Molecular basis for TPR domain-mediated regulation of protein phosphatase 5

Abstract

Protein phosphatase 5 (Ppp5) is a serine/threonine protein phosphatase comprising a regulatory tetratricopeptide repeat (TPR) domain N-terminal to its phosphatase domain. Ppp5 functions in signalling pathways that control cellular responses to stress, glucocorticoids and DNA damage. Its phosphatase activity is suppressed by an autoinhibited conformation maintained by the TPR domain and a C-terminal subdomain. By interacting with the TPR domain, heat shock protein 90 (Hsp90) and fatty acids including arachidonic acid stimulate phosphatase activity. Here, we describe the structure of the autoinhibited state of Ppp5, revealing mechanisms of TPR-mediated phosphatase inhibition and Hsp90- and arachidonic acid-induced stimulation of phosphatase activity. The TPR domain engages with the catalytic channel of the phosphatase domain, restricting access to the catalytic site. This autoinhibited conformation of Ppp5 is stabilised by the C-terminal alphaJ helix that contacts a region of the Hsp90-binding groove on the TPR domain. Hsp90 activates Ppp5 by disrupting TPR-phosphatase domain interactions, permitting substrate access to the constitutively active phosphatase domain, whereas arachidonic acid prompts an alternate conformation of the TPR domain, destabilising the TPR-phosphatase domain interface.

Figure 1

Structure of human Ppp5. Ribbon representation of Ppp5 with the TPR and phosphatase domains coloured blue and pink, respectively. The C-terminal subdomain including the αJ helix is in yellow. Metal ions of the binuclear centre are shown as blue spheres. The figures were produced using PYMOL (http://www.pymol.org).

Figure 2

The TPR domain does not alter the conformation of the phosphatase domain. (A) Superimposition of the isolated phosphatase domain of Ppp5 (cyan) onto the Ppp5 phosphatase domain (salmon) within full-length Ppp5 shows a small shift of the C-terminal subdomain αJ helix (yellow: full-length Ppp5; orange: isolated Ppp5 domain). (B) Superimposition of the TPR domain of full-length (blue) and isolated protein (green).

Figure 3

Phosphatase–TPR domain interactions. (A) The phosphatase domain and C-terminal subdomain are represented with a molecular surface, and the TPR domain as ribbon. Intra-TPR turns form a ridge that inserts into the phosphatase domain catalytic channel, with Glu76 of TPR-2 projecting towards the binuclear metal centre. (B) Glu76 of the TPR-2 interacts with Arg275 and Tyr451 at the catalytic site. Metal ions are indicated as M1 and M2 and side chains of metal ion-binding residues are coloured pink. (C) The αJ helix forms hydrophobic contacts with the TPR domain. Detailed interactions involving Leu493 and Leu494 of the αJ helix with TPR-3 and α7 of the TPR domain are shown. The amide side chain of Gln495 donates hydrogen bonds to the main-chain carbonyls of 489 and 490, stabilising the position of the αJ helix.

Figure 4

Inhibition of Ppp5 by the TPR domain Glu76 residue resembles inhibition of PP1 by toxins and autoinhibition of PP2B. Superimposition of the phosphatase domains of Ppp5, PP1 in complex with toxins microcystin and calyculin A and the autoinhibited state of PP2B shows a conserved anionic group that contacts the invariant catalytic site Arg-phosphate binding and Tyr residues of these phosphatases. For clarity, only the glutamate residue of microcystin and the phosphate group of calyculin A are shown.

Figure 5

Model of Hsp90 activation of Ppp5 based on the structure of the Hop–Hsp70 peptide. The TPR domain (purple) of the Hop–Hsp70 peptide complex is superimposed onto the TPR domain of Ppp5 (blue). Peptide-binding basic residues are conserved in the TPR domains of both proteins, suggesting a related mechanism of peptide binding. The region of the Hsp70 peptide N-terminal to the IEEVD motif overlaps the position of the αJ helix, suggesting that optimal peptide binding to the Ppp5 TPR domain is mutually exclusive with TPR–phosphatase domain interactions. The N- and C-termini of the Hsp70 peptide are indicated by N and C, respectively. The C-terminus of Hsp90 (MEEVD) is predicted to bind to the Ppp5 TPR domain with a similar conformation as seen in the Hop–Hsp70 complex (see text for details).

Figure 6

Hsp90 and Hsp90 peptides activate Ppp5 by binding to its TPR domain. (A) Dose-dependent activation of Ppp5 by full-length yeast Hsp90 and (B) Hsp90 C-terminal peptide. The data plotted are corrected for Ppp5 activity in the absence of activator. ITC plots for Hsp90 peptide interactions with (C) the isolated TPR domain (residues 19–146) and (D) full-length Ppp5.

Figure 7

Arachidonoyl-CoA mediates TPR domain unfolding. Arachidonoyl-CoA affects the folding of the isolated TPR domain. The temperature dependence of the CD signal at 222 nm of amino acids 19–147 Ppp5 was recorded in the presence (closed symbols) and absence (open symbols) of 20 μM arachidonoyl-CoA. The melting temperature is increased from 34.6±0.3 to 39.9±0.2°C by the addition of arachidonoyl-CoA, although the apo-state loses helical content (∼15% of CD signal at 222 nm).