Formation of a β-barrel membrane protein is catalyzed by the interior surface of the assembly machine protein BamA
- Harvard University, United States
Figures
Figure 1 with 1 supplement
The interior wall and lateral gate of the BamA β-barrel form a substrate binding site.
(A–C) Residues at the lateral gate (A), L6 (B), and interior wall (C) of BamA interact with substrate LptD during assembly. MC4100 and lptD4213 (NR698) strains (expressing WT LptD or LptD4213, …
Figure 1—figure supplement 1
Crosslinking of the BamA interior to substrates.
(A) Sequence alignments of all identified photocrosslinking sites in the BamA β-barrel. Residues that interact with substrates are labeled in purple, while residues that do not are labeled in black. …
Figure 2 with 1 supplement
The interior of the BamA β-barrel forms a general substrate binding site.
(A–B) The interior of the BamA β-barrel interacts with a diverse number of wild-type, full-length substrates including (A) LptD and (B) LamB and OmpF. MC4100 strains harboring the amber suppression …
Figure 2—figure supplement 1
Analysis of cellular protein levels of recombinant outer membrane substrates.
(A) Recombinant LptD efficiently assembles into its mature form. Recombinant LamB and OmpF assembles into the mature trimer. MC4100 strains expressing a FLAG-tagged LptD/LamB/OmpF were harvested and …
Figure 3 with 2 supplements
The C-terminal strands of LptD interacts with both BamA and BamD during assembly.
(A) The third-to-last β-strand and final periplasmic loop of substrate LptD interact with BamD during assembly. MC4100 strains harboring both the amber suppression system and expressing a His-tagged …
Figure 3—figure supplement 1
Crosslinking of the C-terminal strands of substrate LptD to BamA and BamD.
(A) Sequence alignment of the C-terminal region of LptD across representative Gram-negative bacteria. Alignments were performed by Clustal Omega using LptD sequences from Escherichia coli (GI: …
Figure 3—figure supplement 2
BamA and BamD bind non-overlapping regions within the C-terminal strands of substrate LptD.
(A) Residues in the third-to-last strand of LptD that interact with BamD during assembly do not interact with BamA. Crosslinking was tested as described in Figure 3. (B) Residues in the last strand …
Figure 4 with 2 supplements
The N-terminal strands of LptD interacts with BamA during assembly.
(A) β-strands four, five, and six of substrate LptD interact with BamA. Crosslinking was tested as described in Figure 3, but with pBPA substitutions in the N-terminal portion of substrate …
Figure 4—figure supplement 1
Crosslinking of the first three strands of substrate LptD to BamA.
(A) β-strands one, two, and three of substrate LptD interact with BamA. Crosslinking was tested as described in Figure 4. (B) Top-down view of the region near the N- and C-termini of LptD showing …
Figure 4—figure supplement 2
Crosslinking of the N-terminal region of substrate LptD to BamA.
(A) Sequence alignment of the N-terminal region of LptD across representative Gram-negative bacteria. Alignments were performed as in Figure 3—figure supplement 1. Residues that crosslink strongly …
Figure 5
The N-terminal strands of the substrate are assembled within the BamA β-barrel.
(A) The lateral gate and interior of the BamA β-barrel interacts with the ends of the LptD substrate. The N-terminal strands of BamA (S439) interacts with the C-terminal strands of substrate LptD, …
Figure 6
Substrate release from the interior wall of BamA allows β-barrel closure, triggering release from the Bam complex.
(A) N274 resides within a large hydrophobic patch that encompasses at least six β-strands at the N-terminal region of the LptD β-barrel. The left panel shows the structure of LptD in cartoon form. …
Figure 7 with 2 supplements
An assembly-defective LptD mutant is rescued by a compensatory mutation in the BamA interior wall.
(A) Expression of LptDΔD330 confers outer membrane permeability defects, while changes in BamA can suppress LptDΔD330 associated-defects. MC4100 or bamAE470G cells were transformed with plasmids …
Figure 7—figure supplement 1
LptDΔD330 partially phenocopies LptD4213-associated assembly defects.
