Structure of dual BON-domain protein DolP identifies phospholipid binding as a new mechanism for protein localisation
- Institute of Microbiology and Infection, University of Birmingham, United Kingdom
- School of Biosciences, University of Birmingham, United Kingdom
- Institute for Cancer and Genomic Sciences, University of Birmingham, United Kingdom
- Infection & Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Australia
- Department of Chemistry, National University of Singapore, Singapore
- Institute for Molecular Bioscience, University of Queensland, Australia
- School of Life Sciences, The University of Warwick, United Kingdom
- Institute of Inflammation and Immunotherapy, University of Birmingham, United Kingdom
- Department of Biochemistry, University of Alberta, Canada
Figures
Figure 1 with 5 supplements
DolP is a conserved BON-domain protein with a distinct role in OM homeostasis.
(A) In E. coli, dolP is located downstream of diaA and encodes a lipoprotein with a signal sequence (orange) and two BON domains (red). The signal sequence is cleaved by LspA, the cysteine at …
Figure 1—figure supplement 1
DolP is an OM lipoprotein.
(A) OM fractions of E. coli BW25113, an isogenic ∆dolP mutant and the complemented mutant were analysed by SDS-PAGE and Western immunoblotting with antibodies to DolP and the known OM lipoproteins …
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Figure 1—figure supplement 1—source data 1
Subcellular localisation of DolP.
- https://cdn.elifesciences.org/articles/62614/elife-62614-fig1-figsupp1-data1-v2.pptx
Figure 1—figure supplement 2
BON domain (Pfam: PF04972) containing proteins.
The Pfam database was interrogated for the presence of proteins containing BON domains. BON domains are widely distributed in bacteria and eight major architectures are noted (Table 1). The …
Figure 1—figure supplement 3
DolP has a distinct function from OsmY and Kbp.
The precise functions of Kbp and OsmY are unknown, though both are induced during adaptation to hyperosmolarity (Yan et al., 2019; Yim and Villarejo, 1992; Weber et al., 2006; Ashraf et al., 2016; Le…
Figure 1—figure supplement 4
Phenotypes of E. coli BW25113 ∆dolP.
(A) Mutants lacking dolP are sensitive to the anionic detergents cholate and deoxycholate (B) Mutants lacking dolP have growth rates that are indistinguishable from wild-type E. coli. (C) Scanning …
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Figure 1—figure supplement 4—source data 1
Comparison of bacterial growth rates of wild type and yraP mutant.
- https://cdn.elifesciences.org/articles/62614/elife-62614-fig1-figsupp4-data1-v2.xlsx
Figure 1—figure supplement 5
Localisation of DolP to the OM is required for function.
The signal sequence and domain architecture of DolP are shown. The sequence changes to pET17b-dolPWT to create the construct targeting DolP to the IM (pET17b-dolPIM) are shown in red. The signal …
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Figure 1—figure supplement 5—source data 1
The influence of signal sequences on DolP localisation.
- https://cdn.elifesciences.org/articles/62614/elife-62614-fig1-figsupp5-data1-v2.pptx
Figure 2 with 6 supplements
Structure of DolP.
(A) Solution structure and topology of DolP, with α helices, β strands and termini labelled. (B) Backbone model of the 20 lowest-energy solution structures of DolP. The core folded domain is …
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Figure 2—source data 1
Influence of site directed mutagenesis of DolP of protein production and stability.
- https://cdn.elifesciences.org/articles/62614/elife-62614-fig2-data1-v2.pptx
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Figure 2—source data 2
S2 order parameter analysis.
- https://cdn.elifesciences.org/articles/62614/elife-62614-fig2-data2-v2.xlsx
Figure 2—figure supplement 1
DolP is monomeric.
(A) DolP, lacking the site of acylation, was purified and subject to analytical ultracentrifugation. DolP demonstrated a uniform sedimentation velocity consistent with a monomeric species. (B) …
Figure 2—figure supplement 2
Structural analysis of the DolP BON domains.
(A) The ensemble of the 20 lowest-energy structures superimposed to DolP BON1 (N47-I111) and BON2 (G120-T185) domain backbones showing how well the domains superimpose as well as the respective …
Figure 2—figure supplement 3
Alignment of DolP sequences from diverse proteobacterial species.
