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The structural basis of PTEN regulation by multi-site phosphorylation

Abstract

Phosphatase and tensin homolog (PTEN) is a phosphatidylinositol-3,4,5-triphosphate (PIP3) phospholipid phosphatase that is commonly mutated or silenced in cancer. PTEN’s catalytic activity, cellular membrane localization and stability are orchestrated by a cluster of C-terminal phosphorylation (phospho-C-tail) events on Ser380, Thr382, Thr383 and Ser385, but the molecular details of this multi-faceted regulation have remained uncertain. Here we use a combination of protein semisynthesis, biochemical analysis, NMR, X-ray crystallography and computational simulations on human PTEN and its sea squirt homolog, VSP, to obtain a detailed picture of how the phospho-C-tail forms a belt around the C2 and phosphatase domains of PTEN. We also visualize a previously proposed dynamic N-terminal α-helix and show that it is key for PTEN catalysis but disordered upon phospho-C-tail interaction. This structural model provides a comprehensive framework for how C-tail phosphorylation can impact PTEN’s cellular functions.

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Fig. 1: Phosphorylation of PTEN influences PIP3 hydrolysis.
Fig. 2: ‘Molecular ruler’ to measure distance requirements for the tail to autoinhibit the enzymatic function of PTEN.
Fig. 3: The N-terminal segment of PTEN folds as a helix.
Fig. 4: Mode of interaction of tetraphosphorylated PTEN C-tail with VSP.
Fig. 5: Mode of interaction of triphosphorylated PTEN C-tail with VSP.
Fig. 6: NMR-driven HADDOCK model of phosphorylated C-tail interactions with VSP.
Fig. 7: Role of N-terminal segment of PTEN in phospho-tail binding.

Data availability

The structural data for n-crPTEN-13sp-T1, 4p-crPTEN-13sp-T2, 4p-crPTEN-20sp-T3, and, 4p-crPTEN-20sp-T3 have been deposited in the Protein Data Bank with the following accession codes: 7JUL, 7JUK, 7JVX and 7JTX. The chemical shift assignments and perturbations of VSP have been deposited in the BMRB under accession code 50381. Additional raw data are deposited in the Dryad database https://doi.org/10.5061/dryad.dfn2z34zh. Source data are provided with this paper.

Code availability

Custom FloppyTail code/flags used in this study can be found in the Supplementary Methods file.

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Acknowledgements

We thank NIH K99GM130961 (D.R.D.), NIH F32GM120855 (D.R.D.), NIH F32 CA259214 (B.A.P.), NIH R01CA74305 (P.A.C.), T32 GM080189 (S.H.), TM32-GM008403 (J.R.J.), R01-GM078221 (J.R.J. and J.J.G.), NIH P50 CA062924 (S.B.G.), and DoD CDMRP BC151831/W81XWH-16-1-0486 (S.B.G.) for generous financial support. H.A. acknowledges funding from the Claudia Adams Barr Program for Innovative Cancer Research and NIH R01GM136859. Maintenance of the NMR equipment was supported by NIH grant no. EB002026. Work at the AMX (17-ID-1) and FMX (17-ID-2) beamlines were supported by the NIH, the National Institute of General Medical Sciences (P41GM111244), the DOE Office of Biological and Environmental Research (KP1605010), and the National Synchrotron Light Source II at Brookhaven National Laboratory is supported by the DOE Office of Basic Energy Sciences under contract number DE-SC0012704 (KC0401040). We acknowledge the use of the Eukaryotic Tissue Culture Facility at JHU and thank its manager Y. Li for her expertise in protein expression using insect cells and M. Miller for her expertise with XDS.

Author information

Authors and Affiliations

Authors

Contributions

D.R.D., T.V., S.B.G., H.A., M.J.E., J.R.J, J.J.G., and P.A.C. designed experiments. D.R.D., T.V., R.I, B.A.P., S.B.G., S.H., Z.C., K.L.P., J.R.J., and E.P. carried out the experiments. All authors contributed to data analysis. D.R.D., T.V., S.B.G., H.A., P.C., and P.A.C. drafted the manuscript. All authors edited and approved the manuscript.

Corresponding authors

Correspondence to Sandra B. Gabelli, Haribabu Arthanari or Philip A. Cole.

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Competing interests

P.A.C. is a consultant for Scorpion Therapeutics. S.B.G. is founder and holds equity of AMS LLC and is a consultant to Cytiva and Genesis Therapeutics.

