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1 Supplementary material Docking procedures PtdIns(4)P headgroup docked onto FAPP1-PH The ligand structure and charges were generated within GHEMICAL (Hassinen and Perakyla, 2001) using minimization (500 steps, gradient cutoff kcal.mol -1.Å -1 ) semi-empirical method PM3 to generate partial charges. The PH domain was prepared with Autodock Tool (ADT) using the coordinates of the minimized ligand and the lowest energy protein structure to which Kollman charges were added. A grid of 60 3 points with a spacing of Å was centered on of the region exhibiting C 6 - PtdIns(4)P induced perturbations. A distance dependent dielectric constant of was used to calculate the maps. The ligand was subjected to 100 docking runs using the Lamarckian Genetic Algorithm. Cluster analysis was performed using a root mean square (RMS) tolerance of 2 Å and interactions were analyzed with ADT. This yielded three clusters (Table SIV). The most frequently populated cluster positioned the 1-phosphate and attached acyl chains near hydrophobic protrusion to allow parallel insertion. In contrast, the other, symmetry-related clusters reversed the 1- and 4-phosphate positions and were antiparallel, and hence were judged to be physiologically unreasonable. DPC micelles docked onto FAPP1-PH Flexibility was introduced for residues K7-R18, E50, D57 and G67-A75 based on their micelle-induced chemical shift changes and solvent accessibility estimated with NACCESS. The docking protocol began with 400 randomly oriented and spatially separated protein and micelle structures taken from the representative ensembles (Dancea et al., 2008).

2 List of Figures and Tables Figure S1: Electrostatic surfaces of FAPP1-PH calculated with APBS, with the orientation at right being identical to that shown in panels 1B and 1C. Figure S2: Comparison of 1 H, 15 N HSQC spectra of FAPP1-PH with and without inorganic phosphate. Collected at 800 MHz with a protein concentration of 200 μm. Figure S3: Chemical shift perturbations in FAPP1-PH resonances upon lipid titrations followed in 15 N HSQC spectra Figure S4: HSQC spectra of FAPP1-PH showing the intensity reduction of H N protons in presence of doxyl derivatives Figure S5: Membrane sheet tubulation assay with mutant Y11L12-GG and Y11L12- EE GST-FAPP2 versions FigureS6: Chemical shift perturbations in FAPP1-PH resonances upon addition of 1mM PtdIns(3,4)P 2 and PtdIns(4,5)P 2 Figure S7: PtdSer specifically enhances FAPP1-PH binding to micelles containing PtdIns(4)P Figure S8: The diffusion coefficient (D) of a sample containing 200 μm FAPP1-PH Figure S9: The purified wild type and C94S mutant proteins yielded similar 1D 1H NMR spectra Table SI. Statistics for the ensemble of FAPP1-PH domain solution structures Table SII. Structural statistics of the best 20 models of FAPP1-PH/DPC micelles Table SIII. Primers used to generate FAPP2-GST mutants

Figure S1: Two views of the electrostatic surface of the representative FAPP1-PH structure calculated with APBS, with the orientation at right being similar to that shown in Figure 2B.

3 Figure S1: Two views of the electrostatic surface of the representative FAPP1-PH structure calculated with APBS, with the orientation at right being similar to that shown in Figure 2B. Figure S2: FAPP1-PH does not bind phosphate detectably despite its exposed basic surfaces. Comparison of HSQC spectra of the 15 N labeled FAPP1-PH domain (200 μm) in the presence (blue) and absence (red) of 1.5 mm inorganic phosphate reveals no significant interaction.

Figure S3: Chemical shift changes induced upon titrations of FAPP1-PH with

The CSPs induced by increasing concentrations of DPC/CHAPS micelles (A) or

containing FAPP1-PH (2000 μm) and DPC/ /CHAPS (4 mm DPC) (C) are shown.

protein using identical solution conditions.

micelle. The normalized CSPs are shown in panels D,E and F, respectively.

the mean plus two standard deviations (E).

(i.e. greater than those shown by the red lines) upon addition of ligand.

4 Figure S3: Chemical shift changes induced upon titrations of FAPP1-PH with micelles or ligand, as monitored in 1 H, 15 N HSQC spectra. The CSPs induced by increasing concentrations of DPC/CHAPS micelles (A) or C 6 -PtdIns(4)P (B) to the apo FAPP1-PH, or by C 8 -PtdIns(4)P to a sample containing FAPP1-PH (2000 μm) and DPC/ /CHAPS (4 mm DPC) (C) are shown. Stock solutions of the indicated ligand were added to the 15 N-labelled protein using identical solution conditions. The micelle concentration indicated in (A) assumes 54 DPC molecules per micelle. The normalized CSPs are shown in panels D,E and F, respectively. The red lines correspond to mean ( ) plus standard deviation (σ) (D) or the mean plus two standard deviations (E). The labels indicate the residues with substantially perturbed resonances (i.e. greater than those shown by the red lines) upon addition of ligand. The Trp indole NH signals are labeled with an e. The extensive CSPs induced by C 8 -PtdIns(4)P in the presence of micelles are mapped on FAPP1-PH structure (G),

5 with residues undergoing slow exchange colored in pink. Residues colored in cyan and blue exhibit CSPs over and 0.07, respectively. Asterisks in panels C and E represent Y11 and T23, which exhibit broad and overlapped peaks. Boxed residues are buried in the interior of the protein. Titration points for DPC/CHAPS titrations and c6-ptdins(4)p are shown for residues L12 and Y29, respectively (H).

