Histidine-Mediated Intramolecular Electrostatic Repulsion for Controlling pH-Dependent Protein–Protein Interaction

Abstract

Protein−protein interactions that can be controlled by environmental triggers have immense potential in various biological and industrial applications. In the current study, we aimed to engineer a pH-dependent protein−protein interaction that employs intramolecular electrostatic repulsion through a structure-guided histidine substitution approach. We implemented this strategy on Streptococcal protein G, an affinity ligand for immunoglobulin G, and showed that even a single point mutation effectively improved the pH sensitivity of the binding interactions without adversely affecting its structural stability or its innate binding function. Depending on the pH of the environment, the protein−protein interaction was disrupted by the electrostatic repulsion between the substituted histidine and its neighboring positively charged residues. Structurally, the substituted histidine residue was located adjacent to a lysine residue that could form hydrogen bonds with immunoglobulin G. Thermodynamically, the introduced electrostatic repulsion was reflected in the significant loss of the exothermic heat of the binding under acidic conditions, whereas accompanying enthalpy−entropy compensation partly suppressed the improvement of the pH sensitivity. Thus, the engineered pH-sensitive protein G could enable antibody purification under mildly acidic conditions. This intramolecular design can be combined with conventional protein−protein interface design. Moreover, the method proposed here provides us with additional design criteria for optimization of pH-dependent molecular interactions.

Neural networks to learn protein sequence-function relationships from deep mutational scanning data

“…We were unable to precisely determine wild-type GB1’s dissociation constant because our assay could not reliably measure binding at IgG concentrations above 100 nM. Our measurements indicate that wild-type GB1’s K d is larger than 100 nM, which is consistent with measurements from the literature that have found this interaction is in the 250-900 nM range [21, 22]. Based on our estimates and other’s previous measurements, we conservatively estimate that Design10 binds human IgG with at least tenfold higher affinity than wild-type GB1.…”

VEGF and VEGFR2 bind to similar pH-sensitive sites on fibronectin, exposed by heparin-mediated conformational changes

“…Based on the known structures of VEGF and ECD, we calculated the pKa values of surface-exposed amino acids (35), and we identified several residues with a pKa value between 5.5 and 7.5, whose protonation state would change between these two pH values (Table 1) and thus, could belong to the fibronectin-binding sites on each molecule. In the case of ECD, most of the candidate residues were His residues, which often function as pH sensors (36)(37)(38). Whereas for ECD almost all candidate residues seem to be free of interactions with the ligand, the majority of the pH-sensitive residues on VEGF were on the receptor-binding domain and were involved in interactions in the VEGF dimer or between VEGF and the receptor, leaving free of interactions only a His residue at the C-terminal domain of VEGF.…”

Discovery and mechanism of a pH-dependent dual-binding-site switch in the interaction of a pair of protein modules

“…3, C and D). Glu 53 and Gln 23 of CaDoc0917 are not at symmetric positions in the sequence (Fig. 1D), but they form similar hydrogen bonds with Asp 130 of CaCohA2 in the low-pH structures of the two orientations.…”

“…The affinity ratio at low and high pH for the interaction between CaCohA2 and CaDoc0917 (R16DE53Q) becomes 26, similar to the ratio (31) for CaCohA2 and CaDoc0917(R49D). Therefore, the Glu 53 and Gln 23 residues at asymmetric positions of CaDoc0917 are responsible for the difference in affinity ratio for its two binding orientations.…”