Engineering and characterization of a pH-sensitive homodimeric antiparallel coiled coil

Structure-based engineering starting from the antiparallel coiled coil domain of the Bcr-Abl oncoprotein (BCR_30-65) afforded a new homodimeric antiparallel coiled coil whose folding is responsive to solution pH. The BCR_30-65 homodimer contains two glutamic acids (Glu52) buried in its hydrophobic core which one would expect to impart pH responsive folding behavior. Surprisingly, BCR_30-65 is resistant to thermal denaturation over a wide pH range. This behavior arises from stabilizing salt bridges formed between solvent-exposed Arg55 and Glu52 that passivates the glutamates’ negative charge within the protein’s core. Replacing Arg55 with alanine imparts pH-responsive folding behavior, but also results in the loss of directional specificity between the coiled coil’s helices. We find that an Ile42Glu mutation preserves the salt bridges and imparts the desired pH responsive folding behavior. The Ile42Glu protein forms an antiparallel homodimeric coiled coil under acidic conditions but unfolds under basic conditions and is only marginally less thermally stable compared to BCR_30-65.