Supplemental Information. Tunnel Formation Inferred from the I-Form. Structures of the Proton-Driven. Protein Secretion Motor SecDF

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1 Cell Reports, Volume 19 Supplemental Information Tunnel Formation Inferred from the I-Form Structures of the Proton-riven Protein Secretion Motor SecF rata Furukawa, Kunihito Yoshikaie, Takaharu Mori, Hiroyuki Mori, Yusuke V. Morimoto, Yasunori Sugano, Shigehiro Iwaki, Tohru Minamino, Yuji Sugita, Yoshiki Tanaka, and Tomoya Tsukazaki

2 C Figure S1 Comparison of the I forms of SecF and the pseudosymmetrical transmembrane segments, Related to Figure 1 () The crystal structures of SecF (4.0 Å structure, orange;, green; Mol, pink; Mol, purple) were superposed based on the Cterminal TM segments TM7 12. The introduced S-S bond is coloured red. () The membrane region of (coloured) is cross-sectioned at the middle of the TM region viewed from the periplasm (Peri). The asterisk indicates the pseudo-symmetrical axis. The triangular frames indicate the pseudo-symmetrical units. (C) TM1-6 (red), P1-base and TM7-12 (purple), and P4 of are superimposed with the structurally corresponding Cα atoms of the transmembrane regions. The TM regions are numbered. Cyto, cytoplasm. () P1 domain superimposition. The P1-head domain in I form fluctuates even in the crystal (4.0 Å structure, orange;, green; Mol, pink; Mol, purple, P1 domain fragment (P I 3QO), yellow). () RMSs of the transmembrane segments among Mol, and C.

3 Figure S2 Sequence similarities among SecFs, Related to Figure 1 () Sequence alignment of rsecf, TtSecF, and csec. () Sequence alignment of rsecf, TtSecF, and csecf. The T-Coffee sequence alignment of 11 bacterial Secs and SecFs* is shown in CLUSTL W format (Thompson et al., 1994). The alignment at TM1 was manually modified based on the crystal structures. Secondary structures of rsecf are indicated by a for α-helices and b for β-sheets. Residues indicated by asterisks (red) and colons (orange) are perfectly and highly conserved, respectively. Transmembrane regions are shown in the same colours used in Figure 1. The closed triangles indicate conserved 365 and Y662. * mino acid sequences of the Secs and SecFs of the following bacteria were aligned by T-Coffee: einococcus radiodurans, Thermus thermophilus, scherichia coli, quifex aeolicus, acillus subtilis, Haemophilus influenzae, Pseudomonas aeruginosa, Salmonella choleraesuis, Staphylococcus aureus, Vibrio cholera, and Yersinia pestis.

4 C Figure S3 Polypeptide interaction in the cavity of the SecF P1-head, Related to Figure 1 () Close-up view of the cavities of the P1-head. The corresponding residues in. coli SecF are indicated in parentheses. The Fo-Fc electron density map (3.0 σ) in the cavity is shown in blue. () Formation of translocation intermediates (upper) using pp mutants and a 35S-labelled substrate, proomp(l59), which has two cysteine residues that form an intracellular disulfide bond. The loop hampers protein translocation, as shown in (C). Translocated regions of proomp intermediate were detected as protease-resistant bands. ccumulation of the full-length pp derivatives in the cells (lower). *Unrelated samples. (C) Schematic depiction of the photo-crosslinking and reaction schemes. (), () Photo-crosslinking between Sec with pp and 35S-labelled proomp. Samples were either directly analysed by SSPG and phosphor-imaging () or first immunoprecipitated (IP) with an α-sec antibody before electrophoresis () without proteinase K. Solid triangles indicate specifically observed crosslinked products.

5 I form (Mol) Occupied F form (3QP) Reduced C P1 domain fragment (3QO) mpty Ligand Q253(R407) I form (Mol) mpty I form () mpty F Mol Tunnel Open G Mol Tunnel Closed H Tunnel Closed I 3QP Tunnel Closed 5 H406 (Sec T560) 6 N439 (Sec 593) (Sec 519) Y662 (SecF Y209) 10 Figure S4 Cut-away models of the I and F forms of SecF, Related to Figure 2 () () Cut-away models of the surface representations of the P1-head cavities along the dotted line in Figure S3. Mol in the I form (); F form (P I code 3QP) (); P1 domain fragment (P I code 3QO) (C); Mol in the I form () and in the I form (). PG captured by the cavity in Mol is coloured red; Q253, corresponding to csecf R407, which is crosslinked to the substrate in Figure S3, is coloured green. The corresponding positions of PG and Q253 on the 3QP, 3QO, Mol, and P1 domain fragment structures are coloured red (with 50% transparency as a ghost model) and green, respectively, and were generated by the superimposition of each P1- head region. (F) (I) Cut-away models of the surface representations of the tunnel through the TM region along the plane including Cα of 365, viewed from the periplasmic side. Mol (F); Mol (G); (H) and F form (P I code 3QP) (I). Residues located at the tunnel surface at the cut-away point are shown. The corresponding residues in. coli SecF are indicated in parentheses. TM4, 5, 6, and 10 are coloured as in Figure 1.

