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Structural basis of Sec-independent membrane protein insertion by YidC


Newly synthesized membrane proteins must be accurately inserted into the membrane, folded and assembled for proper functioning. The protein YidC inserts its substrates into the membrane, thereby facilitating membrane protein assembly in bacteria; the homologous proteins Oxa1 and Alb3 have the same function in mitochondria and chloroplasts, respectively1,2. In the bacterial cytoplasmic membrane, YidC functions as an independent insertase and a membrane chaperone in cooperation with the translocon SecYEG3,4,5. Here we present the crystal structure of YidC from Bacillus halodurans, at 2.4 Å resolution. The structure reveals a novel fold, in which five conserved transmembrane helices form a positively charged hydrophilic groove that is open towards both the lipid bilayer and the cytoplasm but closed on the extracellular side. Structure-based in vivo analyses reveal that a conserved arginine residue in the groove is important for the insertion of membrane proteins by YidC. We propose an insertion mechanism for single-spanning membrane proteins, in which the hydrophilic environment generated by the groove recruits the extracellular regions of substrates into the low-dielectric environment of the membrane.

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Figure 1: Overall structure of YidC.
Figure 2: In vivo functional analyses of YidC.
Figure 3: Substrate binding to the hydrophilic groove.
Figure 4: Proposed model for membrane insertion of a single-spanning membrane protein.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates and structure factors for YidC27–266 and YidC27–267 have been deposited in the Protein Data Bank under accession numbers 3WO6 and 3WO7, respectively.


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We wish to thank K. Watanabe from Shoko Scientific for assistance with the SEC-MALLS experiments; T. Nishizawa, T. Higuchi, H. E. Kato, M. Hattori, R. Ishii and H. Nishimasu for discussions; A. Kurabayashi, H. Nakamura, S. Hibino, T. Takino and C. Tsutsumi for technical support; A. Nakashima and R. Yamazaki for secretarial assistance; the RIKEN BioResource Center for providing B. halodurans genomic DNA; the RIKEN Integrated Cluster of Clusters (RICC) for providing computational resources; and the beamline staff members at BL32XU of SPring-8 for technical assistance during data collection. The synchrotron radiation experiments were performed at BL32XU of SPring-8 (proposal no. 2011A1125, 2011A1139, 2011B1062, 2011B1280, 2012A1093, 2012A1201, 2012B1146, 2012B1162 and 2013A1128), with approval from RIKEN. This work was supported by the Platform for Drug Discovery, Informatics and Structural Life Science by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), by JSPS KAKENHI (grant no. 20247020, 20523517, 24687016, 24102503, 24121704, 24227004, 24657095, 25291006, 25291009 and 25660073), by the FIRST program, by PRESTO, by the JST, by a Grant-in-Aid for JSPS Fellows, by a grant for the HPCI STRATEGIC PROGRAM Computational Life Science and Application in Drug Discovery and Medical Development from MEXT, and by grants from the Private University Strategic Research Foundation Support Program (MEXT), the Nagase Science and Technology Foundation, and the Astellas Foundation for Research on Metabolic Disorders.

Author information

Authors and Affiliations



K.K. performed the crystallization and structure determination. S.C. performed the genetic analyses. K.K., A.F., K.-I.N., Y. Sugano, A.D.M., Y.T., H.M. and T.T. performed the functional analysis. M.T., T.M., Y. Sugita and R.I. performed the molecular dynamics simulation. K.K., N.D. and F.A. identified the molecular mass. K.H., Y.N.-N., R.I., T.T. and O.N. assisted with the structure determination. K.K., S.C., K.I., R.I., T.T. and O.N. wrote the manuscript. T.T. and O.N. directed and supervised all of the research.

Corresponding authors

Correspondence to Tomoya Tsukazaki or Osamu Nureki.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Multiple amino acid sequence alignment of YidC proteins.

