Alignment of the protein substrate hairpin along the SecA two-helix finger primes protein transport in Escherichia coli

Mapping of PhoA signal peptide and early mature region on to the SecA–SecYEG complex. (A) Schematic of the SecA–PhoA chimera construct in which the PhoA substrate peptide is genetically fused to SecA after a Gly–Ser linker (not drawn to scale). Cys residues were introduced for dye labeling at the indicated positions and are depicted in blue, green, yellow, and red. (B) Representative fluorescence spectra of the doubly labeled SecA–PhoA chimeras in the presence of SecYEG and ADP, with the donor dye positioned at different points within PhoA and the acceptor dye positioned at SecA residue 321. The spectrum generated with the donor dye at PhoA position 22 has the lowest intensity and highest transfer efficiency. Data were acquired and analyzed as described in SI Materials and Methods. (C) Time-resolved fluorescence decay spectra of the SecA–PhoA chimera labeled with the donor dye at PhoA position 22 and with the acceptor dye at SecA residue 321. A donor-only decay is shown in dark violet and a donor–acceptor decay is shown in light violet. The instrument response function (IRF) is given in gray. The donor–acceptor decay yields a shorter lifetime indicative of energy transfer. Data were acquired and analyzed as described in SI Materials and Methods. (D and E) The T. maritima SecA–SecYEG complex (PDB ID code 3DIN) is shown as a ribbon diagram with SecA and SecYEG in light and dark gray, respectively. The locations of T. maritima residues homologous to E. coli SecA37, SecA321, and SecY292 are shown by magenta-, violet-, or cyan-colored spheres, respectively. Mapped location of the PhoA substrate within the SecA–SecYEG complex in the presence of ADP (D) or ATP-γS (E). The region of overlap of the structure with the FRET data for PhoA residue 2, 22, 37, or 45 is shown in blue, green, yellow, or red, respectively. Overlap regions of PhoA residues 22, 37, and 45 are shown in olive, and overlap regions of 37 and 45 are shown in orange. Figures on the Right are rotated by ≈180°.

Verification of normal ligand binding in the presence of higher salt concentration. (A) SecA binding to the PhoA signal peptide or extended signal peptide labeled with dye at position 22 (SP22 or SP41, respectively) at 300 mM KCl is depicted using a fluorescence anisotropy binding assay as described in SI Materials and Methods. (B) SecA binding to SecYEG in the presence of 300 mM KCl and 0.1% DDM is depicted using a FRET assay that relies on PPXD-HWD separation (given in angstroms) induced by complex formation. For this purpose, 20 nM SecA labeled at residues 321 and 721 within PPXD and HWD, respectively, with appropriate donor and acceptor dyes was mixed with SecYEG at the final indicated concentration in high-salt TKM buffer containing 0.1% DDM at 20 °C. The calculated distances were determined as described in SI Materials and Methods.

Fluorescence spectra of donor–acceptor labeled SecA–PhoA–SecYEG complexes generated with a 488-nm excitation wavelength. For all three sets of FRET pairs examined, the FRET efficiency is not linearly proportional to the position of the label on the PhoA portion of the chimera, consistent with a hairpin loop configuration. (A) All spectra were obtained in the presence of ADP. (Top) Spectra were generated with SecA37–AF647 as the acceptor, and the donor dye (AF488) was located at four different positions on the PhoA portion of the chimera: PhoA2 (blue), PhoA22 (green), PhoA37 (orange), or PhoA45 (red). (Middle) Spectra were generated with SecA321–AF647 as the acceptor and donor dye (AF488) positioned at either PhoA2 (blue), PhoA22 (green), PhoA37 (orange), or PhoA45 (red). (Bottom) The donor dye was located at SecY292–AF488, and the acceptor dye (AF647) was located at either PhoA2 (blue), PhoA22 (green), PhoA37 (orange), or PhoA45 (red). (B) Same as Fig. S2A, except all spectra were generated in the presence of ATP-γS. Spectral acquisition and analysis are described in SI Materials and Methods. FRET efficiencies, distances, and the degree of labeling are given in Tables S2–S4.