Additional information

Peer review information Nature Structural & Molecular Biology thanks John Burke and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Anke Sparmann and Florian Ullrich were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Engineering and evaluating a chimeric version of VSP and PTEN.

a, Primary and secondary structure overlay of our new PTEN structure and VSP (PDB: 3V0D, chain B) using ESPript. b, SDS-PAGE gel showing EPL product of 4p-crPTEN-13sp-T2. c, SDS-PAGE gel showing EPL product 4p-VSP (VSP/PTEN chimera). d, gel filtration result for 15N-VSP C363S used for NMR analysis. e, Sequence of phosphorylated peptides used in inhibition experiments. f, Bar graph of the initial velocity of PTEN in the presence of changing concentration of either 4p-T15 or 4p-T16 peptide inhibitor. Assay condition: 50 mM Tris pH 8.0, 100 mM NaCl, 10 mM 2-mercaptoethanaol. g, Bar graph of the initial velocity of PTEN in the presence of changing concentration of 3p-T14 peptide inhibitor. Assay condition: 50 mM Tris pH 8.0, 10 mM 2-mercaptoethanaol. Uncertainty reported as ± SEM, n = 2 biologically independent experiments.

Source data

Extended Data Fig. 2 N-terminal helix and its relationship to symmetry related molecules.

a, 2fofc Electron density map of n-crPTEN-13sp-T1 (PDB: 7JUL) at 1σ (grey mesh) with the refined structure in blue sticks and symmetry related molecule as C-alpha (magenta). Residue E7 of the n-terminal helix is at hydrogen bonding distance of R173. b, Ribbon diagram of n-crPTEN-13sp-T1 with the n-terminal helix in blue and the rest of the molecule in green. Adjacent symmetry related PTEN molecules in the top and bottom are shown in magenta with the helix shown in blue. Symmetry related molecules further away are shown in yellow.

Extended Data Fig. 3 N-terminal helix 1 extends a positively charged patch on PTEN.

a, b, c, d, structures of ncrPTEN-13sp-T1, 4p-crPTEN-13sp-T2, 4p-crPTEN-20sp-T3, 4p-crPTEN-22sp-T3 in ribbons with the width of the tube proportional to b-factors. The N-terminal helix, loop of catalytic domain, and CBRIII display the highest spread. e, surface representation of PTEN 1D5R colored as the electrostatic potential with the N-terminal helix area of 4p-crPTEN-13sp-T2 (PDB ID 7JUK) marked as a yellow box. The tartrate molecule observed in 1D5R bound to active site is shown as orange sticks. f, surface representation of 4p-crPTEN-13sp-T2 colored as the electrostatic potential displays how the N-terminal helix closes up the tartrate binding site observed in 1D5R. Tartrate is not a component of the crystallization mix of 7JUL.

Extended Data Fig. 4 VSP as a surrogate to understand the structural basis for phospho-tail interactions with PTEN.

a, New PTEN crystal structure of n-crPTEN-13sp-T2 showing N-terminal helix (blue) that ranges from aa7–14 in the N-terminal segment. b, VSP crystal structure (PDB:3V0D, chain B) showing similar N-terminal helix in blue as our new PTEN crystal structure. c, Domain architecture overlay of PTEN and VSP showing VSP lacking the 50 amino acid C-tail of PTEN. d, EPL strategy for producing chimeric VSP fused to the C-tail of PTEN. e, Phosphatase sensitivity assay of 4p-PTEN and 4p-VSP using 1 U of alkaline phosphatase to evaluate VSP’s ability to mimic PTEN’s conformational closed state. Half-life for 4p-PTEN = 73 ± 10 min and 4p-VSP = 71 ± 10 min, n = 3 biologically independent experiments. f, Binding constants determined by fluorescence anisotropy for various ligands with either tPTEN (aa1-379,Y379C) or VSP-C363S n = 6 biologically independent experiments. g, Cartoon showing competition of PIP2 with tetraphosphorylated C-tail for binding to VSP/PTEN. Uncertainty reported as ± SEM, n = 6 biologically independent experiments.

Source data

Extended Data Fig. 5 Assigned NMR spectrum of VSP with tetra-phosphorylated C-tail.

a, Assigned 15N-1H-HSQC spectrum VSP with tetra-phosphorylated C-tail. b, Overlay of 15N-1H-HSQC spectra of VSP alone (blue), VSP in the presence of 4 molar equivalent of PTEN C-tail (red), semi-synthetic VSP chimera with non-phosphorylated PTEN C-tail (green), semi-synthetic VSP chimera with tetraphosphorylated PTEN C-tail (orange) and SUMO (purple). Extra peaks (impurities) in the spectra of semi-synthetic proteins are attributed to the SUMO tag. Peaks of VSP without or with non-phosphorylated tails overlay and peaks of VSP with tetraphosphorylated tails in inter- or intra-molecular interaction overlay (except for a few residues in the C-term of VSP). This proves that the intermolecular experiments faithfully report on the interaction in a single chain protein.

