Investigating the Aggregation of the Basic Leucine Zipper (bZIP) Domain of Activating Transcription Factor 5 (ATF5)

Abstract

Protein aggregation is a major problem for biopharmaceuticals. Aggregate formation in a drug formulation can have serious health implications for the patient. While the control of protein aggregation is critically important for the future of protein pharmaceuticals, the mechanism is still poorly understood. In particular, the role that protein structure plays in aggregate assembly is not well understood. We have selected the basic leucine zipper (bZIP) domain of activating transcription factor 5 (ATF5) as model system with which to investigate the relationship between protein structure and aggregate assembly. This domain contains three regions with differing structural propensity: a disordered N-terminal polybasic region possessing transient helicity; a central, helical leucine zipper region; and a C-terminal, extended valine zipper region with a propensity to form beta structure. Additionally, a centrally positioned cysteine residue readily forms an intermolecular disulfide bond. We have modulated solution conditions and engineered mutations that affect the structure and aggregation of this domain in order to characterize how different structural elements participate in aggregate formation. Specifically, we have evaluated the impact of intermolecular disulfide bond formation on ATF5 structure and stability. We have also investigated how removal of the C-terminal valine zipper region affects ATF5 structure and aggregation. Our results indicate that intermolecular disulfide bond formation facilitates the retention of helical structure and reduces the growth of thermally induced protein aggregates. Additionally, the C-terminal valine zipper region is critical for the formation of α-helical structure. Removal of this region results in a change in protein structure and a change in the mechanism of protein self-association. The structure and stability of this truncated mutant are largely unaffected by intermolecular disulfide bond formation. When compared to the wild-type ATF5 protein, this mutant displays increased self-association at low temperature but improved resistance to aggregate growth upon temperature elevation.