(A) Residue D330 in LptD resides in β-strand seven, and its side chain is oriented into the β-barrel interior. The first and last β-strands are colored in tan, the region deleted in LptD4213 is …
Figure 7—figure supplement 2
Compensatory mutations in BamA can rescue outer membrane permeability defects associated with stalled LptD substrates.
Changes in BamA can suppress LptDΔD330-associated defects. A BamA depletion strain was transformed with a plasmid encoding a BamA variant (including bamAE470G) and another plasmid encoding either WT …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain (Escherichia coli) | MC4100 | (Casadaban, 1976); PMID: 781293 | CGSC#: 6152 | See Supplementary file 1 |
| Strain (Escherichia coli) | NR698 | (Wu et al., 2005); PMID: 15851030 | See Supplementary file 1 | |
| Strain (Escherichia coli) | NR1134 | (Lee et al., 2018); PMID: 29463713 | See Supplementary file 1 | |
| Strain (Escherichia coli) | JCM166 | (Wu et al., 2005); PMID: 15851030 | See Supplementary file 1 | |
| Strain (Escherichia coli) | DEK1 | This paper | See Supplementary file 1 | |
| Strain (Escherichia coli) | DH5α λpir | (Metcalf et al., 1994; Simon et al., 1983) | See Supplementary file 1 | |
| Antibody | Mouse monoclonal Anti-Penta His HRP conjugate | Qiagen | Cat#34460 | (1:5000) |
| Antibody | Mouse monoclonal Anti-FLAG M2 HRP conjugate | Sigma | Cat#A8592; RRID:AB_439702 | (1:50,000) |
| Antibody | Rabbit polyclonal Anti-BamA primary | (Kim et al., 2007); PMID: 17702946 | (1:5000) | |
| Antibody | Rabbit polyclonal Anti-BamD primary | (Kim et al., 2007); PMID: 17702946 | (1:5000) | |
| Antibody | Rabbit polyclonal Anti-LptD primary | (Narita et al., 2013); PMID: 24003122 | (1:5000) | |
| Antibody | Rabbit polyclonal Anti-LptE primary | (Chng et al., 2010); PMID: 22936569 | (1:5000) | |
| Antibody | Rabbit IgG, HRP-linked whole Ab (from donkey) | GE Healthcare | Cat#: NA935 | (1:5000) |
| Recombinant DNA reagent | Plasmids used | This paper | See Supplementary file 2 | |
| Commercial assay | Amersham ECL Western Blotting Detection Kit | GE Healthcare | Cat#:RPN2232 | |
| Chemical compound | p-Benzoylphenylalanine | Bachem | Cat#:4017646 | |
| Chemical compound | 10X Casein blocking buffer | Sigma | Cat#:B6429 | |
| Chemical compound | Anzergent 3–14 | Anatrace | Cat#:AZ314 | |
| Chemical compound | Ni-NTA Superflow | Qiagen | Cat#:30450 | |
| Chemical compound | BMB (1,4-bismaleimidobutane) | ThermoFisher | Cat#:22331 | |
| Chemical compound | L-cysteine hydrochloride | Alfa Aesar | Cat#:L06328 | |
| Chemical compound | TCEP-HCl | VWR | Cat#:97064 | |
| Chemical compound | S35 Methionine | American Radiolabeled Chemicals | Cat#:ARS 104A | |
| Chemical compound | N-ethylmaleimide | Sigma | Cat#:E3876 | |
| Chemical compound | Anti-FLAG M2 magnetic beads | Sigma | Cat#:M8823 | |
| Software | PyMol | Schrodinger | http://pymol.org; RRID:SCR_000305 |
Additional files
-
Supplementary file 1
List of strains used.
- https://cdn.elifesciences.org/articles/49787/elife-49787-supp1-v2.docx
-
Supplementary file 2
List of plasmids used.
- https://cdn.elifesciences.org/articles/49787/elife-49787-supp2-v2.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/49787/elife-49787-transrepform-v2.docx
