Engineering pH-dependent protein binding using combinatorial libraries

While protein engineering efforts typically aim to enhance a protein's binding affinity or increase its thermal stability, a relatively unexplored aim involves engineering new function, such as the regulation of protein binding. The origins of biological regulation frequently involve linked-binding events. Proton-linkage is one example, where the protonation of an ionizable group is linked to macromolecular binding, which produces a pH-dependent binding event. Here, we explore two novel, combinatorial library methods to introduce pH-dependent binding into a protein-protein complex, using a model antibody/antigen system.;The pH sensitivity of a protein binding event is related to the number of ionizable groups participating in linked-protonation. Using a unique combinatorial histidine scanning and selection approach, we were able to incorporate multiple histidine residues throughout an anti-RNase A VHH antibody interface, which resulted in several VHH variants displaying highly pH sensitive binding. In general, the anti-RNase A VHH interface was highly tolerant of histidine incorporation, with half of the VHH interface residues capable of incorporating a histidine residue. Three interface positions were identified as "hot spots" as they incorporated histidine in ~50% of the antibody variants sequenced. In addition, approximately one-third of the variants showed histidine residues "paired" in either adjacent sequence or space. Two anti-RNase A VHH variants were analyzed to better understand the structural and biophysical origins of the pH sensitivity. One of the variants possesses all three hot-spot histidines, while the other incorporated five, the max number of histidines. Both variants showed significant pH-dependence, with two to three contributing histidine residues, while maintaining near wild-type binding affinity.;A variant possessing histidine residues at the three "hot spot" positions was examined to better understand the thermodynamic basics of pH-sensitivity. The variant's binding thermodynamics were dissected through individual single and double histidine mutants. Individual protonation constants (pKa values) were determined using both ITC and NMR. The pKa values for the single and double mutants suggested non-independent protonation constants for the three histidine hot spot variant. A crystal structure of the hot spot anti-RNase VHH variant/RNase A complex suggested that the pH-dependent binding occurs without any major structural changes, when compared to the original wt-VHH/RNase A complex.;The shape of a pH-dependent binding profile is ultimately governed by the participating ionizable group(s). To test this idea, a related interface scanning approach was explored to develop a reverse pH-switch. A library was generated to sample aspartate, glutamate, and wild-type residues within the anti-RNase A VHH interface. This library displayed a high level of non-specific binding at low pH values. After exploring several strategies to minimize non-specific binding, selection was performed at physiological pH, where non-specific binding was low. Four unique variants were identified, which displayed near wild-type affinity and possessed aspartate/glutamate residues in up to 80% of the sampled CDR interface positions. Notably, despite using a higher pH for selection, a reverse pH-switch variant was identified.