Structure of a 14-3-3 coordinated hexamer of the plant plasma membrane H+ -ATPase by combining X-ray crystallography and electron cryomicroscopy.
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.
Ottmann
Christian
C
Zentrum für Molekularbiologie der Pflanzen, Pflanzenphysiologie, Universität Tübingen, Auf der Morgenstelle 5, 72076 Tübingen, Germany.
Marco
Sergio
S
Jaspert
Nina
N
Marcon
Caroline
C
Schauer
Nicolas
N
Weyand
Michael
M
Vandermeeren
Caroline
C
Duby
Geoffrey
G
Boutry
Marc
M
Wittinghofer
Alfred
A
Rigaud
Jean-Louis
JL
Oecking
Claudia
C
D048948:14-3-3 Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D020816:Amino Acid Motifs; D001665:Binding Sites; D020285:Cryoelectron Microscopy; D018360:Crystallography, X-Ray; D006027:Glycosides / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D008954:Models, Biological; D008958:Models, Molecular; D009154:Mutation; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D014026:Nicotiana / Q000378:metabolism
D048948:14-3-3 Proteins; D006027:Glycosides; D008565:Membrane Proteins; D010940:Plant Proteins; C007808:fusicoccin; D006180:Proton-Translocating ATPases; C430720:PMA4 protein, Nicotiana plumbaginifolia