Structure of a 14-3-3 Coordinated Hexamer of the Plant Plasma Membrane H+-ATPase by Combining X-Ray Crystallography and Electron Cryomicroscopy

Abstract

Regulatory 14-3-3 proteins activate the plant plasma membrane H(+)-ATPase by binding to its C-terminal autoinhibitory domain. This interaction requires phosphorylation of a C-terminal, mode III, recognition motif as well as an adjacent span of approximately 50 amino acids. Here we report the X-ray crystal structure of 14-3-3 in complex with the entire binding motif, revealing a previously unidentified mode of interaction. A 14-3-3 dimer simultaneously binds two H(+)-ATPase peptides, each of which forms a loop within the typical 14-3-3 binding groove and therefore exits from the center of the dimer. Several H(+)-ATPase mutants support this structure determination. Accordingly, 14-3-3 binding could result in H(+)-ATPase oligomerization. Indeed, by using single-particle electron cryomicroscopy, the 3D reconstruction of the purified H(+)-ATPase/14-3-3 complex demonstrates a hexameric arrangement. Fitting of 14-3-3 and H(+)-ATPase atomic structures into the 3D reconstruction map suggests the spatial arrangement of the holocomplex.

Structural Basis for the 14-3-3 Protein-dependent Inhibition of the Regulator of G Protein Signaling 3 (RGS3) Function

“…S1). Furthermore, the N-terminal part of RGS3 contains a phosphorylated 14-3-3 binding motif (sequence RRRTHpS 264 EGS), which is presumably docked in the ligand binding groove of 14-3-3, as has been observed in other structures of 14-3-3 protein complexes (21,22,24,25,44). Therefore, the rigid body modeling of the 14-3-3⅐pRGS3 complex was performed using the crystal structures of 14-3-3 (21) and RGS domain of RGS3 (27) (Protein Data Bank (PDB) codes 1qjb and 2oj4, respectively).…”

“…Despite the large number of 14-3-3 binding partners, only two crystal structures of 14-3-3 complexes with the protein ligand of a size that exceeds the consensus recognition motifs have been solved: the 14-3-3⅐serotonin N-acetyltransferase (AANAT) complex (24) and the 14-3-3⅐C-terminal region of the plant plasma membrane H ϩ -ATPase complex (25). These structures revealed that the primary interactions between 14-3-3 and its protein ligand take place within the ligand binding groove where the segment containing the recognition motif binds, similarly as seen in complexes of 14-3-3 isoforms with short phosphopeptides (21,22,44).…”

Molecular Mechanism of 14-3-3 Protein-mediated Inhibition of Plant Nitrate Reductase

“…In most cases, 14-3-3 proteins are believed to trigger conformational changes in their targets, leading to alterations in activity, stability, oligomerization, or cellular localization of binding partners (36). Although numerous crystal structures of 14-3-3 proteins have been determined in both the apopeptide-bound and the phospho-peptide-bound state (37), to date, their specific mode of action has been established in only two cases: the increase in substrate affinity of serotonin N-acetyltransferase in the 14-3-3 complexed state (33) and the oligomerization of plant plasma membrane ATPase (34).…”

β2 integrin phosphorylation on Thr758 acts as a molecular switch to regulate 14-3-3 and filamin binding

“…Thus, 14-3-3 could act by inducing integrin clustering, in a similar way that has been observed for 14-3-3s to induce hexamer formation of a P-type H ϩ -ATPase. 38 On the other hand, 14-3-3 could induce some further interactions of the integrin cytoplasmic domain, for instance by favoring certain conformations of the tail. It is apparent that Rac1/Cdc42 activation is downstream of 14-3-3-integrin interaction.…”