The study of pH-dependent stability shows that the TPLH-mediated hydrogen-bonding network is important for the conformation and stability of human gankyrin.

Ankyrin repeat (AR) proteins possess a distinctive modular and repetitive architecture, and their global folds are maintained by short-range interactions in terms of the primary sequence. In this work, we extended our previous study on the highly conserved TPLH tetrapeptide and investigated the impact of a solvent-exposed histidine residue on the pH-dependent stability of gankyrin, providing further insight into the contribution of the TPLH motif to the tertiary fold of AR proteins. Consisting of seven ARs, gankyrin has five histidine residues in TPLH motifs or its variants, all of which adopt a H(ε2)-tautermeric form and are shielded from solvent. By truncating the C-terminal ankyrin repeat (AR7), we exposed H177 in the (174)TPLH(177) of AR6 (the second C-terminal AR) to an aqueous environment. We showed that this truncated gankyrin mutant, namely, Gank(1-201), was well-folded at a neutral pH with a slightly lower stability with respect to gankyrin wild type (WT). However, unlike gankyrin WT, the stability of Gank(1-201) was markedly decreased together with a loss of conformation at a pH slightly below 6.0. It was rationalized that the protonation of the H177 imidazole ring triggered the disruption of the TPLH-mediated hydrogen-bonding network, which in turn led to the loss of conformation and stability. These results together with the work on Q210H mutant nicely explain that the C-terminal AR7 has a (207)TPLQ(210) variant and are in support of the notion that a string of TPLH/variant, which may arguably act like a zip lock to the elongated AR proteins via intra-/inter-repeat hydrogen-bonding, is important in maintaining the tertiary fold. Additionally, we made rational mutagenesis to introduce extra surface charge on AR7 of gankyrin and demonstrated that G214E and I219D mutations increased the stability of gankyrin while the function remained intact. Taken together, our results indicate that the TPLH-mediated hydrogen-bonding network is important for the conformation and stability of human gankyrin, and the C-terminal AR contributes to the conformational stability of gankyrin (AR proteins in general) through shielding this TPLH network from solvent as well as making the surface area more accessible to solvent.