(A) Schematic domain structure of human SHIP2. SH2, Src homology domain 2; PH-R, pleckstrin homology related domain; 5-Ptase, 5-phosphatase; PR, proline rich; SAM, sterile-α-motif. (B) Ribbon …
(A–H) Details of the interdomain interactions are shown for the 8 Ptase-C2 WT molecules A-H. Ptase residues are labeled in brown, C2 residues in blue. Hydrophobic interactions are indicated as red …
(A–C) Ptase-C2 interactions in the FLDD mutant. Details of the interdomain interactions are shown for the Ptase-C2 FLDD molecule in the I2 (A) and the two molecules in the P21 crystal form (B–C). …
(A) Structure-based sequence alignment of the C2 domains of SHIP2, dysferlin, phospholipase A2 (PLA2) and protein kinase Cα (PKCα) and alignment of the corresponding sequence of SHIP1. Structurally …
Source data for plots in Figure 2C-insert and 2D.
A protein lipid overlay (PLO) assay was employed to probe for SHIP binding to indicated amounts of PS-diC16 or PC-diC16 lipids spotted on a nitrocellulose membrane in presence or absence of Ca2+. …
Source data for plot in Figure 2—figure supplement 1.
(A–F) Enzyme activity for SHIP2 Ptase and Ptase-C2 was measured using a Malachite Green assay, with IP4 or PI(3,4,5)P3-diC8 as substrates. Substrate titrations of wild type SHIP2 (A–B) and the …
Source data for plots in Figure 3A–F.
Values are in [PO4]*[E]−1*s−1. The values in red were excluded. Numbers (#i) above data indicate independent experiment number. Equation used to fit data in Figure 3A–F: Y = Bo + Vm*X/(X + Km); Variables: Vmax, Km, Bo = baseline. Software used: Graphpad Prism.
(A–B) Activity was measured in presence or absence of 100 μM PC-diC8 or PS-diC8 and as substrate 150 μM IP4 (A) or PI(3,4,5)P3-diC8 (B). Enzyme concentrations used were 400 nM for measurements with …
Source data for graphs in Figure 4A–F.
Values are in [PO4]*[E]−1*s−1. The values in red were excluded. Numbers (#i) above data indicate independent experiment number. p-Values are calculated using a two-tailed unpaired Student t test assuming Gaussian distribution. Exact p-values are shown if >0.001.
(A) Root mean square fluctuations (RMSF) during MD simulations of the Ptase (brown) or Ptase-C2 (blue) are plotted for Cα atoms in the Ptase domain. Peaks correspond to loop regions (L1–L4) and the …
(A–C) RMSF plots of Ptase (brown) and Ptase-C2 (blue) simulations are shown, separated into conformations where R682 is unbound (A), R682 is singly bound (B) or R682 is doubly bound to D613/D615 (C).…
(A) The SHIP2 Ptase-PI(3,4,5)P3-diC8 complex is modeled based on crystal structures of the homologue INPP5B crystal structure bound to PI(4)P (pdbs: 3MTC). L4 is in the ‘in’ conformation and R682 …
(A) Electron density for one Mg2+ and one phosphate group is clearly visible in 6 of the eight molecules (shown is molecule H). 2Fo-Fc electron density is shown in blue countered at a σ level of …
(A) Residues mutated in the SHIP2 Ptase domain are shown as sticks and are labeled. (B–E) For mutants that display significant activity (D613A/D615A and R665A), substrate titration curves are shown. …
Source data for plots in Figure 7B–E.
Values are in [PO4]*[E]−1*s−1. The values in red were excluded. Numbers (#i) above data indicate independent experiment number. Equation used to fit data in Figure 3A–F: Y = Bo + Vm*X/(X + Km); Variables: Vmax, Km, Bo = baseline. Software used: Graphpad Prism.
(A–B) Far UV CD spectra of SHIP2 Ptase (A) and Ptase-C2 (B) are shown for WT and all purified mutants.
Full-length SHIP2 proteins were transiently expressed in HEK293 cells and resulting Akt-pT308 levels monitored. Shown are transfections of empty vector (-), wild type SHIP2 (WT), the FLDD mutant and …
Source data for graph in Figure 8.