Figure S4: PRE NMR data identifying the buried

The intensity reductions of H N signals of FAPP1-PH

DPC/ /CHAPS micelles was mapped ontoo the FAPP1-PH

Backbone and sidechain atoms for which the H N

addition of 14-doxyl to the micelles are indicated

micelles containing equimolar 5-doxyl or 14-doxyl

6 Figure S4: PRE NMR data identifying the buried wedge. The intensity reductions of H N signals of FAPP1-PH induced by adding 14- (A) and 5-doxyl PC (B) into DPC/ /CHAPS micelles was mapped ontoo the FAPP1-PH surface (C). Backbone and sidechain atoms for which the H N signal intensity was significantly reduced upon addition of 14-doxyl to the micelles are indicated as red spheres and traces, respectively, on the ribbon structure (D). The 15 N HSQC spectra of FAPP1-PH mixed with micelles containing equimolar 5-doxyl or 14-doxyl (E) derivative are superimposed with control spectra containing DPPC. Residues whose signal intensities are dramatically reduced by the presence of doxyl derivatives in the micelle are labeled.

7 Figure S5: FAPP2-mediated tubulation of flat membrane sheets requires an intact wedge. Abolishment of tubulation activity of mutant Y11L12-GG and Y11L12-EE forms of a GST fusion of full length human FAPP2 protein on lipid membrane sheets containing POPC:PtdIns(4)P (98:2 mol%) shown by DIC. Previous studies have demonstrated the PtdIns(4)P-dependent membrane tubulation activity of FAPP2 using the same assay conditions (Cao et al, 2009). Screen shots were taken from Movies. Tubulation was initiated by injection of 5 μl FAPP2 (1 mg/ml) into the reaction chamber.

8 Figure S6: Polyphosphorylated PIs bind similarly to the FAPP1-PH. Normalized CSPs induced in 15 N-labelled FAPP1-PH (200 μm) upon addition of submicellar concentrations (1 mm) of C6-PtdIns(3,4 6 )P 2 (top panel) and C 6 -PtdIns(4,5)PP 2 (bottom panel). The 15 N, 1 H HSQC spectra were collected at 800 MHz in the NMR buffer.

Figure S7: PtdSer specifically enhances FAPP1-PH binding to micelles containing PtdIns(4)P.

5mM) with the PtdIns(4) )P binding pocket is comparatively weak based on the normalized CSPs induced in 15 N-labelled FAPP1-PH

Upon addition of C 6 -PtdSer (80 μm) to the DPC/ /CHAPS complexed FAPP1PH, relatively small additional CSPs were induced (middle

9 Figure S7: PtdSer specifically enhances FAPP1-PH binding to micelles containing PtdIns(4)P. The interaction of soluble C 6 6-PtdSer (1.5mM) with the PtdIns(4) )P binding pocket is comparatively weak based on the normalized CSPs induced in 15 N-labelled FAPP1-PH (2000 μm) (top panel). Upon addition of C 6 -PtdSer (80 μm) to the DPC/ /CHAPS complexed FAPP1PH, relatively small additional CSPs were induced (middle panel). However addition of C 6 -PtdSer (100 μm) to a FAPP1-PH sample whichh had been pre-bound to DPC-CHAPS micelles containing PtdIns(4)P (200 μm) induced substantial additional CSPs (bottom panel). Thus C 6 -PtdSer binding to FAPP1-PH appears to particularly stabilize its PtdIns(4)P micelle complex. The 15 N, 1 H HSQC spectra were collected at 800 MHz in the NMR buffer.

10 FAPP1-PH forms a monomeric state The oligomeric state of the wild type and C94S mutant forms were evaluated by NMR and confirmed by analytical ultracentrifugation in presence of β-mercoptoethanol. Figure S8: The diffusion coefficient (D) of a sample containing 200 μm FAPP1-PH was deduced from NMR diffusion experiments (Nesmelova et al., 2004) at 800 MHz by varying the amplitude of the field gradients. The monomeric state of the protein in solution was deduced from D = x m 2.s -1 which corresponds to a radius of Å for a spherical protein ln I(g 2 )/I(0) -2-3 k 2 (cm 2.s -1 ) Where, ln (A(g 2 )/A(0))=-Dk 2 and k 2 =(γ H ) 2 δ 2 g 2 (Δ-δ/3) The delay and values used for these experiments are described elsewhere (Nesmelova et al., 2004). The field strength g was varied from 5.6 to 53.3 G.cm -1 and the molecular size was deduced from the Stokes-Einstein equation for spherical particles.

