Structural Mechanism Underlying the Specific Recognition between the Arabidopsis State-Transition Phosphatase TAP38/PPH1 and Phosphorylated Light-Harvesting Complex Protein Lhcb1

Abstract

During state transitions, plants regulate energy distribution between photosystems I and II through reversible phosphorylation and lateral migration of the major light-harvesting complex LHCII. Dephosphorylation of LHCII and the transition from state 2 to state 1 requires a thylakoid membrane-associated phosphatase named TAP38 or PPH1. TAP38/PPH1 specifically targets LHCII but not the core subunits of photosystem II, whereas the underlying molecular mechanism of their mutual recognition is currently unclear. Here, we present the structures of Arabidopsis thaliana TAP38/PPH1 in the substrate-free and substrate-bound states. The protein contains a type 2C serine/threonine protein phosphatase (PP2C) core domain, a Mn 2+ (or Mg 2+ ) binuclear center and two additional motifs contributing to substrate recognition. A 15-mer phosphorylated N-terminal peptide of Lhcb1 binds to TAP38/ PPH1 on two surface clefts enclosed by the additional motifs. The first segment of the phosphopeptide is clamped by a pair of tooth-like arginine residues at Cleft 1 site. The binding adopts the lock-and-key mechanism with slight rearrangement of the substrate binding residues on TAP38/PPH1. Meanwhile, a more evident substrate-induced fitting occurs on Cleft 2 harboring the extended part of the phosphopeptide. The results unravel the bases for the specific recognition between TAP38/PPH1 and phosphorylated Lhcb1, a crucial step in state transitions.

A trapped human PPM1A–phosphopeptide complex reveals structural features critical for regulation of PPM protein phosphatase activity

“…For PPM phosphatases, the closely-spaced M1-M2 cluster provides a highly specific binding environment for phosphate ions, pSer-and pThr-peptides, and phosphoprotein substrates (6,12,21). The requirements for specific binding of phosphoproteins are distinct from requirements for catalytic activity, as specific binding of substrates was observed by catalytically inactive mutants that preserve the M1-M2 cluster or in the presence of Ca 2+ ions, as reported previously (29,33,37) and in the present work. The M1 and M2 ions jointly coordinate a bridging water molecule/hydroxide ion that has been proposed to function as the nucleophile in the S N 2 phosphate monoester hydrolase reaction (6,12,21).…”

“…The structural comparison of the free and substrate-bound forms of the PPM1A catalytic domain and the analysis of sequence variation among metazoan homologues support the importance of the first few N-terminal residues, the α2-β5 bridge, and the Flap subdomain in interactions between the enzyme and substrate. Recently, crystal structures of the Arabidopsis thaliana PP2C phosphatase TAP38/PPH1 in the free state and as the D180E mutant in complex with a phosphothreonine-peptide substrate were described (37). Similar to conformational changes of PPM1A residues R33 and R186 described above, the conformations of the TAP38/PPH1 residues R69 and R225 are altered upon binding of the substrate (37).…”

“…PPM1A cat D146E did not exhibit detectable activity for either substrate. The motif 5 aspartate residue (aligning to PPM1A D146) is highly conserved and intolerant of substitution, supporting its proposed role in coordinating a metal ion essential for catalysis (29,(34)(35)(36)(37).We sought a tighter-binding substrate to increase the stability of potential protein-phosphopeptide complexes. Based on the p38α activation loop sequence, the cyclic peptide c(MpSIpYVA) (Fig.…”

“…The absence of detectable enzymatic activity without supplementation with millimolar concentrations of divalent Mg 2+ or Mn 2+ is one of the defining characteristics of the PPM phosphatases (7,35). In eukaryotic and eukaryoticlike bacterial PPM phosphatases, detection of a third metal ion in the active site is correlated with the presence of the sequence GDS in motif 5, corresponding to aa [145][146][147]35,36); mutation of the motif 5 aspartate residue in these phosphatases results in loss of activity (24,(26)(27)(28)(29)37). The location of the D146/D239 subsite suggests that M3 is needed to facilitate protonation of the serine/threonine leaving group by positioning an activated, metal-bound water molecule near the fissile phosphate monoester bond (6,37).…”

Activation of the Stt7/STN7 Kinase through Dynamic Interactions with the Cytochrome b6f Complex

“…Under these conditions the redox state of the photosynthetic PQ pool is altered and the Stt7/STN7 is instrumental in maintaining a proper plastid redox poise. Moreover, this kinase appears to be the major regulator of the phosphorylation of the LHCII proteins because there is no evidence that the activity of the PPH1/TAP38 phosphatase, the counteractor of STN7, is regulated, suggesting that it is constitutively active (Shapiguzov et al, 2010;Wei et al, 2015). Our study has revealed new features of the Stt7/STN7 protein kinase that have to be taken into account for modeling this activation process.…”