Values are pAkt/totalAkt intensities, scaled relative to empty vector (-).The values in red were excluded. Numbers (#i) above data indicate independent experiment number. p-Values are calculated using a two-tailed unpaired Student t test assuming Gaussian distribution. Exact p-values are shown if >0.001. *Replicas from experiment #2 were loaded on different gels and scaled relative to empty vector (i.e. all scale to 1). Therefore empty vector values of experiment #2 are excluded in the statistical evaluation.
HEK293 cells were transiently transfected with the empty vector (-) or vector expressing GST fused full-length SHIP2 constructs (WT, FLDD or ∆C2). Expression levels were assessed by quantifying …
Source data for graph in Figure 8—figure supplement 1.
Values are SHIP2/Vinculin intensities, scaled relative to wild type-SHIP2 (WT).The values in red were excluded. Numbers (#i) above data indicate independent experiment number. p-Values are calculated using a two-tailed unpaired Student t test assuming Gaussian distribution. Exact p-values are shown.
(A) Model of SHIP2 Ptase-C2 docked to the membrane and with PI(3,4,5)P3-Mg2+ bound to the active site. Basic and hydrophobic side chains of K531, I534, L538, K568, R571, R581, R588 and L590 in the …
(A) Emission scan resulting from excitation at 280nm of dansyl vesicles alone (blue trace) or vesicles in presence of SHIP2 Ptase (red trace). SHIP2 Ptase induces a reduction of the emission signal …
Movements according to the first four eigenvectors from the principle component analysis of the Ptase simulation are shown. The starting conformation in the doubly bound L4-out conformation, based …
Movements according to the first four eigenvectors from the principle component analysis of the Ptase-C2 simulation are shown. The starting conformation in the doubly bound L4-out conformation, …
The first 100 ns of the trajectory from the simulation of Ptase bound to PI(3,4,5)P3-diC8 and Mg2+ are shown.
The first 100 ns of the trajectory from the simulation of Ptase-C2 bound to PI(3,4,5)P3-diC8 and Mg2+ are shown.
The conformational changes between L4-out doubly bound, L4-out singly bound and L4-in conformations are shown as morphs between observed crystal structures and assembled in sequence according to the …
Diffraction and refinement statistics.
Ptase-C2 WT | Ptase-C2 FLDD | Ptase-C2 FLDD | Ptase-C2 D607A | |
---|---|---|---|---|
Space group | P212121 | P21 | I2 | P212121 |
Cell dimensions | ||||
a, b, c (Å) | 136.0, 175.8, 176.9 | 44.0, 81.1, 128.9 | 43.7, 73.4, 158.0 | 137.1, 177.1, 177.4 |
α, β, γ (°) | 90.0, 90.0, 90.0 | 90.0, 92.9, 90.0 | 90.0, 90.7, 90 | 90.0, 90.0, 90.0 |
Resolution (Å)* | 48.97–1.96 (1.99–1.96) | 81.12–1.94 (1.99–1.94) | 78.98–1.85 (1.89–1.85) | 49.17–2.65 (2.70–2.65) |
Rmerge* | 8.0 (88.1) | 13.3 (110.7) | 5.9 (59.5) | 16.1 (134.4) |
Rmeas* | 8.6 (95.6) | 14.4 (120.0) | 7.0 (70.4) | 17.0 (142.5) |
Rpim* | 3.3 (36.8) | 5.5 (45.9) | 3.8 (37.2) | 5.5 (46.7) |
CC (1/2)* | 0.999 (0.743) | 0.998 (0.787) | 0.999 (0.768) | 0.996 (0.700) |
Mean (I/σ(I))* | 14.8 (2.1) | 11.8 (2.3) | 11.6 (2.1) | 13.4 (2.2) |
Completeness (%)* | 100.0 (100.0) | 100.0 (100.0) | 99.3 (99.1) | 100.0 (100.0) |
Multiplicity * | 6.8 (6.7) | 6.8 (6.7) | 3.4 (3.5) | 9.0 (8.9) |
Refinement | ||||
Resolution (Å) | 48.96–1.96 | 81.12–1.94 | 78.98–1.85 | 49.17–2.65 |
