Enhancing the Conformational Stability of the cl-Par-4 Tumor Suppressor via Site-Directed Mutagenesis

Abstract

Intrinsically disordered proteins play important roles in cell signaling, and dysregulation of these proteins is associated with several diseases. Prostate apoptosis response-4 (Par-4), an approximately 40 kilodalton proapoptotic tumor suppressor, is a predominantly intrinsically disordered protein whose downregulation has been observed in various cancers. The caspase-cleaved fragment of Par-4 (cl-Par-4) is active and plays a role in tumor suppression by inhibiting cell survival pathways. Here, we employed site-directed mutagenesis to create a cl-Par-4 point mutant (D313K). The expressed and purified D313K protein was characterized using biophysical techniques, and the results were compared to that of the wild-type (WT). We have previously demonstrated that WT cl-Par-4 attains a stable, compact, and helical conformation in the presence of a high level of salt at physiological pH. Here, we show that the D313K protein attains a similar conformation as the WT in the presence of salt, but at an approximately two times lower salt concentration. This establishes that the substitution of a basic residue for an acidic residue at position 313 alleviates inter-helical charge repulsion between dimer partners and helps to stabilize the structural conformation.

Evidence of direct interaction between cisplatin and the caspase‐cleaved prostate apoptosis response‐4 tumor suppressor

“…We previously reported that at pH 4 with low salt, cl‐Par‐4 is highly helical and stable against aggregation (Clark et al, 2018 ; Pandey et al, 2023 ). Therefore, in an attempt to avoid the scattering phenomenon seen at pH 7, we repeated the 10:1 C:P binding studies at pH 4 with low salt (50 mM NaCl).…”