(A) The amino acid sequences of the experimentally derived BON domains of DolP and OmpATb are aligned with the predicted amino acid sequences of the BON domains from Kbp and OsmY. The position of …
Figure 2—figure supplement 4
Additional SAXS analysis of DolP.
(A) Zoom in of the low s region of the small-angle X-ray scattering curve of DolP shown in Figure 2 highlighting the closeness of fit to the DolP solution structure. (B) Residuals plot between the …
Figure 2—figure supplement 5
Representation of DolP interdomain interactions highlighting the location of interdomain NOEs identified.
38 interdomain NOEs were identified via Cyana (Table 3). Due to the ambiguity between chemically equivalent hydrogens within the same group, multiple NOEs are displayed to all equivalent hydrogens …
Figure 2—figure supplement 6
SAXS processing analysis.
(A) The linear region of the Guinier plot measured from the raw SAXS data for DolP. Values for Rg and I(0) are shown calculated using AutoRG in program Primus. (B) Pair-wise distance distribution …
Figure 3 with 4 supplements
DolP BON2:α1 binds phospholipid.
(A) DolP ribbon structure highlighting residues exhibiting substantial CSPs (Δδave) upon DHPG micelle interaction. The histogram shows the normalised perturbations induced in each residue’s amide …
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Figure 3—source data 1
Chemical shift perturbations for lipid titration results.
- https://cdn.elifesciences.org/articles/62614/elife-62614-fig3-data1-v2.xlsx
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Figure 3—source data 2
Data for HADDOCK calculations of micelle-DolP interactions.
- https://cdn.elifesciences.org/articles/62614/elife-62614-fig3-data2-v2.xlsx
Figure 3—figure supplement 1
dolP has genetic interactions with bamB and bamE but no detectable physical interaction.
(A) dolP genetically interacts with the genes encoding the non-essential BAM complex accessory lipoproteins. Strains were arrayed on LB Lennox agar plates using a Biomatrix six replicator. Genetic …
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Figure 3—figure supplement 1—source data 1
Genetic interactions with DolP.
- https://cdn.elifesciences.org/articles/62614/elife-62614-fig3-figsupp1-data1-v2.pptx
Figure 3—figure supplement 2
Loss of DolP affects membrane fluidity, but does not affect membrane lipid profiles.
(A) SDS-PAGE gel showing separation of LPS preparations from E. coli BW25113 and E. coli BW25113 harbouring pET20b-wbbL which restores O-antigen expression on the bacterial cell surface. (B) …
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Figure 3—figure supplement 2—source data 1
LPS production in a dolP negative background.
- https://cdn.elifesciences.org/articles/62614/elife-62614-fig3-figsupp2-data1-v2.pptx
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Figure 3—figure supplement 2—source data 2
Phospholipid content of membranes isolated from a dolP mutant.
- https://cdn.elifesciences.org/articles/62614/elife-62614-fig3-figsupp2-data2-v2.xlsx
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Figure 3—figure supplement 2—source data 3
Comparison of hepta- and hexa-acylated LPS levels.
- https://cdn.elifesciences.org/articles/62614/elife-62614-fig3-figsupp2-data3-v2.xlsx
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Figure 3—figure supplement 2—source data 4
Raw data for membrane fluidity assay.
- https://cdn.elifesciences.org/articles/62614/elife-62614-fig3-figsupp2-data4-v2.xlsx
Figure 3—figure supplement 3
DolP phosphatidylglycerol binding HSQC spectra.
(A) 1H,15N HSQC spectra of 15N-DolP (300 μM) in the presence (red) and absence (black) of 40 mM 1,2-dihexanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DHPG) highlighting the large chemical shift …
Figure 3—figure supplement 4
Kd estimation from HSQC titration data.
Kd estimation was performed using the sum of the average chemical shift distance plotted against ligand concentration and fit using a standard ligand binding curve. Representative fits for G120, …
Figure 4 with 2 supplements
DolP specifically recognises anionic phospholipid via BON2:α1.
(A) Histograms showing the normalised CSP values observed in 15N-labelled DolP (300 μM) amide signals in the presence of 20 mM 1,2,-dihexanoyl-sn-glycero-3-phosphethanolamine, 20 mM …
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Figure 4—source data 1
Effect of site-directed mutations on DolP function.
