Joel Chia's research while affiliated with Nanyang Technological University and other places

FKBP38 regulates apoptosis through unique interactions with multiple regulators including Bcl-2. Interestingly, the peptidylprolyl isomerase activity of FKBP38 is only detectable when it binds to calcium-saturated calmodulin (CaM/Ca(2+)). This, in turn, permits the formation of a complex with Bcl-2. FKBP38 thereby provides an important link between isomerase activity and apoptotic pathways. Here, we show that the N-terminal extension (residues 1-32) preceding the catalytic domain of FKBP38 has an autoinhibitory activity. The core isomerase activity of FKBP38 is inhibited by transient interactions involving the flexible N-terminal extension that precedes the catalytic domain. Notably, CaM/Ca(2+) binds to this N-terminal extension and thereby releases the autoinhibitory contacts between the N-terminal extension and the catalytic domain, thus potentiating the isomerase activity of FKBP38. Our data demonstrate how CaM/Ca(2+) modulates the catalytic activity of FKBP38.

The amide region of the peptides. 1D proton NMR was collected as described in the “Materials and methods”. The superimposed spectra in the amide proton region are shown. Peptide and phosphorylated peptide are shown as blue and red, respectively. The residue numbers are shown on the spectra. (TIF)

Phosphorylation of Bcl-2 in the loop domain by ERK2 and JNK. (A–B) Different peptides (200 µM) were added into the reaction mixture with either (A) JNK (10 µM) or (B) ERK2 (10 µM). After reaction at 30°C for 1 h, the samples were heated at 90°C to inactivate the kinase and loaded onto C-18 column for reverse phase-HPLC analysis. (C) Point mutations of Bcl-2 (T56A and S87A) were used for the kinase reaction as described in “Materials and methods”, and the phosphorylation reactions were analyzed by using γ-32P-ATP and followed by autoradiography. (D) Western blotting shows the expression levels of Bcl-2 and its point mutants used in the kinase reactions. (E) The wild-type Bcl-2 and the flexible loop-deletion Bcl-2 mutant Bcl-2, Δ(V35–V89):6A were subject to the phosphorylation reactions by ERK2 and JNK. 1, Bcl-2 with ERK2; 2, Bcl-2Δ(V35–V89):6A with ERK2; 3, Bcl-2 with JNK; 4, Bcl-2Δ(V35–V89):6A with JNK. (TIF)

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.

Clustering analyses of MD simulation data based on RMSD of the pS87 Bcl-2 phosphopeptide. Highest number of conformations is populated in Cluster-4. (TIF)

Model for the interaction between Pin1 and pS87 (A) S87 peptide. (B) The pS87 peptide, which contains the pSer-Pro motif, undergoes conformational changes after phosphorylation. (C) Structural model of the Pin 1 WW domain complexed with pS87 was generated using GOLD v3.1.1 (Cambridge Crystallographic Data Centre, UK). (D) The structure of the Pin1 WW domain complexed with Tau peptide (PDB 1I8G). The pSer-Pro motif is shown in red. The model shows that phosphorylation of the Bcl-2 S87 peptide undergoes a conformational change and the resulting structure resembles the complex structure of the WW domain and the Tau peptide. (TIF)

CSI analysis of S87 and pS87. Chemical shift index analysis for the peptides. The Hα chemical shifts were compared with that of the random coil of each residue. (TIF)

Resonance assignment of peptides S87 and pS87. (DOC)

Structures of the peptides from Bcl-2. Ensembles of 10 lowest energy NMR structures for T74 (A), T56 (B), p74 (C) and pT56 peptides (D) are shown, respectively. Residues Thr is highlighted with sticks. The 15N-labeled Pin1 was recorded in the presence of regular/phosphorylated T74 (E) and T56 (F). 2D 1H-15N HSQC spectra with or without peptide are shown in red and black, respectively. (TIF)

The immunosuppressive drug FK506 binds its targets FK506-binding protein (FKBP) family and modulates cellular processes. Recent studies demonstrated that FK506 shows anti-malaria effects. Newly identified FK506-binding protein 35 from Plasmodium falciparum (PfFKBP35) is assumed to be the molecular target of FK506 in the parasite. Currently, molecular and structural basis of growth inhibition of the parasite by FK506 remains unclear. In this study, to examine characteristics of PfFKBP35 and also understand its molecular mechanism of the inhibition by FK506, we have cloned, expressed, and purified the full-length PfFKBP35 and its FK506-binding domain (FKBD). We demonstrate that the full-length PfFKBP35 and the FKBD were properly folded, and suitable for biochemical and biophysical studies. PfFKBP35 showed a basal activity in inhibiting the phosphatase activity of calcineurin in the absence of FK506, but the presence of FK506 greatly enhanced its calcineurin-inhibitory activity. Our NMR data indicate that the FKBD binds FK506 with a high affinity.