6 C Peri. TM5 Mol M (365) Mol M (365P) Cyto. Mol Mol M simulation (eprotonated 365) M simulation (Protonated 365) eprotonated 365 Protonated 365 M 200 ns (run1) Mol Y ( ) Mol F Crystal structures G X ( ) Sec precursor MP mature Omp precursor mature MP translocation (%) Omp translocation (%) eprotonated 365 Sec Cys less Y209F Y209N Y209Q Y209 Protonated 365 Figure S5 Structural dynamics of SecF, Related to Figure 3 () Mol, Mol, and were superimposed based on the transmembrane segments and are coloured as in Figure S1 (left). () SecF (deprotonated 365) structure after 200-ns M simulation, (C) SecF (protonated 365) structure after 200-ns M simulation. The M structures are shown in red and superposed on. Close-up views of TM5 (right). The distance between the Cα atoms of S409 (TM5) in and Mol or in each M simulation structure at 200 ns is shown. Cyto, cytoplasm; Peri, periplasm. () Comparison between the crystal structures Mol (red) and (gray). The membrane regions of Mol and are viewed from the periplasm. () Projection of the trajectories of Ca atoms in TM4, 5, 6, 7, and 10 onto the XY plane, where TM4, 6, 7, and 10 were fitted to the initial structure. Magenta and green dots indicate the instantaneous position of Ca atoms in the 365 deprotonation and 365 protonation simulations (every 40 ps), respectively. TM helices in the X-ray crystal structures are illustrated with black () and red (Mol) solid lines. (F) Functional analysis of mutations in csecf(y209), corresponding to rsecf(y662). (upper) Complementation of sec1 (Cs) growth by the indicated mutants. Cells were spotted onto L-agar plates supplemented with 0.4% glucose and 50 mm Na-citrate ph 5.0. The accumulation of SecF was detected by immunoblotting after cultivation in L medium supplemented with 0.4% glucose and 50 mm Na-citrate ph 5.0. (middle) Translocation analysis of csecf mutants. Cells [sec1(cs)] expressing H-Sec variants were subjected to the procedures described in XPRIMNTL PROCURS. (lower) ccumulation of Sec in the cells was detected using anti-h immunoblotting. (G) Fluctuation of conserved Y662 residue conformation in the SecF tunnel in the M simulations. The crystal structures of Mol and Mol are superimposed onto that of. lue dots represent the trajectory of the oxygen atom in the side chain of Y662, obtained from the 200-ns M simulations for deprotonated and protonated 365. These dots were output every 10ps by fitting the TM helices. In the M simulations, the side chain of Y662 underwent a conformational transition between two orientations as observed in the crystal structures.