- https://cdn.elifesciences.org/articles/62614/elife-62614-fig4-data1-v2.pptx
Figure 4—figure supplement 1
Electrostatic analysis of DolP.
(A) Electrostatic surface map of DolP BON domains 1 and 2 calculated using DelPhi (Li et al., 2012) at a pH of 6 and 0.05M ionic strength (which approximates the experimental conditions). The −3kT/e …
Figure 4—figure supplement 2
Analysis of DolP mutants.
(A) E. coli BW25113 ∆dolP mutants were complemented with plasmids expressing a wild-type copy of DolP or a mutant version. Each strain was serially diluted and plated on LB-agar containing either …
Figure 5
Phospholipid binding is required for DolP recruitment to division sites.
(A) Fluorescence microscopy of ΔdolP cells expressing either DolPWT::mCherry or DolPW127E::mCherry from the pET17b plasmid after growth to mid-exponential phase (OD600 ~0.4–0.8). Scale bars …
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Figure 5—source data 1
Effect of DolP-anionic phospholipid interactions on DolP localisation.
- https://cdn.elifesciences.org/articles/62614/elife-62614-fig5-data1-v2.pptx
Tables
Table 1
Taxonomic distribution of BON family domain architectures.
| Cluster number* | UniRef100† | Total number of proteins ‡ | Major domain architecture in cluster§ | α | β | γ | δ | ε | ζ | Aci†† | Act†† | Bac†† | Chl†† | Chl†† | Chl†† | Cya†† | Dei†† | Fib†† | Fir†† | Gem†† | Nit†† | Pla†† | Spi†† | Syn†† | The†† | The†† | The†† | Ver†† |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 1280 | 2723 | OsmY-like and 1 x BON | 41 (89)¶,** | 176 (533) | 1484 (1830) | 33 (56) | 12 (12) | 1 (1) | 6 (12) | 2 (3) | 5 (5) | 3 (11) | 3 (4) | 43 (65) | 1 (1) | 13 (13) | 1 (2) | 1 (1) | 14 (30) | 9 (9) | 1 (1) | 1 (1) | 7 (19) | ||||
| 2 | 833 | 2395 | DolP-like | 97 (103) | 330 (335) | 1892 (1919) | 15 (17) | 2 (2) | 1 (1) | 1 (2) | 1 (1) | |||||||||||||||||
| 3 | 579 | 690 | three x BON + 1 x BON | 95 (187) | 108 (255) | 35 (36) | 18 (28) | 7 (23) | 14 (25) | 14 (30) | 2 (2) | 3 (21) | 6 (10) | 5 (7) | 1 (1) | 32 (32) | 1 (2) | 12 (27) | 1 (1) | |||||||||
| 4 | 476 | 537 | BON + secretin | 207 (276) | 77 (80) | 70 (117) | 32 (34) | 4 (4) | 1 (1) | 3 (3) | 10 (11) | 1 (1) | 7 (7) | 1 (1) | ||||||||||||||
| 5 | 409 | 1570 | Kbp-like | 66 (66) | 131 (132) | 1323 (1328) | 1 (1) | 1 (1) | 31 (31) | 5 (5) | 1 (1) | 1 (1) | ||||||||||||||||
| 6 | 282 | 300 | CBS + CBS + BON | 82 (136) | 17 (29) | 4 (4) | 53 (127) | 4 (4) | ||||||||||||||||||||
| 7 | 220 | 318 | BON + BON + OmpA | 157 (161) | 55 (57) | 9 (11) | 62 (64) | 1 (1) | 19 (23) | 1 (1) | ||||||||||||||||||
| 8 | 70 | 75 | BON + Mschannel | 31 (32) | 1 (1) | 24 (25) | 2 (3) | 1 (1) | 8 (13) | |||||||||||||||||||
| 9 | 52 | 52 | one x BON | 1 (1) | 42 (51) | |||||||||||||||||||||||
| 10 | 43 | 80 | one x BON and 1 x DUF2204 | 1 (1) | 1 (1) | 77 (77) | 1 (1) | |||||||||||||||||||||
| 11 | 33 | 87 | 1–2 X Forkhead + BON | 2 (2) | 4 (4) | 2 (2) | 78 (79) | |||||||||||||||||||||
| 12 | 30 | 33 | one x BON | 26 (27) | 3 (3) | 1 (1) | 1 (1) | 1 (1) | ||||||||||||||||||||
| smaller cluster/unclustered: | ||||||||||||||||||||||||||||
| 83 | 109 | 22 (29) | 19 (19) | 25 (25) | 9 (9) | 1 (1) | 4 (12) | 2 (2) | 1 (1) | |||||||||||||||||||
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* The main twelve clusters were analysed, all proteins falling into smaller clusters were summarised into the single category ‘smaller cluster’.
