Allosteric control of ligand-binding affinity using engineered conformation-specific effector proteins

Abstract

We describe a phage display methodology for engineering synthetic antigen binders (sABs) that recognize either the apo or the ligand-bound conformation of maltose-binding protein (MBP). sABs that preferentially recognize the maltose-bound form of MBP act as positive allosteric effectors by substantially increasing the affinity for maltose. A crystal structure of a sAB bound to the closed form of MBP reveals the basis for this allosteric effect. We show that sABs that recognize the bound form of MBP can rescue the function of a binding-deficient mutant by restoring its natural affinity for maltose. Furthermore, the sABs can enhance maltose binding in vivo, as they provide a growth advantage to bacteria under low-maltose conditions. The results demonstrate that structure-specific sABs can be engineered to dynamically control ligand-binding affinities by modulating the transition between different conformations.

Figure 1. Phage display selection strategy

Maltose binding protein (MBP) undergoes a conformational change through a hinge-bending motion upon binding to maltose (red). Carrying out the phage display selection in the absence of maltose generates sABs (yellow sphere) that bind preferentially to the open form of MBP, whereas selection in the presence of maltose results in closed-specific sABs (green sphere). Note: placement of spheres indicate postulated binding modes of sABs to the different forms of MBP.

Figure 2. Influence of sABs on maltose binding

(A) The change in fluorescence of the MBP-233C Alexa 488 conjugate (solid line) upon addition of 1 mM maltose (dashed line). (B) Fluorescence maltose binding curves of MBP-233C Alexa 488 in the absence (●) or presence of 200 nM sAB MCS1 (◇) or 200 nM sAB MCS4 (○). (C) Intrinsic tryptophan fluorescence of MBP in the absence (solid line) or presence of 1 mM maltose (dashed line). (D) Fluorescence maltose binding curves of MBP the absence (●) or presence of 200 nM sAB MOS1 (○).

Figure 3. Scatchard analysis of maltose binding

Data points from figure 2 were re-plotted as r vs. r/[Maltose], where r is the fractional saturation of MBP with maltose. (A) Binding of maltose to MBP in the absence of sABs showing no co-operativity. (B) Maltose binding in the presence of 200 nM sAB MOS1 showing negative co-operativity. (C) Maltose binding in the presence of 200 nM sAB MCS1 or (D) 200 nM sAB MCS4 showing positive co-operativity.

Figure 4. Crystal structure of MBP-MCS2 complex

MBP (blue) is in the closed form, bound to 1 molecule of maltose (red). The sAB (Heavy chain: green, Light chain: yellow) interacts with MBP at the opposite side of the binding pocket forming a wedge that favors the closed form.

Figure 5. The “wedge” formed by the CDR loops of MCS2

(A) A group of bulky side-chain residues within CDRH-3 of the sAB (green sticks) form the wedge structure, which interacts with a region within MBP (blue) that is only exposed in the closed, maltose-bound conformation. (B) Sequence of the CDR loops of MCS2. Bold letters indicate residues that interact with the MBP molecule. (C) An overlay of the MCS2-MBP complex with the open form of MBP (purple, PDB code: 1OMP 12) indicates that the apo form of MBP clashes with the sAB CDR loops.

Figure 6. Rescuing binding function of an MBP mutant

The effect of the binding pocket mutation W62F on affinity of MBP for maltose was assessed using the emission change of Alexa 488 attached to a cysteine at position 233. In the absence of sAB (●), the mutant exhibits low binding affinity. The affinity is restored by addition of either 200 nM sAB MCS4 (○) or 200 nM sAB MCS1 (◇). The binding curve of MBP-233C with no binding pocket mutations is shown (dashed line) as a reference.

Figure 7. Alloesteric activity of sABs in vivo

E. coli cells expressing sABs in the periplasm were grown in minimal media containing maltose as the sole carbon source. Cells expressing sAB MCS1 (○) or sAB MCS4 (◇) show no change in the growth rate at low maltose concentrations. Control cells expressing sAB-27 (■), which is an actin binding sAB (from reference14), show a lower growth rate at low maltose concentrations.