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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.

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Abbreviations

3D:

Three dimensional

CTF:

Contrast transfer function

DDM:

n-Dodecyl-β-d-maltoside

DF:

Dark field

EM:

Electron microscopy

FSC:

Fourier shell correlation

MSA–HAC:

Multivariate statistical analysis in combination with hierarchical ascendant classification

RND:

Resistance-nodulation-cell division

SIRT:

Simultaneous iterative reconstruction technique

SPA:

Single particle analysis

STEM:

Scanning transmission electron microscopy

TEM:

Transmission electron microscopy

TM:

Transmembrane

TSecDF:

Thermus thermophilus SecDF

wt:

Wild type

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Acknowledgments

This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas, Structural basis of cell-signaling complexes mediating signal perception, transduction and responses, by grants from PREST and CREST, from the Ministry of Economy, Trade and Industry, and from the Ministry of Education, Culture, Sports, Science, and Technology.

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Correspondence to Kazuhiro Mio, Osamu Nureki or Chikara Sato.

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Mio, K., Tsukazaki, T., Mori, H. et al. Conformational variation of the translocon enhancing chaperone SecDF. J Struct Funct Genomics 15, 107–115 (2014). https://doi.org/10.1007/s10969-013-9168-4

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Keywords

  • Conformational change
  • EM
  • SecDF
  • Single particle analysis
  • Translocon