10 figures, 5 videos, 3 tables and 1 additional file

Figures

Figure 1 with 2 supplements
Structure of SHIP2 Ptase-C2.

(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 …

https://doi.org/10.7554/eLife.26640.002
Figure 1—figure supplement 1
Details of interactions between the Ptase and C2 domains in Ptase-C2 WT.

(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 …

https://doi.org/10.7554/eLife.26640.003
Figure 1—figure supplement 2
Details of interactions between the Ptase and C2 domains in Ptase-C2 FLDD.

(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). …

https://doi.org/10.7554/eLife.26640.004
Figure 2 with 1 supplement
The C2 domain of SHIP2 and lipid binding.

(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 …

https://doi.org/10.7554/eLife.26640.006
Figure 2—source data 1

Source data for plots in Figure 2C-insert and 2D.

https://doi.org/10.7554/eLife.26640.007
Figure 2—figure supplement 1
Lipid binding by protein lipid overlay.

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+. …

https://doi.org/10.7554/eLife.26640.008
Enzyme kinetics of SHIP2.

(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 …

https://doi.org/10.7554/eLife.26640.010
Figure 3—source data 1

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.

https://doi.org/10.7554/eLife.26640.011
Effect of PS binding on SHIP activity.

(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 …

https://doi.org/10.7554/eLife.26640.014
Figure 4—source data 1

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.

https://doi.org/10.7554/eLife.26640.015
Figure 5 with 1 supplement
SHIP2 dynamics.

(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 …

https://doi.org/10.7554/eLife.26640.016
Figure 5—figure supplement 1
SHIP dynamics for different L4 states.

(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).…

https://doi.org/10.7554/eLife.26640.017
Figure 6 with 1 supplement
Model of substrate bound SHIP2.

(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 …

https://doi.org/10.7554/eLife.26640.020
Figure 6—figure supplement 1
Structure of Ptase-C2 D607A, crystallized in presence of PI(3,4,5)P3-diC8 and Mg2+.

(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 …

https://doi.org/10.7554/eLife.26640.021
Figure 7 with 1 supplement
SHIP2 mutational analysis.

(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. …

https://doi.org/10.7554/eLife.26640.024
Figure 7—source data 1

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.

https://doi.org/10.7554/eLife.26640.025
Figure 7—figure supplement 1
Circular dichroism analysis of purified SHIP proteins.

(A–B) Far UV CD spectra of SHIP2 Ptase (A) and Ptase-C2 (B) are shown for WT and all purified mutants.

https://doi.org/10.7554/eLife.26640.026
Figure 8 with 1 supplement
SHIP2 cellular activity.

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 …

https://doi.org/10.7554/eLife.26640.029
Figure 8—source data 1

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.

https://doi.org/10.7554/eLife.26640.030
Figure 8—figure supplement 1
Quantification of SHIP expression levels.

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 …

https://doi.org/10.7554/eLife.26640.031
Figure 8—figure supplement 1—source data 1

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.

https://doi.org/10.7554/eLife.26640.032
Model of SHIP2 catalytic cycle.

(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 …

https://doi.org/10.7554/eLife.26640.033
Author response image 1
Tryptophan-dansyl FRET assay.

(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 …

https://doi.org/10.7554/eLife.26640.036

Videos

Video 1
Principal component analysis of Ptase simulation.

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 …

https://doi.org/10.7554/eLife.26640.018
Video 2
Principal component analysis of Ptase-C2 simulation.

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, …

https://doi.org/10.7554/eLife.26640.019
Video 3
Simulation of Ptase with substrate.

The first 100 ns of the trajectory from the simulation of Ptase bound to PI(3,4,5)P3-diC8 and Mg2+ are shown.

https://doi.org/10.7554/eLife.26640.022
Video 4
Simulation of Ptase-C2 with substrate.

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.

https://doi.org/10.7554/eLife.26640.023
Video 5
The SHIP2 catalytic cycle.

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 …

https://doi.org/10.7554/eLife.26640.034

Tables

Table 1

Diffraction and refinement statistics.

https://doi.org/10.7554/eLife.26640.005
Ptase-C2
WT
Ptase-C2
FLDD
Ptase-C2
FLDD
Ptase-C2
D607A
Space groupP212121P21I2P212121
Cell dimensions
a, b, c (Å)136.0, 175.8, 176.944.0, 81.1, 128.943.7, 73.4, 158.0137.1, 177.1, 177.4
α, β, γ (°)90.0, 90.0, 90.090.0, 92.9, 90.090.0, 90.7, 9090.0, 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)48.96–1.9681.12–1.9478.98–1.8549.17–2.65
 No. reflections2870026379040283119204
 Rwork/Rfree18.0/20.819.0/22.917.8/20.520.6/24.5
 No. atoms
 Protein281426901342327555
 Ligand2253916237
 Water1564288139284
 B- factors
 Protein46.7727.3734.0161.54
 Ligand/ion42.6634.6442.1066.88
 Water44.0129.8238.3045.57
 R.m.s. deviation
 Bond lengths (Å)0.0090.0100.0080.009
 Bond angles (°)1.3061.3541.2601.267
  1. *Highest resolution range shown in parentheses

Table 2

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 …

https://doi.org/10.7554/eLife.26640.012
PtasePtase-C2
IP4PI(3,4,5)P3IP4PI(3,4,5)P3
kcat (s−1)KM (μM)kcat (s−1)KM (μM)kcat (s−1)KM (μM)kcat (s−1)KM (μM)
WT1.32 ± 0.0298 ± 70.69 ± 0.0243 ± 42.02 ± 0.04115 ± 87.83 ± 0.26*94 ± 9*
FLDD1.20 ± 0.0582 ± 134.44 ± 0.17*61 ± 8*1.39 ± 0.0673 ± 134.79 ± 0.49*122 ± 31*
R649A1.64 ± 0.05126 ± 131.35 ± 0.0659 ± 81.79 ± 0.05110 ± 106.37 ± 0.42*100 ± 18*
Table 2—source data 1

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.

https://doi.org/10.7554/eLife.26640.013
Table 3

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 …

https://doi.org/10.7554/eLife.26640.027
PtasePtase-C2
IP4PI(3,4,5)P3IP4PI(3,4,5)P3
kcat (s−1)KM (μM)kcat (s−1)KM (μM)kcat (s−1)KM (μM)kcat (s−1)KM (μM)
R691ANANANANANDNDNDND
D613A,
D615A
NANANANA1.45 ± 0.0576 ± 117.10 ± 0.57*100 ± 22*
R682ANDNDND*ND*0.27 ± 0.04ND1.74 ± 0.2393 ± 35
N684ANDNDNDNDNDNDNDND
R665A0.53 ± 0.03119 ± 190.23 ± 0.0143 ± 102.07 ± 0.0994 ± 157.02 ± 0.37*119 ± 16*
D607ANANANANANDNDNDND
Table 3—source data 1

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.

https://doi.org/10.7554/eLife.26640.028

Additional files

Supplemental file 1

Thermal melting (Tm) of SHIP2 WT and mutants.

Tm values are determined by thermofluor. NA, not analyzed; WS, weak signal.

https://doi.org/10.7554/eLife.26640.035

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