Tetramer formation by the caspase‐activated fragment of the Par‐4 tumor suppressor

Abstract

The prostate apoptosis response‐4 (Par‐4) tumor suppressor can selectively kill cancer cells via apoptosis while leaving healthy cells unharmed. Full length Par‐4 has been shown to be predominantly intrinsically disordered in vitro under neutral conditions. As part of the apoptotic process, cellular Par‐4 is cleaved at D131 by caspase‐3, which generates a 24 kDa C‐terminal activated fragment (cl‐Par‐4) that enters the nucleus and inhibits pro‐survival genes, thereby preventing cancer cell proliferation. Here, the structure of cl‐Par‐4 was investigated using CD spectroscopy, dynamic light scattering, intrinsic tyrosine fluorescence, and size exclusion chromatography with mutli‐angle light scattering. Biophysical characterization shows that cl‐Par‐4 aggregates and is disordered at low ionic strength. However, with increasing ionic strength, cl‐Par‐4 becomes progressively more helical and less aggregated, ultimately forming largely ordered tetramers at high NaCl concentration. These results, together with previous results showing induced folding at acidic pH, suggest that the in vivo structure and self‐association state of cl‐Par‐4 may be strongly dependent upon cellular environment.

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

“…Previously, we reported concentration-dependent effects of salts on the CD spectra of WT cl-Par-4. An intense spectrum characteristic of ~80% alpha-helix content (with two intensity minima at 208 and 222 nm wavelengths) was found in the presence of 500 mM or higher concentrations of either of the monovalent cations sodium or potassium ( Figure 2 A) [ 38 , 39 ]. The spectral intensity decreases with decreasing salt concentration, with an intermediate intensity at 250 mM salt.…”

“…Sequence analysis suggests disorder in 60–85% of cl-Par-4 depending upon the prediction tool used (see Figure 1 ), with the highest order propensity in the coiled coil region, which comprises less than 40% of cl-Par-4. However, CD spectra and scattering data under in vitro conditions of acidic pH or high salt show an approximately 80% helical and compact conformation of dimers or tetramers, respectively [ 37 , 38 , 39 ]. Structural models of the dimer and tetramer conformations are provided in Figure 6 .…”

“…We previously found that cl-Par-4 at pH 7.0 requires a high salt concentration to form compact helical tetramers [ 38 , 39 ]. For NMR analysis, high salt and tetramer formation are problematic in terms of the signal-to-noise ratio and line width.…”

“…However, the analysis shown in Figure 1 indicates that the cl-Par-4 fragment has higher order propensity than the PAF fragment. Most notably, the C-terminal 80 residues of cl-Par-4 are known to form a coiled coil (CC), at least under certain conditions [ 14 , 37 , 38 , 39 ]. Even in the CC, the order propensity is not strong.…”

“…Our previous studies have shown that cl-Par-4 achieves a compact, highly helical (approximately 80% helix), and stable conformation in the presence of either high salt or acidic pH [ 37 , 38 ]. We have also demonstrated that cl-Par-4 shows a similar conformation in the presence of divalent cations as it does in the presence of monovalent cations, but at a five times lower cation concentration [ 39 ].…”