11 Figure S9: The purified wild type and C94S mutant proteins yielded similar 1D 1 H NMR spectra. Both proteins were monomeric under reducing conditions based on analytical ultracentrifugation in a Beckman XL-I Protein Characterization System (Beckman Coulter) equipped with an An-50-Ti rotor. Samples were prepared in Tris buffer 20mM ph 7, NaCl 100mM, β-mercapto ethanol 9.6mM and loaded in double sector cells. Velocity sedimentation was performed at 20 C at rotations per minute. The sample was detected at a wavelength of 280nm, and data were processed in SEDFIT (Brown and Schuck, 2006) resulting in frictional ratios for the wild type and C94S mutant of 1.39S and 1.71S, respectively.

12 Table SI. Primers for GST-FAPP2 mutants Y11L12-EE Forward 5 -TGT ACA AGT GGA CCA ACG AAG AGA GCG GTT GGC AGC CTA GAT GG-3 Reverse 5 -CCA TCT AGG CTG CCA ACC GCT CTC TTC GTT GGT CCA CTT GTA CA-3 Y11L12-GG Forward 5 -TAC AAG TGG ACC AAC GGT GGG AGC GGT TGG CAG CCT AGA TGG-3 Reverse 5 -CCA TCT AGG CTG CCA ACC GCT CCC ACC GTT GGT CCA CTT GTA-3

13 Table SII. Statistics for the ensemble of FAPP1-PH domain solution structures Experimental restraints NOE distance restraints 1560 Unambiguous 893 Long range 170 Medium 21 Short 66 Intra residue 428 Sequential 208 Ambiguous 629 HB constraints 38 Talos dihedral constraints 103 Residual experimental violations a NOE > 0.5 Å 0 NOE > 0.3 Å 3 Dihedral restraints > 5 Å 2 Energies (Kcal.mol -1 ) Total ± E noe ± 4.70 E cdih 8.60 ± 1.31 E bond ± 1.47 E improper ± E angle ± 7.85 E vdw ± E dihe ± 5.46 Deviation from idealized geometry Bonds / Angles 0.53 ± 0.02 Impropers 1.33 ± 0.13 Atomic pairwise rmsd (Å) b Backbone atoms 0.26 Heavy atoms 0.80 Ramachandran statistics c Residues in core regions 82.6% Residues in allowed regions 14.4% Residues in generous regions 2.7% Residues in disallowed regions 0.3% a Sum of violations for the ensemble of structures b RMSD is calculated for secondary structural elements including residues 1-7,16-22,26-30,43-44,50-53,61-64,69-73 and c Non glycine residues

14 Table SIII. Structural statistics of the best 20 models of FAPP1-PH/DPC micelles Experimental parameters a Ambiguous distance restraints Number of flexible residues b Atomic pairwise rmsd (Å) All backbone Flexible interface backbone 10 including NH of N10, L12, T13,G14, NHε of W8,W15,Q16 and NH δ of N10 23 (K7 to R18, E50, D57 and G67-A75) Å Å Intermolecular energies (kcal.mol -1 ) E vdw ± E elec ± E restraints 3.73 ± 3.40 Buried surface area ± Distance / insertion angle Micelles center coordinates in Protein inertia frame r (micelle-protein centres) ± 0.85 Å θ (deg) ± 4.01 ψ (deg) ± a deduced from intensity reductions observed in presence of 14-doxyl derivative b according to their surface accessibility and the chemical shift perturbation in presence of DPC/CHAPS

15 Table SIV. Interactions of the Ins(1,4)P 2 head group docked into the FAPP1-PH domain. The sum of VDW radii were used to identify the interacting residues in ADT (scaling factor 1). Amino acid residues involved in intermolecular hydrogen bonds for more than 50% of the docked structures are indicated in bold. Cluster number Population Binding free energy (kcal.mol -1 ) Interacting residues K7,W8,T9,N10,R18,F20,H70,F K7,W8,T9,R18,Y29,H70,F K7,W8,T9,N10,H70,F71

16 References Brown PH and Schuck P. (2006) Macromolecular size-and-shape distributions by sedimentation velocity analytical ultracentrifugation. Biophys J, 90, Dancea F, Kami K and Overduin M. (2008) Lipid interaction networks of peripheral membrane proteins revealed by data-driven micelle docking. Biophys J, 94, Hassinen T and Perakyla M. (2001) New energy terms for reduced protein models implemented in an off-lattice force field. J Comput Chem, 22, Nesmelova IV, Idiyatullin D and Mayo KH. (2004) Measuring protein self-diffusion in protein-protein mixtures using a pulsed gradient spin-echo technique with WATERGATE and isotope filtering. J Magn Reson, 166,

17 Supplementary movies Membrane deformation monitored by DIC FAPP1-PH.avi

Supplementary Table 3. 3 UTR primer sequences. Primer sequences used to amplify and clone the 3 UTR of each indicated gene are listed.

Supplementary Table 3. 3 UTR primer sequences. Primer sequences used to amplify and clone the 3 UTR of each indicated gene are listed. Supplemental Figure 1. DLKI-DIO3 mirna/mrna complementarity. Complementarity between the indicated DLK1-DIO3 cluster mirnas and the UTR of SOX2, SOX9, HIF1A, ZEB1, ZEB2, STAT3 and CDH1with mirsvr and PhastCons