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Enhancing the Conformational Stability of the cl-Par-4 Tumor Suppressor via Site-Directed Mutagenesis
Samjhana Pandey1, Krishna K Raut2, Andrea M Clark2
1Biomedical Sciences Program, Old Dominion University, Norfolk, VA 23529, USA.
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.
Insights
Prostate apoptosis response-4 (Par-4) is a tumor suppressor. A D313K mutant of cleaved Par-4 (cl-Par-4) stabilizes its structure at lower salt concentrations, aiding cancer research.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Intrinsically disordered proteins (IDPs) are crucial in cell signaling, with dysregulation linked to diseases.
- Prostate apoptosis response-4 (Par-4) is a predominantly IDP and a proapoptotic tumor suppressor, often downregulated in cancers.
- The active caspase-cleaved fragment of Par-4 (cl-Par-4) exhibits tumor suppressor activity by inhibiting cell survival pathways.
Purpose of the Study:
- To investigate the structural conformational changes of a site-directed mutant (D313K) of cl-Par-4.
- To compare the biophysical properties of the D313K mutant with wild-type (WT) cl-Par-4.
Main Methods:
- Site-directed mutagenesis was used to generate the cl-Par-4 D313K point mutant.
- The expressed and purified D313K protein was characterized using biophysical techniques.
- Structural conformation was analyzed in the presence of varying salt concentrations at physiological pH.
Main Results:
- The D313K mutant attained a stable, compact, helical conformation similar to WT cl-Par-4.
- This stabilization occurred at approximately half the salt concentration required for WT cl-Par-4.
- The substitution of an acidic residue with a basic residue at position 313 reduces inter-helical charge repulsion, enhancing structural stability.
Conclusions:
- The D313K mutation enhances the structural stability of cl-Par-4.
- This finding provides insights into the structural dynamics of intrinsically disordered proteins and their role in cancer.
- Understanding these structural properties can inform therapeutic strategies targeting cancer-associated protein dysregulation.
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