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Structural basis of protein phosphatase 2A stable latency.

Jiang L, Stanevich V, Satyshur KA, Kong M, Watkins GR, Wadzinski BE, Sengupta R, Xing Y - Nat Commun (2013)

Bottom Line: This structure suggests that α4 binding to the full-length PP2Ac requires local unfolding near the active site, which perturbs the scaffold subunit binding site at the opposite surface via allosteric relay.These changes stabilize an inactive conformation of PP2Ac and convert oligomeric PP2A complexes to the α4 complex upon perturbation of the active site.Our results show that α4 is a scavenger chaperone that binds to and stabilizes partially folded PP2Ac for stable latency, and reveal a mechanism by which α4 regulates cell survival, and biogenesis and surveillance of PP2A holoenzymes.

View Article: PubMed Central - PubMed

Affiliation: McArdle Laboratory, Department of Oncology, University of Wisconsin-Madison, School of Medicine and Public Health, Madison, Wisconsin 53706, USA.

ABSTRACT
The catalytic subunit of protein phosphatase 2A (PP2Ac) is stabilized in a latent form by α4, a regulatory protein essential for cell survival and biogenesis of all PP2A complexes. Here we report the structure of α4 bound to the N-terminal fragment of PP2Ac. This structure suggests that α4 binding to the full-length PP2Ac requires local unfolding near the active site, which perturbs the scaffold subunit binding site at the opposite surface via allosteric relay. These changes stabilize an inactive conformation of PP2Ac and convert oligomeric PP2A complexes to the α4 complex upon perturbation of the active site. The PP2Ac-α4 interface is essential for cell survival and sterically hinders a PP2A ubiquitination site, important for the stability of cellular PP2Ac. Our results show that α4 is a scavenger chaperone that binds to and stabilizes partially folded PP2Ac for stable latency, and reveal a mechanism by which α4 regulates cell survival, and biogenesis and surveillance of PP2A holoenzymes.

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Exclusive binding of α4 and the scaffold subunit.(a) Illustration of allosteric relay from the α4-binding site, the helix switch, the active site loops and the first central β-sheet, to the scaffold subunit-binding sites at the opposite end of the β-sheet. Hypothetical shift of the central β-sheet is shown in green. A portion of the interface between PP2A scaffold and catalytic subunits is shown (right). Structural presentations and colour scheme are similar to Fig. 4a. The scaffold subunit is shown in surface and coloured purple. Residues of the catalytic subunit are in cylinder and coloured green. (b) Time-dependent conversion of PP2A core enzyme to the α4-PP2Ac complex in the presence and absence of 50 μM Mn++ or 2 mM PPi, determined by pull-down via GST-α4. The bound protein samples were visualized on SDS–PAGE by Coomassie blue staining. (c) Time-dependent conversion of stable B′γ1 holoenzyme to the α4-PP2Ac complex in the presence and absence of 2 mM PPi. Experiments were performed similar to b. For panels b and c, experiments were repeated three times; representative results are shown. (d) Schematic diagram of the structural basis for conversion of active PP2A oligomeric complexes to inactive α4 complex. Disturbance of the active site and dislodging of catalytic metal ions (red spheres) lead to relaxation of the helix switch and loop switch, which allows α4 binding and alters the scaffold subunit-binding site at the opposite surface through allosteric changes propagated through central structures. A, B and C stand for PP2A scaffold, regulatory, and catalytic subunits, respectively.
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f5: Exclusive binding of α4 and the scaffold subunit.(a) Illustration of allosteric relay from the α4-binding site, the helix switch, the active site loops and the first central β-sheet, to the scaffold subunit-binding sites at the opposite end of the β-sheet. Hypothetical shift of the central β-sheet is shown in green. A portion of the interface between PP2A scaffold and catalytic subunits is shown (right). Structural presentations and colour scheme are similar to Fig. 4a. The scaffold subunit is shown in surface and coloured purple. Residues of the catalytic subunit are in cylinder and coloured green. (b) Time-dependent conversion of PP2A core enzyme to the α4-PP2Ac complex in the presence and absence of 50 μM Mn++ or 2 mM PPi, determined by pull-down via GST-α4. The bound protein samples were visualized on SDS–PAGE by Coomassie blue staining. (c) Time-dependent conversion of stable B′γ1 holoenzyme to the α4-PP2Ac complex in the presence and absence of 2 mM PPi. Experiments were performed similar to b. For panels b and c, experiments were repeated three times; representative results are shown. (d) Schematic diagram of the structural basis for conversion of active PP2A oligomeric complexes to inactive α4 complex. Disturbance of the active site and dislodging of catalytic metal ions (red spheres) lead to relaxation of the helix switch and loop switch, which allows α4 binding and alters the scaffold subunit-binding site at the opposite surface through allosteric changes propagated through central structures. A, B and C stand for PP2A scaffold, regulatory, and catalytic subunits, respectively.

Mentions: Previous studies suggested that α4 and the scaffold subunit bind mutually exclusively to PP2Ac (5). Strikingly, structures of the nPP2Ac–α4 complex and PP2A core enzyme22 show that α4 and the scaffold subunit bind to opposite sides of PP2Ac and do not overlap (Fig. 5a). We hypothesize that exclusive binding of α4 and the scaffold subunit is mediated by allosteric changes through PP2A inner structures (Fig. 5a). The first central β-sheet of PP2Ac connects to the active site loops and the helix switch at one end, and to the scaffold-binding site at the other end22. Unfolding of helix switch and perturbation of active site loops would lead to shift of the central β-sheet that in turn alters the scaffold subunit-binding site at the opposite end. These changes favour α4 binding but weaken scaffold subunit binding.