Extended Data Fig. 6 Mode of interaction of PIP2 with VSP.

a, Overlay of 15N-1H-HSQC spectra of VSP alone (blue) and VSP in the presence of 25 μM PIP2 (red). b, Combined chemical shift perturbations corresponding to the spectra in (a) and plotted against VSP primary sequence. Dashed red line corresponds to the standard deviation to the mean, excluding outliers (higher than 3xStDev). Notable regions of VSP for their strong interaction or biological relevance are shown in colored areas. c, Peak intensity ratios corresponding to the spectra in (a) and plotted against VSP primary sequence. Notable regions of VSP for their strong interaction or biological relevance are shown in colored areas.

Source data

Extended Data Fig. 7 Modeling of tail:core interactions.

a, Fraction of phosphorylated residues S380, T382, T383, S385 interacting with core residue X in regions of interest. The red line indicates the mean number of interactions across all models. Models were generated using the standard FloppyTail protocol. The phosphorylated residues show an above average preference for interacting with the CBRIII loop and the Cα-2 segment. b, Fraction of phosphorylated residues S380, T382, T383, S385 interacting with core residue X in regions of interest. The red line indicates the mean number of interactions across all models. Models were generated using fragments and constraints. The phosphorylated residues show an above average preference for interacting with the CBRIII loop and Cα-2 segment. c-d, Model of the complex created by HADDOCK (started from PDB:3V0G). The C2 domain is depicted in light blue and the catalytic domain in light green. Biologically important regions of VSP are pointed out. Top scoring HADDOCK cluster is shown in red (panel c) and the other in purple (four best structures, panel d). Their respective scores and statistical parameters are shown.

Extended Data Fig. 8 Lowest energy docking model of phospho-peptide interacting with VSP.

a, Molecular docking of PTEN phosphorylated C-tail (aa 353–403) with VSP (PDB:3V0G) showing the lowest energy model. For VSP structure: Cα2 segment in cyan, CBRIII loop in green, active site P-loop in red, and site of previous determined crosslinking in yellow. For phosphorylated C-tail, the black region represents segment of tail not used in NMR experiments whereas magenta is the phospho-cluster, dark blue is the second acidic patch spanning residues 386–390, and orange is the third acidic patch spanning residues 391–394. b, Surface representation of VSP and tetra-phosphorylated PTEN C-tail from HADDOCK model colored as electrostatic potential showing complementarity of the surfaces for binding. Basic surfaces in blue, neutral in white, and acidic in red. c, Residues in the Cα2 segment including Lys553, Lys555 and Lys558 which correspond to Lys330, Lys332 and Arg335 in PTEN that are important for tail binding from Haddock model, respectively (PDB:3V0G). d, Residues in the catalytic domain including Lys364 and Lys367 which correspond to Lys125 and Lys128 in PTEN that are important for tail binding, respectively.

Extended Data Fig. 9 PRE-validation of docking model.

Overlay of 15N-1H-HSQC spectra of VSP in presence of 0.9 molar equivalent 4p-T20(TEMPO) in the oxidized (red) and reduced (blue) forms for select peaks of the C2 domain and corresponding intensity ratios plotted against the primary sequence. Green line indicates a ratio of 1, expected for residues away from the spin label. Red line indicates 1*StDev. Segments of 2 or more residues with intensities below the red line are expected in a 20–25 Å radius from the spin label. Notable regions of VSP for their strong interaction or biological relevance are shown in grey areas.

Source data

Extended Data Fig. 10 Role of N-terminal segment of PTEN in phospho-tail and PIP2 binding.

a, Overlay of 1D 15N-1H-HMQC spectra of 15N-aa1-16-ssPTEN (PTEN N-term, blue) and free 16-mer peptide (green). Signal of PTEN N-term is broadened and more dispersed indicating that this segment is stably interacting with the core of PTEN and adopts a more structured conformation. b, Combined chemical shift perturbations plotted for assigned residues of 15N-aa1-16-ssPTEN in absence (blue) and presence (red) of 2 molar equivalent tetra-phosphorylated C-tail, referenced to the peak position of the free 16-mer peptide. Tetra-phosphorylated tail drives PTEN N-term in a conformation/binding mode more closely resembling a free, disordered peptide. c, Overlay of 15N-1H-HMQC spectra of 15N-aa1-16-ssPTEN (PTEN N-term) in absence (blue) and presence (orange) of ~0.5 molar equivalent PIP2. Available assignments are shown. PTEN-N-term is sensitive to the presence of PIP2, as seen from small chemical shift perturbations and large peak broadening. d, Binding constants for PTEN and VSP N-terminal mutants with fluorescein-labeled tetraphosphorylated C-tail determined by measuring the change in fluorescence anisotropy. Uncertainty reported as ± SEM, n ≥ 3.

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Dempsey, D.R., Viennet, T., Iwase, R. et al. The structural basis of PTEN regulation by multi-site phosphorylation. Nat Struct Mol Biol 28, 858–868 (2021). https://doi.org/10.1038/s41594-021-00668-5

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