Technical Reference #1609

1609.

Structure-function correlation on human programmed cell dealth 5 protein Hongwei Yao; Lanjun Xu; Yingang Feng; Dongsheng Liu; Yingyu Chen; Jinfeng Wang, Chinese Academy of Sciences, Archives of Biochemistry and Biophysics, 486(1609), (2009)

Abstract:
Human programmed cell death 5 (PDCD5) is a translocatory protein playing an important role in theapoptotic process of cells. Although there are accumulated data about PDCD5 function the correlationof the structure with the function of PDCD5 has not b

Keywords:
PDCD5; NMR; Structure-function relationship; Cell translocation; fragments of PDCD5

Materials & Methods:
Preparation of deletion mutants of PDCD5 PDCD5 fragments namely PDCD5(1–112) PDCD5(1–104) PDC D5(34–125) PDCD5(34–112) and PDCD5(34–104) and PDCD 5(20–104) containing residues 1–112 1–104 34–125 34–112 34– 104 and 20–104 respectively were used in this study. The fusion expression system used for expression of the intact human PDCD5 was adopted for expression of deletion mutants of PDCD5 except PDCD5(20–104) [13]. The target genes were amplified from the plasmid pET-3d-HR52-PDCD5 and cloned into the pET-3d-HR52 vector with the KpnI and BamHI restriction sites to generate expression plasmids. In each of these expression plasmids there is a thrombin cleavage site (LVPR;GS) between the fusion partner HR52 a His6-tagged N-terminal 52-residue fragment of staphylococcal nuclease R and the target PDCD5 fragment for removing the fusion partner. For the expression ofPDCD5(20–104) the target gene was amplified from the plasmid pET-3d-HR52-PDCD5 and cloned into the NdeI and EcoRI restriction sites of the vector pGBO [14] to generate expression plasmid pGBO-PDCD5(20–104). The expression and purification of PDCD5(20–104) were carried out using similar method as described in the previous study for isolation of PDCD5 protein [11]. As a result of thrombin cleavage residues Gly-Ser replaced the N-terminal residue M1 in the sequences of PDCD5(1–112) and PDCD5(1–104) and were appended to the N-terminal residue H34 of PDCD5(34–125) PDCD5(34–112) and PDCD5(34–104). Also four residues Gly-Ser-His-Met are appended to the N-terminal residue K20 of PDCD5(20–104) as a result of the thrombin cleavage. Uniformly 15N and/or 13C-labeled PDCD5 fragments for NMR studies were obtained by the growth in M9 minimal media containing 15NH4Cl and/or [13C]-glucose as the sole nitrogen and/or carbon sources respectively. The purity of proteins was checked by SDS–PAGE to ensure a single band. Preparation of FITC–PDCD5 complex Fluorescein isothiocyanate (FITC) conjugated recombinant PDCD5 fragment that is FITC–PDCD5 fragment complex was prepared for fluorescence microscopic studies. FITC (Merk) labeling of recombinant PDCD5 fragment was prepared as described previously [15]. Briefly different recombinant PDCD5 fragments (2 mg/ ml) were mixed with DMSO lysed FITC (0.1 mg) and incubated for 16 h at 4 C. Then the FITC–PDCD5 fragment complex was further purified using a Sephadex G-50 gel filtration column. The purified FITC–PDCD5 fragment complex was stored in PBS buffer containing 0.1% NaN3 and 0.1% BSA and ready for use. NMR spectroscopy Samples of uniformly 13C- 15N- or 13C/15N-labelled PDCD5(1– 112) for determination of 3D solution structure by NMR method were prepared as follows: 1.0–2.0 mM 15N- or 13C/15N-labelled PDCD5(1–112) in 90% H2O/10% D2O containing 100 mM deuterated acetate buffer (pH 4.7) and 100 mM NaCl; 1.0–2.0 mM 13C-labelled PDCD5(1–112) in 99.996% D2O containing 100 mM deuterated acetate buffer (pH 4.7) and 100 mM NaCl. The sample for backbone dynamic studies was 1.0 mM 15N-labelled PDCD5(20–104) in 90% H2O/10% D2O containing 100 mM deuterated acetate buffer (pH 4.7) and 100 mM NaCl. The sample for hydrogen exchange experiments was 1.0 mM 15N-labelled PDCD5 protein dissolved in 90% H2O/10% D2O containing 50 mM phosphate buffer (pH 6.5) and 200 mM NaCl. All the NMR samples contain 0.01% 22-dimethyl-2-silapentane-5-sulfonate (DSS) and 0.01% NaN3. The heteronuclear NMR experiments were carried out for isotope- labeled PDCD5(1–112) at 308 K on a Bruker DMX600 spectrometer equipped with a z-gradient triple-resonance cryo-probe. The 3D 1H–13C–15N HNCA HN(CA)CO HNCACB CBCA(CO)NH H(CC)(CO)NH and HNCO 3D 1H–15N TOCSY-HSQC (sm = 60 ms) and 3D 1H–13C HC(C)H-COSY and HC(C)H-TOCSY (sm = 12 ms) experiments [16] were performed for backbone and side chain resonance assignments. The 3D 1H–15N NOESY-HSQC and 1H–13C NOESY-HSQC experiments with mixing time sm = 200 ms were performed for obtaining the NOE distance constraints. The measurements of 1H–15N relaxation parameters were performed for 15N-labeled PDCD5(20–104) at 308 K using