Structural basis of protein phosphatase 2A stable latency.

Jiang L, Stanevich V, Satyshur KA, Kong M, Watkins GR, Wadzinski BE, Sengupta R, Xing Y - Nat Commun (2013)

Exclusive binding of α4 and the scaffold subunit.(a) Illustration of allosteric relay from the α4-binding site, the helix switch, the active site loops and the first central β-sheet, to the scaffold subunit-binding sites at the opposite end of the β-sheet. Hypothetical shift of the central β-sheet is shown in green. A portion of the interface between PP2A scaffold and catalytic subunits is shown (right). Structural presentations and colour scheme are similar to Fig. 4a. The scaffold subunit is shown in surface and coloured purple. Residues of the catalytic subunit are in cylinder and coloured green. (b) Time-dependent conversion of PP2A core enzyme to the α4-PP2Ac complex in the presence and absence of 50 μM Mn++ or 2 mM PPi, determined by pull-down via GST-α4. The bound protein samples were visualized on SDS–PAGE by Coomassie blue staining. (c) Time-dependent conversion of stable B′γ1 holoenzyme to the α4-PP2Ac complex in the presence and absence of 2 mM PPi. Experiments were performed similar to b. For panels b and c, experiments were repeated three times; representative results are shown. (d) Schematic diagram of the structural basis for conversion of active PP2A oligomeric complexes to inactive α4 complex. Disturbance of the active site and dislodging of catalytic metal ions (red spheres) lead to relaxation of the helix switch and loop switch, which allows α4 binding and alters the scaffold subunit-binding site at the opposite surface through allosteric changes propagated through central structures. A, B and C stand for PP2A scaffold, regulatory, and catalytic subunits, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3644067&req=5

f5: Exclusive binding of α4 and the scaffold subunit.(a) Illustration of allosteric relay from the α4-binding site, the helix switch, the active site loops and the first central β-sheet, to the scaffold subunit-binding sites at the opposite end of the β-sheet. Hypothetical shift of the central β-sheet is shown in green. A portion of the interface between PP2A scaffold and catalytic subunits is shown (right). Structural presentations and colour scheme are similar to Fig. 4a. The scaffold subunit is shown in surface and coloured purple. Residues of the catalytic subunit are in cylinder and coloured green. (b) Time-dependent conversion of PP2A core enzyme to the α4-PP2Ac complex in the presence and absence of 50 μM Mn++ or 2 mM PPi, determined by pull-down via GST-α4. The bound protein samples were visualized on SDS–PAGE by Coomassie blue staining. (c) Time-dependent conversion of stable B′γ1 holoenzyme to the α4-PP2Ac complex in the presence and absence of 2 mM PPi. Experiments were performed similar to b. For panels b and c, experiments were repeated three times; representative results are shown. (d) Schematic diagram of the structural basis for conversion of active PP2A oligomeric complexes to inactive α4 complex. Disturbance of the active site and dislodging of catalytic metal ions (red spheres) lead to relaxation of the helix switch and loop switch, which allows α4 binding and alters the scaffold subunit-binding site at the opposite surface through allosteric changes propagated through central structures. A, B and C stand for PP2A scaffold, regulatory, and catalytic subunits, respectively.
Mentions: Previous studies suggested that α4 and the scaffold subunit bind mutually exclusively to PP2Ac (5). Strikingly, structures of the nPP2Ac–α4 complex and PP2A core enzyme22 show that α4 and the scaffold subunit bind to opposite sides of PP2Ac and do not overlap (Fig. 5a). We hypothesize that exclusive binding of α4 and the scaffold subunit is mediated by allosteric changes through PP2A inner structures (Fig. 5a). The first central β-sheet of PP2Ac connects to the active site loops and the helix switch at one end, and to the scaffold-binding site at the other end22. Unfolding of helix switch and perturbation of active site loops would lead to shift of the central β-sheet that in turn alters the scaffold subunit-binding site at the opposite end. These changes favour α4 binding but weaken scaffold subunit binding.

Bottom Line: This structure suggests that α4 binding to the full-length PP2Ac requires local unfolding near the active site, which perturbs the scaffold subunit binding site at the opposite surface via allosteric relay.These changes stabilize an inactive conformation of PP2Ac and convert oligomeric PP2A complexes to the α4 complex upon perturbation of the active site.Our results show that α4 is a scavenger chaperone that binds to and stabilizes partially folded PP2Ac for stable latency, and reveal a mechanism by which α4 regulates cell survival, and biogenesis and surveillance of PP2A holoenzymes.

View Article: PubMed Central - PubMed

Affiliation: McArdle Laboratory, Department of Oncology, University of Wisconsin-Madison, School of Medicine and Public Health, Madison, Wisconsin 53706, USA.

ABSTRACT
The catalytic subunit of protein phosphatase 2A (PP2Ac) is stabilized in a latent form by α4, a regulatory protein essential for cell survival and biogenesis of all PP2A complexes. Here we report the structure of α4 bound to the N-terminal fragment of PP2Ac. This structure suggests that α4 binding to the full-length PP2Ac requires local unfolding near the active site, which perturbs the scaffold subunit binding site at the opposite surface via allosteric relay. These changes stabilize an inactive conformation of PP2Ac and convert oligomeric PP2A complexes to the α4 complex upon perturbation of the active site. The PP2Ac-α4 interface is essential for cell survival and sterically hinders a PP2A ubiquitination site, important for the stability of cellular PP2Ac. Our results show that α4 is a scavenger chaperone that binds to and stabilizes partially folded PP2Ac for stable latency, and reveal a mechanism by which α4 regulates cell survival, and biogenesis and surveillance of PP2A holoenzymes.

Show MeSH
Related in: MedlinePlus