Computational design of a pH-sensitive IgG binding protein

Significance Protein–protein interactions are part of almost every biological process; hence, the ability to manipulate and design protein binding has widespread applications. Here, we describe an approach to designing pH-dependent protein interfaces and use it to design a protein that binds antibodies at high pH but poorly at low pH. The designed protein should be useful for antibody affinity purification and diagnostic devices. Computational design provides the opportunity to program protein–protein interactions for desired applications. We used de novo protein interface design to generate a pH-dependent Fc domain binding protein that buries immunoglobulin G (IgG) His-433. Using next-generation sequencing of naïve and selected pools of a library of design variants, we generated a molecular footprint of the designed binding surface, confirming the binding mode and guiding further optimization of the balance between affinity and pH sensitivity. In biolayer interferometry experiments, the optimized design binds IgG with a Kd of ∼4 nM at pH 8.2, and approximately 500-fold more weakly at pH 5.5. The protein is extremely stable, heat-resistant and highly expressed in bacteria, and allows pH-based control of binding for IgG affinity purification and diagnostic devices.