the standard methods [17]. For 15N T1 measurements the delay times were set to 10 30 80 160 280 380 530 760 1200 and 1600 ms whereas for T2 measurements the relaxation delays were 16.96 33.92 50.88 67.84 84.80 101.76 118.72 152.64 186.56 220.48 and 254.40 ms. In the 2D 1H–15N NOE experiments a delay of 3 s was followed by 1H saturation for 6 s whereas the saturation period was replaced by a delay of equivalent duration in the control experiment. Two experiments were run in an interleaved manner. For amide proton H/D exchange measurements 2D 1H–15N HSQC spectra of intact PDCD5 were recorded successively after every 245 s at 298 K. Each 2D spectrum required 240 s to complete. All NMR data were processed and analyzed using FELIX98 (Accelrys Inc.). 1H chemical shifts were referenced to internal DSS. 15N and 13C chemical shifts were referenced indirectly [18]. Detecting the cell translocation of PDCD5 fragments For detecting the structural correlation of internalization of PDCD5 the deletion mutants of PDCD5 were employed. The FITC conjugated PDCD5 fragment complexes: FITC–PDCD5(1–125) FITC–PDCD5(1–112) FITC–PDCD5(1–104) FITC–PDCD5(34–125) FITC–PDCD5(34–112) and FITC–PDCD5(34–104) were constructed. The fluorescence microscopy study was performed by adding these six complexes into cell culture medium. HL-60 cells were cultured in 35-cm of specialized glass-bottom microwell dishes (MatTek Corp.) with RPMI-1640 medium supplemented with 10% fetal bovine serum (FCS). For fluorescent protein treatments 1 lM of different FITC–PDCD5 fragment was added. Five-h later cells were rinsed twice with PBS buffer and fixed with 4% paraformaldehyde in PBS for 15 min at 4 C. Fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min washed and stained with DAPI (0.25 lg/ml) for 15 min. Cells were rinsed and imaged using a TCS-SP laser-scanning confocal microscope (Leica Microsystems Mannheim Germany). Detecting the effects of different PDCD5 fragments on cell apoptosis The promoting-apoptosis effects of intact and different fragments of PDCD5 can be detected by determining the exposure of PS at the surface of apoptotic cells. For this the apoptotic HL-60 cells were analyzed by flow cytometry using fluorescence-labeled annexin-V [1920]. HL-60 cells were cultured as described above. For induction of apoptosis HL-60 cells were washed and adjusted to 2 105/ml in serum-free RPMI-1640. Cell suspensions were added into 24- well plates (0.5 ml for each well) and then 20 lg of different recombinant PDCD5 fragment were added. About 20 lg of BSA and etoposide were used as negative and positive controls respectively. The cells were harvested after treatment for 20 h and washed twice with PBS followed by resuspending in 100 ll of annexin- V-binding buffer (10 mM Hepes 140 mM NaCl 2 mM MgCl2 5 mM KCl and 2.5 mM CaCl2 pH 7.4). FITC-conjugated annexin-V (10 ll) (Beijing Biosea Biotechnology Co.) was added according to the manufacturer’s protocol. After incubation for 20 min at room temperature in the dark another 400 ll of binding buffer was added and samples were immediately analyzed on a FACSCalibur. Cells (1 104) were collected and analyzed with CELLQuest software (BD Bioscience). Apoptotic cells are expressed as a percentage of total cells. This experiment was done at least three times. Structure calculations Initial structures of PDCD5(1–112) were generated using CANDID module of CYANA software [21]. The NOE assignments given by CANDID were checked manually and the structures were refined in explicit water using CNS software [22] and RECOORDScript [23]. Dihedral angel restrains were obtained using the program TALOS [24]. A family of 100 structures was generated and the 20 structures with lowest energies were selected for analysis. Structural analysis and statistics were obtained using the programs MOLMOL [25] and PROCHECK-NMR [26]. The molecular figures were generated with MOLMOL. Analysis of 15N relaxation parameters R1 and R2 relaxation rates for each residue were determined by fitting peak intensities of the spectra acquired at various relaxation delay times to an exponential decay function I/I0 = exp( R12 t) where I0 is the intensity at t = 0 and I is the intensity after a time delay t. The steady-state 1H–15N NOEs were calculated from the ratio of peak intensities in the NOE spectra obtained with and without proton saturation. The uncertainties of the R1 and R2 values were estimated from the signal-to-noise ratios. The root-meansquare (RMS) value of the noise of background regions in the spectrum was used to estimate the standard deviation of NOE values.

Microscopic Technique
Fluorescence Microscopy, Laser Scanning Confocal Microscopy

Cell Type(s)
HL-60