Sequence alignment of Bacillus halodurans YidC2 (BhYidC2), B. halodurans YidC1 (BhYidC1), Bacillus subtilis SpoIIIJ (BsSpoIIIJ), B. subtilis YidC2 (BsYidC2) and Escherichia coli YidC (EcYidC). The secondary structure of YidC27–266 is indicated above the sequences. The α-helices (as described in the main text) and β-strands (ES1 and ES2 in the E2 region) are indicated by cylinders and arrows, respectively. Strictly conserved residues among the five molecules are highlighted in red boxes, and highly conserved residues are indicated by red letters. The hydrophilic and bulky residues that were mutated and the pBpa positions introduced into B. halodurans YidC2 are indicated by grey, green and blue triangles, respectively. The spoIIIJ K248stop derivative has a stop codon introduced at position 248, as indicated, and thereby expresses a SpoIIIJ mutant that lacks the C-terminal 14 residues.

Extended Data Figure 2 Electron density map of B. halodurans YidC.

Stereo view of the 2mFO – DFC electron density map of the TM2 helix, contoured at 1.1 σ, where m is the figure of merit and D is the SIGMA-A weighting factor. FO and FC are the observed and the calculated structure factor amplitudes, respectively.

Extended Data Figure 3 Monomeric B. halodurans YidC.

a, The crystal packing of YidC27–266, viewed from the plane of the membrane. The molecule in the asymmetric unit is coloured red. b, The crystal packing of YidC27–267, viewed from the plane of the membrane. Two molecules (Mol A in light pink and Mol B in light blue) are in the asymmetric unit. c, The chromatograms show the ultraviolet (UV), refractive index (RI) and light scattering (LS) detector readings. The volume delays of UV between the other detectors were corrected. The traces were normalized to the peak maxima. The green and blue lines in the LS chromatogram indicate the calculated molecular masses of the protein–detergent complex and the protein, respectively. d, The RI of the mixture was measured in response to five concentration steps. The refractive index increment (dn/dc) of the mixture of DDM and CHS was determined using linear regression of the RI versus the concentration. e, The molecular mass values determined by SEC-MALLS and calculated from the amino acid sequence.

Extended Data Figure 4 Structural flexibility of the hydrophilic groove and C1 region.

a, b, Superimposition of the crystal structures of YidC27–266 (coloured) and Mol B of YidC27–267 (grey), viewed from the plane of the membrane (a) and from the extracellular space (b). The conformational changes observed in CH1 and CH2 are indicated by black arrows. c, Close-up view of the C1 region. The side chains of P111 are shown by stick models. In the YidC27–267 structure, the arrangement of the C1 region with respect to the core region is rotated by 35° compared with that in the YidC27–266 structure. As a result, the tip of the C1 region is displaced by 10 Å in the YidC27–267 structure. d, Close-up views of the hydrophilic groove (left, YidC27–266; right, Mol B of YidC27–267). The distances between the Cα atoms of C136 and M221 are indicated by dashed lines.

Extended Data Figure 5 The hydrophobic core of B. halodurans YidC.

a, The crystallographic B-factors are coloured in a gradient varying from blue to red, representing 30 to 140 Å2. b, c, Stereo views of the hydrophobic core, showing the side chains of the hydrophobic residues.

Extended Data Figure 6 Molecular dynamics simulation of B. halodurans YidC for 1,000 ns in a lipid bilayer.

a, Snapshots of the structure over the time course of the simulation at 400 ns intervals: 0 ns (blue), 400 ns (magenta) and 800 ns (light green). b, Root mean square fluctuation (r.m.s.f.) of YidC during the simulation. The secondary structure of YidC is indicated below the line. c, The water probability density map in the simulation, contoured at 0.001 molecules Å−3 ns−1.