7 P1-head Peri. P4 P1-base L268C (Sec L422C) I143C (Sec I272C) Peri. 585 (SecF 132) R285 (Sec R439) Cyto. Cyto. TT + TT S-S form 90 Sec 64 S-S form SecF (rsecf) Cys-less (R439C)F F(132C) (R439C)F(132C) Cys-less (R439C)F F(132C) (R439C)F(132C) Cys-less (R439C)F F(132C) (R439C)F(132C) C WT 365N * * MP translocation (%) SecF (csecf) WT Sec(519N)F I143C,L268C Sec(I272C, L422C)F Y662 SecF(Y209) Y662F SecF(Y209F) WT WT 365N I143C,L268C Sec(I272C, L422C)F F β-m ph9 precursor MP mature Omp precursor mature MP translocation (%) Omp translocation (%) S-S form Sec I ph Cys-less (R439C) F(132C) 47 Figure S6 Importance of P1 and P4 dynamics, Related to Figure 4 () (C) Proton influx activity and the importance of P1 flexibility. The introduced Cys residues on the crystal structure (Mol) (). The corresponding residues in. coli SecF are indicated in parentheses. The intracellular ph of. coli L21 cells overexpressing rsecf was determined at external ph 5.5 using the fluorescence of phluorin, a ratiometric ph indicator (). The cellular ph of SecF overexpressed cells was sensitive for lower ph environment. The inactive mutant 365N (Tsukazaki et al., 2011) was used as a negative control. rror bars indicate the S.. TT was added to reduce the disulphide bond between I143C and L268C, which were introduced to fix the flexible P1-head domain. ccumulation of SecF in the cells was detected using anti-poly-his immunoblotting. WT, wild-type. The protein translocation efficiency using corresponding. coli SecF mutants (Tsukazaki et al., 2011) is shown as a reference (C). * The protein translocation efficiency of Y209 mutants was measured in Figure S5. () (F) Importance of P4 and P1-base dynamics. Close-up view of the boundary between P1-base and P4 (Mol) (). The displayed residues replaced with Cys are shown as red representations. The corresponding residues in. coli SecF are indicated in parentheses. Complementation of the sec1(cs) growth defect by double-cys mutants (). Cells were spotted onto L-agar plates supplemented with 1 mm IPTG, 50 mm Tris-HCl (ph 7.0 or 9.0) and 50 µm FeSO 4 (for ph 9.0). isulphide bond formation and expression of SecF were detected by immunoblotting after cultivation in L medium supplemented with 1 mm IPTG, 50 mm Tris-HCl (ph 7.0 or 9.0) and 50 µm FeSO 4 (for ph 9.0). SS-PG was performed under nonreducing (+iodoacetamide (I)) or reducing (+β-m) conditions. The S-S bond formation prevented SecF activity. Protein translocation analysis of csecf mutants (F). Cells [sec1(cs)] expressing H-Sec variants were subjected to the procedures described in XPRIMNTL PROCURS (upper). TT was added to reduce the disulphide bond. isulphide bond formation in vivo is shown (lower). Cross linked signals are indicated with black dots.

8 Movie S1 Formation of the hydrogen-bonded single-file water chain along the tunnel of SecF, Related to Figure 3 Water chain formation was observed three times in the 200-ns M simulation for deprotonated 365 (see Figure 3). The movie (56-65 ns), corresponds to the first event, which occurred around 62 ns. References Lovell, S.C., avis, I.W., rendall, W.., 3rd, de akker, P.I., Word, J.M., Prisant, M.G., Richardson, J.S., and Richardson,.C. (2003). Structure validation by Calpha geometry: phi,psi and Cbeta deviation. Proteins 50, Thompson, J.., Higgins,.G., and Gibson, T.J. (1994). CLUSTL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic cids Res. 22, Tsukazaki, T., Mori, H., chizen, Y., Ishitani, R., Fukai, S., Tanaka, T., Perederina,., Vassylyev,.G., Kohno, T., Maturana,.., et al. (2011). Structure and function of a membrane component SecF that enhances protein export. Nature 474,

9 Table S1. ata collection and refinement statistics of SecF crystals, Related to Figure 1 ata Collection rsecf (WT) rsecf (S-S mutant ) () Wavelength Space group P2 1 C2 P Cell dimensions rsecf (S-S mutant) (Mol, Mol) a, b, c ( ) 93.3, 62.3, , 69.8, , 73.8, α, β, γ ( ) 90.0, 101.7, , 94.9, , 90.0, 90.0 Resolution ( ) ( ) ( ) ( ) Unique reflections 6,301(127) 28,645 (1,409) 47,548 (2,054) Total reflections 182, , ,654 Completeness (%) 36.0 (14.5) 99.0 (98.9) 97.8 (86.4) I/σ (I) 5.5 (0.48) 15.1 (2.2) 12.0 (1.7) Redundancy 2.5 (2.1) 10.2 (6.5) 6.4 (5.0) R sym * 0.16 (0.82) 0.22 (0.83) 0.14 (0.56) CC(1/2) 85.4 (37.9) 92.8 (73.0) 95.5 (81.3) Refinement Resolution ( ) ( ) ( ) ( ) No. reflections 6,267 28,635 45,733 R work / R free 0.333/0.402 (0.374/0.489) 0.203/0.258 (0.224/0.321) 0.212/0.268 (0.243/0.317) No. atoms Protein 9,852 5,545 11,035 Water Others factors ( 2 ) Protein Water Others R.M.S. deviations ond lengths ( ) ond angles ( ) Ramachandran plot statistics (%) Favored regions llowed regions isallowed regions The X-ray diffraction data were processed using HKL2000 (HKL Research). Ramachandran plots were calculated with RMPG (Lovell et al., 2003). The values in parentheses are for highest resolution shell. *R sym =Σ I avg -I i /ΣI i