title: Conformational variation of the translocon enhancing chaperone SecDF.
abstract: The Sec translocon facilitates transportation of newly synthesized polypeptides from the cytoplasm to the lumen/periplasm across the phospholipid membrane. Although the polypeptide-conducting machinery is formed by the SecYEG-SecA complex in bacteria, its transportation efficiency is markedly enhanced by SecDF. A previous study suggested that SecDF assumes at least two conformations differing by a 120° rotation in the spatial orientation of the P1 head subdomain to the rigid base, and that the conformational dynamics plays a critical role in polypeptide translocation. Here we addressed this hypothesis by analyzing the 3D structure of SecDF using electron tomography and single particle reconstruction. Reconstruction of wt SecDF showed two major conformations; one resembles the crystal structure of full-length SecDF (F-form structure), while the other is similar to the hypothetical structural variant based on the crystal structure of the isolated P1 domain (I-form structure). The transmembrane domain of the I-form structure has a scissor like cleft open to the periplasmic side. We also report the structure of a double cysteine mutant designed to constrain SecDF to the I-form. This reconstruction has a protrusion at the periplasmic end that nicely fits the orientation of P1 in the I-from. These results provide firm evidence for the occurrence of the I-form in solution and support the proposed F- to I-transition of wt SecDF during polypeptide translocation.
authors.affiliation: BRI and ITRI, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, 305-8568, Japan, kazu.mio@aist.go.jp.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Mio
authors.forename: Tomoya
authors.initials: T
authors.lastname: Tsukazaki
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Mori
authors.forename: Masaaki
authors.initials: M
authors.lastname: Kawata
authors.forename: Toshio
authors.initials: T
authors.lastname: Moriya
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Sasaki
authors.forename: Ryuichiro
authors.initials: R
authors.lastname: Ishitani
authors.forename: Koreaki
authors.initials: K
authors.lastname: Ito
authors.forename: Osamu
authors.initials: O
authors.lastname: Nureki
authors.forename: Chikara
authors.initials: C
authors.lastname: Sato
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000648:ultrastructure*; D018360:Crystallography, X-Ray; D055032:Electron Microscope Tomography; D008565:Membrane Proteins / Q000235:genetics / Q000648:ultrastructure*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000648:ultrastructure*; D009154:Mutation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D016963:Thermus thermophilus / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C075767:secD protein, Bacteria; C075768:secF protein, Bacteria