No. reflections | 287002 | 63790 | 40283 | 119204 |
Rwork/Rfree | 18.0/20.8 | 19.0/22.9 | 17.8/20.5 | 20.6/24.5 |
No. atoms | ||||
Protein | 28142 | 6901 | 3423 | 27555 |
Ligand | 225 | 39 | 16 | 237 |
Water | 1564 | 288 | 139 | 284 |
B- factors | ||||
Protein | 46.77 | 27.37 | 34.01 | 61.54 |
Ligand/ion | 42.66 | 34.64 | 42.10 | 66.88 |
Water | 44.01 | 29.82 | 38.30 | 45.57 |
R.m.s. deviation | ||||
Bond lengths (Å) | 0.009 | 0.010 | 0.008 | 0.009 |
Bond angles (°) | 1.306 | 1.354 | 1.260 | 1.267 |
*Highest resolution range shown in parentheses
Enzymatic parameters are calculated by fitting the Michaelis-Menten equation to substrate titrations. Enzyme concentrations used for activity measurements were 400 nM, or if this caused saturated …
Ptase | Ptase-C2 | |||||||
---|---|---|---|---|---|---|---|---|
IP4 | PI(3,4,5)P3 | IP4 | PI(3,4,5)P3 | |||||
kcat (s−1) | KM (μM) | kcat (s−1) | KM (μM) | kcat (s−1) | KM (μM) | kcat (s−1) | KM (μM) | |
WT | 1.32 ± 0.02 | 98 ± 7 | 0.69 ± 0.02 | 43 ± 4 | 2.02 ± 0.04 | 115 ± 8 | 7.83 ± 0.26* | 94 ± 9* |
FLDD | 1.20 ± 0.05 | 82 ± 13 | 4.44 ± 0.17* | 61 ± 8* | 1.39 ± 0.06 | 73 ± 13 | 4.79 ± 0.49* | 122 ± 31* |
R649A | 1.64 ± 0.05 | 126 ± 13 | 1.35 ± 0.06 | 59 ± 8 | 1.79 ± 0.05 | 110 ± 10 | 6.37 ± 0.42* | 100 ± 18* |
Source data for values shown in Table 2.
All values are [PO4] (in μM). Numbers (#i) above data indicate independent experiment number. Most kinetic parameters (kcat, KM) are extracted from curves shown in Figure 3, for which source data are available with this figure. Below source data are shown for cases where experiments were repeated at higher enzyme concentration (400 nM) to extract reliable kinetic parameters. Equation used to extract Vmax and KM: Y = Bo + Vm*X/(X + KM); Variables: Vmax, KM, Bo = baseline. Software used: Graphpad Prism.
Enzymatic parameters are calculated by fitting the Michaelis-Menten equation to substrate titrations. Enzyme concentrations used for activity measurements were 400 nM, or if this caused saturated …
Ptase | Ptase-C2 | |||||||
---|---|---|---|---|---|---|---|---|
IP4 | PI(3,4,5)P3 | IP4 | PI(3,4,5)P3 | |||||
kcat (s−1) | KM (μM) | kcat (s−1) | KM (μM) | kcat (s−1) | KM (μM) | kcat (s−1) | KM (μM) | |
R691A | NA | NA | NA | NA | ND | ND | ND | ND |
D613A, D615A | NA | NA | NA | NA | 1.45 ± 0.05 | 76 ± 11 | 7.10 ± 0.57* | 100 ± 22* |
R682A | ND | ND | ND* | ND* | 0.27 ± 0.04 | ND | 1.74 ± 0.23 | 93 ± 35 |
N684A | ND | ND | ND | ND | ND | ND | ND | ND |
R665A | 0.53 ± 0.03 | 119 ± 19 | 0.23 ± 0.01 | 43 ± 10 | 2.07 ± 0.09 | 94 ± 15 | 7.02 ± 0.37* | 119 ± 16* |
D607A | NA | NA | NA | NA | ND | ND | ND | ND |
Source data for values shown in Table 3.
All values are [PO4] (in μM). Numbers (#i) above data indicate independent experiment number. Most kinetic parameters (kcat, KM) are extracted from curves shown in Figure 6, for which source data are available with this figure. Below source data are shown for cases where no curve is shown or where experiments were repeated at higher enzyme concentration (400 nM) to extract reliable kinetic parameters. Equation used to extract Vmax and KM: Y = Bo + Vm*X/(X + KM); Variables: Vmax, KM, Bo = baseline. Software used: Graphpad Prism.
Thermal melting (Tm) of SHIP2 WT and mutants.
Tm values are determined by thermofluor. NA, not analyzed; WS, weak signal.