Fluorescence intensity decay spectra of SecA–PhoA–SecYEG complexes obtained with 490-nm excitation. Donor-only decays are shown in darker colors, and lighter-colored decays depict decays obtained in the presence of acceptor. In all cases, the presence of the acceptor leads to a faster decay consistent with energy transfer. (Top) The donor dye (AF488) is located at PhoA22, and the acceptor dye is located at SecA37 (AF647). Donor only is shown in dark magenta and the donor–acceptor decay is given in light magenta. Decays were obtained in the presence of ADP. (Middle) The donor dye (AF488) is located at PhoA22, and the acceptor dye is located at SecA321 (AF647). Donor-only is shown in dark violet, and the donor–acceptor decay is given in light violet. Decays were obtained in the presence of ADP. (Bottom) The donor dye was located at SecY292–AF488, and the acceptor dye (AF647) was located at PhoA22. The donor only decay is shown in dark cyan and donor–acceptor decay is shown in cyan. Both decays were obtained in the presence of ADP. The donor–acceptor decay shown in light cyan was obtained in the presence of ATP-γS. For all spectra depicted, the instrument response function (IRF) is shown in gray. Parameters obtained from the fitting of the decays are given in Table S5.

Depiction of the spherical shells (pink dots) used to identify the FRET overlap within the SecA–SecYE structure for PhoA residue 22 within the SecA–PhoA chimera. The shells were generated using distance values obtained in the presence of ATP-γS and are shown on the SecA–SecYE cocrystal structure PDB ID 5EUL. SecA is shown in light gray, SecYE is shown in dark gray, and the OmpA peptide is shown in pink. The width of the shells corresponds to the uncertainty in the FRET distance measurement (given in Tables S2–S4). (A) The shell determined from SecA residue 37 (magenta sphere) within NDB-1, (B) the shell determined from SecA residue 321 (violet sphere) within the PPXD, and (C) the shell determined from SecY residue 292 (cyan sphere) at the bottom of the channel. The intersection of the three spherical shells defines the region ascribed to PhoA residue 22 (green) in this case. The script for determining this intersection in given in SI Materials and Methods.

The B. subtilis SecA–G. thermodenitrificans SecYE cocrystal structure (PDB ID code 5EUL). SecA is shown in light gray, SecYE is in dark gray, and the OmpA peptide substrate inserted at the end of the THF is shown in pink. For clarity, the nanobody crystallized with the complex has been omitted (10). The FRET-mapped regions in the presence of ATP-γS of the beginning portions of the PhoA substrate for residues 2, 22, 37, or 45 are shown in blue, green, yellow, or red, respectively. Mapping was done as described in Fig. S4. Overlap regions of PhoA residues 22, 37, and 45 are shown in olive, and overlap regions of 37 and 45 are shown in orange.

(A) FRET-mapped regions projected on the B. subtilis SecA–Geobacillus thermodenitrificans SecYE cocrystal structure (PDB ID code 5EUL). SecA is shown in light gray, SecYE is in dark gray, and the OmpA peptide substrate inserted at the end of the THF is shown in pink. For clarity, the nanobody crystallized with the complex has been omitted (10). Generation of FRET-mapped regions and their associated colors in the presence of ATP-γS was done as described in Fig. 2. Circled in red is the peptide substrate (residues 749–791) (shown in cyan) excised from the original 5EUL PDB structure and modeled into the mapped regions without any alteration of the original structure. (B) Enlarged view of the modeled peptide (cyan) and mapped locations. Residues 2 (Lys), 22 (Tyr), and 37 (Gly) of the OmpA peptide are shown in a stick representation in blue, green, and yellow, respectively, and exhibit excellent agreement with the PhoA-mapped locations. Note the adjacent C-terminal portion of SecY discussed in the text.

Schematic representation of SecA priming of protein transport. Stage A represents ADP-bound SecA binding to SecYEG, thus forming an inactive SecA–SecYEG binary complex; stages B and C correspond to the recognition and binding of the preprotein substrate to SecA–SecYEG to form the ternary complex whereby the signal peptide (in orange) and early mature region (in dark blue) of the substrate bind to the THF (in cyan) of SecA to adopt their hairpin structure characteristic of the preinsertion state; stages D and E depict the activation of the ternary complex whereby nucleotide exchange and ATP hydrolysis at SecA allows insertion of the substrate hairpin into the SecY channel to adopt the postinsertion state. Subsequent ATP hydrolytic cycles promote SecA ratcheting function, which along with Brownian motion, drive substrate proteins across the SecY channel.