The natively disordered loop of Bcl-2 undergoes phosphorylation-dependent conformational change and interacts with Pin1

Abstract

Bcl-2 plays a central role in the regulation of apoptosis. Structural studies of Bcl-2 revealed the presence of a flexible and natively disordered loop that bridges the Bcl-2 homology motifs, BH3 and BH4. This loop is phosphorylated on multiple sites in response to a variety of external stimuli, including the microtubule-targeting drugs, paclitaxel and colchicine. Currently, the underlying molecular mechanism of Bcl-2 phosphorylation and its biological significance remain elusive. In this study, we investigated the molecular characteristics of this anti-apoptotic protein. To this end, we generated synthetic peptides derived from the Bcl-2 loop, and multiple Bcl-2 loop truncation mutants that include the phosphorylation sites. Our results demonstrate that S87 in the flexible loop of Bcl-2 is the primary phosphorylation site for JNK and ERK2, suggesting some sequence or structural specificity for the phosphorylation by these kinases. Our NMR studies and molecular dynamics simulation studies support indicate that phosphorylation of S87 induces a conformational change in the peptide. Finally, we show that the phosphorylated peptides of the Bcl-2 loop can bind Pin1, further substantiating the phosphorylation-mediated conformation change of Bcl-2.

Figure 1. CD analysis of Bcl-2.

The purified Bcl-2 was concentrated to 1 mg/ml and exchanged to the phosphate buffer containing 20 Na-PO₄, pH 7.4, 100 mM NaCl, and subject to CD analysis of Bcl-2 (A) and pBcl-2 (B).

Figure 2. Conformational change of S87 after phosphorylation.

(A) Trajectories of radius of gyration (Rg) for peptide S87 in an unphosphorylated (black line) and a phosphorylated (red line) state. (B) Representative structure of the largest-member cluster of unphosphorylated S87 peptide from the final 5 ns simulation trajectory. (C) Representative structure of the largest-member cluster (84%) of phosphorylated S87 peptide from the final 5 ns simulation trajectory. (D) NMR structure ensemble of the 10 lowest energy structures for S87 peptide. (E) NMR structure ensemble of the 10 lowest energy structures for pS87 peptide. Serine and phosphoserine groups are highlighted with sticks.

Figure 3. Pin 1 binds to the phosphopeptide derived from the flexible loop of Bcl-2.

The ¹⁵N-labeled Pin1 (0.1 mM) was recorded with (A) 0.8 mM S87 and (B) phosphorylated S87 (pS87). The ¹H-¹⁵N-HSQC spectra with or without peptide are show in blue and red, respectively. (C, D) Some of the amino acid chemical shifts change with the increasing peptide concentration. The arrows show the direction of chemical shift perturbations with an increasing concentration of ligand. (E, F) The relationship between chemical shift change and the ratio between ligand and protein was drawn. Values of K_D were calculated from the graphs using the equation described in “Materials and methods”.

Figure 4. Model building and MD simulation result of Pin1-pS87 peptide.

(A) Crystal structure of the Pin1 WW domain with C-terminal domain (CTD) of RNA polymerase II phosphopeptide. (B) Initial model of Pin1 WW domain with pS87 Bcl-2 phosphopeptide. Both the tryptophan residues and the phosphoserine-interacting arginine residue are highlighted as sticks, and labeled accordingly. Both phosphopeptides are highlighted with yellow (CTD peptide) and blue (pS87 Bcl-2 peptide), and the pSer-Pro motif in both the phosphopeptides is represented as sticks. (C) Initial and representative frames of MD simulation. Protein is represented with gray and cyan; pS87 peptide is highlighted with purple and pink for the initial and final frames, respectively. (D) Hydrogen bond interaction patterns of the phosphoserine with the WW domain residues. All three residues (2 serines and 1 arginine) are highlighted with sticks and labeled. Hydrogen bonds between the phosphoserine and the WW domain residues are represented with dashed lines.

Figure 5. Pin1 WW domain-pS87 MD simulation analyses.

(A) Radius of gyration for pS87 Bcl-2 phosphopeptide in the WW domain-pS87 simulation. (B) Hydrogen bond interaction analysis between pS87 and the WW domain residues. (C) Short range columbic interaction analysis between the WW domain and the pS87, as well as the WW domain and the pSer alone. (D) Electrostatic potential calculations of the WW domain and the pS87 phosphopeptide complex. The arginine residue, which forms a charged/hydrogen bond interaction with pSer, is highlighted as sticks, and the other two arginine residues are represented with lines. The hydrophobic pocket residues (Y23 and W34) are also shown in stick representation. The pSer-Pro motif is also highlighted.