Extended Data Figure 7 Gene structures of the strains and YidC mutants used for in vivo genetic analyses.

a, Schematic representations of the gene structures of the yidC2–lacZ reporter strains used for the MifM-based assay. spoIIIJ*-flag indicates either wild type or mutant spoIIIJ. yidC2-lacZ represents a translational gene fusion with the lacZ sequence in-frame after the sixth codon of yidC2. The native mifM-yidC2 on the chromosome remained intact. b, Deleted regions of SpoIIIJ, viewed from the extracellular side. Residue numbers in SpoIIIJ are indicated. Δ92–126-GG represents a mutant in which the entire C1 region has been replaced by a glycine–glycine linker. Δ97–103/Δ114–120 represents a mutant in which both the CH1 and CH2 helices have been shortened by seven residues. c, Schematic representations of the gene structures used for growth complementation assays. SpoIIIJ becomes essential for the growth of B. subtilis when yidC2 is disrupted. Cells with a disruption of chromosomal yidC2 were transformed with the rescue plasmid pCH1805, which expresses wild-type spoIIIJ-flag under the control of the IPTG-inducible Pspac promoter. The native spoIIIJ on the chromosome was replaced by either wild-type or mutant spoIIIJ (spoIIIJ*-flag). In the absence of IPTG, spoIIIJ-flag is not expressed from the plasmid, making the chromosomal spoIIIJ*-flag the only source of cellular YidC. The complementation test measures the global role of SpoIIIJ in inserting a wide range of membrane proteins, including single-spanning and multi-spanning membrane proteins.

Extended Data Figure 8 Schematic explanation of β-galactosidase activity assay and MifM insertion activity.

a, b, MifM is a single-spanning membrane protein, and its membrane insertion is considered to be mediated by YidC (SpoIIIJ)21. To evaluate the MifM insertion activity of SpoIIIJ, we performed a genetic analysis using B. subtilis. In B. subtilis, SpoIIIJ is constitutively expressed, whereas YidC2 is expressed only when the SpoIIIJ activity is compromised, by the following mechanism. The expression of yidC2 is regulated by the upstream cis regulator open reading frame of mifM, which is co-transcribed with yidC2. During the synthesis of MifM, the C-terminal region of nascent MifM interacts with the peptide exit tunnel of the ribosome and causes translational arrest40,50. When the SpoIIIJ activity is normal, the translational arrest is released by the SpoIIIJ-dependent membrane insertion of MifM. Therefore, the translational arrest is transient or does not occur (a). By contrast, when SpoIIIJ activity is compromised, MifM is not inserted into the membrane, and its translation is arrested, which causes ribosome stalling. The stalled ribosome disrupts the downstream stem–loop structure and exposes the Shine–Dalgarno (SD) translation initiation signal sequence of the yidC2 messenger RNA (b). Thus, we can estimate the in vivo SpoIIIJ activity by measuring the expression of the introduced yidC2-lacZ fusion (Extended Data Fig. 7a): the reduction of MifM insertion efficiency by SpoIIIJ elevates the LacZ activity21,50.

Extended Data Figure 9 Effects of N-terminal negatively charged residues of substrates on insertion.

a, Schematic representations of the N-terminal negatively charged residues of MifM and the Pf3–MifM chimaeric protein. b, Membrane insertion efficiencies of MifM mutants and Pf3–MifM mutants. The efficiencies were determined by the LacZ activities (mean ± s.d., n = 3). The N-terminal negatively charged residues of MifM and Pf3–MifM and the numbers of the charged residues are shown at the bottom (EDD, wild-type MifM; DD, wild-type Pf3–MifM). Mutations of the acidic residues in the Pf3 coat protein had less pronounced effects than mutation of those in MifM, probably because the membrane insertion is facilitated by multiple interacting factors depending on the amino acid sequence.

Extended Data Table 1 Data collection and refinement statistics

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Kumazaki, K., Chiba, S., Takemoto, M. et al. Structural basis of Sec-independent membrane protein insertion by YidC. Nature 509, 516–520 (2014).

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