Supplementary Data S1.

Figure 3

In vivo switching activity conferred by the MBP317‐347 gene. (A) A heat map representation of the ratio of the MICs of ampicillin (+maltose/‐maltose) (MIC_Amp) for each member of the MBP317‐347 focused library. (B) MIC_Amp for cells plated in the absence of maltose. (C) MIC_Amp for cells plated in the presence of 256 mM maltose. For (A)–(C), white indicates the value conferred by MBP317‐347 lacking any mutation. (D) Average MIC_Amp ratio for types of residues as categorized in [Fig. 2(B)]. All MICs and their corresponding ratios can be found in Supplementary Data S1.

Figure 4

Western dot blot analysis of MBP317‐347 abundance in cells cultured in the (A) presence of 256 mM maltose and (B) absence of maltose as a function of the mutation in the MBP domain. (C) Average abundance of MBP317‐347 in the presence of maltose for types of residues as categorized in [Fig. 2(B)]. Expression data can be found in Supplementary Data S1.

Figure 5

Kinetic properties of switches. (A) Relationship between the switch ratio and the apparent K _d for maltose. The switch ratio is the ratio of initial rates for hydrolysis of 100 µM nitrocefin in the presence of maltose to that in the absence of maltose. The solid circle labeled WT indicates the values of MBP317‐347. Select mutants are labeled. (B) Mutations that improve the switch ratio by greater than twofold. (C) The rates of hydrolysis with and without maltose for the 15 switches with an improved switch ratio. (D) Positively charged residues introduced at position Y155 decrease switching by severely diminishing the catalytic rate in the presence of maltose. K _d's and the switching ratios can be found in Supporting Information Table S2 and Supporting Information Data S1.

Figure 6

Sites of mutations with interesting properties mapped onto the structure of MBP. (A) Sites of mutations (red) that improve switching in vitro by reducing the activity in the absence of maltose (white) (PDB ID: 1ANF34). (B) Sites of mutations (red) that disrupt transport in cells yet have minimal effects on maltose affinity. Shown is a detail of the pre‐translocation complex of MBP and membrane proteins MalG and MalF (PDB ID: 3PV035). Residue Q256 of MalG forms a hydrogen bond with maltose and is key for maltose transport. Maltose is shown in white.