†, ‡, §, ¶ Shown are the number of UniRef100 used in the clustering approach†, the corresponding number of proteins derived from the HMMER search‡, the observed major domain architecture§ and the number of unique protein sequences (in brackets)¶ as well as the number of unique organisms mapped to the bacterial (Sub)Phyla**.
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†† Acidobacteria, Actinobacteria, Bacteroidetes, Chlamydiae, Chlorobi, Chloroflexi, Cyanobacteria, Deinococcus-Thermus, Fibrobacteres, Firmicutes, Gemmatimonadetes, Nitrospirae, Planctomycetes, Spirochaetes, Synergistetes, Thermobaculum, Thermodesulfobacteria, Thermotogae, Verrucomicrobia.
Table 2
Structural statistics of the ensemble of 20 DolP solution structures.
| DolP | |
|---|---|
| Completeness of resonance assignments† | |
| Aromatic completeness | 74.14% |
| Backbone completeness | 98.42% |
| Sidechain completeness | 84.84% |
| Unambiguous CH2 completeness | 100% |
| Unambiguous CH3 completeness | 100% |
| Unambiguous sidechain NH2 completeness | 100% |
| Conformationally restricting restraints‡ | |
| Distance restraints | |
| Total NOEs | 2930 (2762) |
| Intra residue (i = j) | 408 (374) |
| Sequential (| i – j |=1) | 869 (783) |
| Medium range (1 < | i - j |<5) | 773 (741) |
| Long range (| i – j |≥5) | 880 (866) |
| Interdomain | 38 |
| Dihedral angle restraints | 258 |
| Hydrogen bond restraints | 128 |
| No. of restraints per residue | 16.6 (20.9) |
| No. of long range restraints per residue | 5.0 (6.5) |
| Residual restraint violations‡ | |
| Average No. of distance violations per structure | |
| 0.2 Å-0.5 Å | 3.55 |
| >0.5 Å | 0 |
| Average No. of dihedral angle violations per structure | |
| >5o | 0 (max 4.8) |
| Model quality‡ | |
| Global (residues 46–190) | |
| Rmsd backbone atoms (Å)§ | 0.5 |
| Rmsd heavy atoms (Å)§ | 0.9 |
| Domain 1 (Residues 46–112) | |
| Rmsd backbone atoms (Å) | 0.3 |
| Rmsd heavy atoms (Å) | 0.7 |
| Domain 2 (Residues 118–190) | |
| Rmsd backbone atoms (Å) | 0.3 |
| Rmsd heavy atoms (Å) | 0.8 |
| Rmsd bond lengths (Å) | 0.005 |
| Rmsd bond angles (o) | 0.6 |
| MolProbity Ramachandran statistics‡.§ | |
| Most favoured regions (%) | 95.1 |
| Allowed regions (%) | 4.3 |
| Disallowed regions (%) | 0.7 |
| Global quality scores (raw/Z score)‡ | |
| Verify 3D | 0.38 /- 1.28 |
| Prosall | 0.52 /- 0.54 |
| Procheck (phi-psi)d | −0.28 /- 0.79 |
| Procheck (all)d | −0.75 /- 4.44 |
| Molprobity clash score | 47.99 /- 6.71 |
| Model Contents | |
| Ordered residue ranges§ | 45–193 |
| Total number of residues | 178 |
| BMRB accession number | 19760 |
| PDB ID code | 7A2D |
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* Structural statistics computed for the ensemble of 20 deposited structures.
† Computed using AVS software (Moseley et al., 2004) from the expected number of resonances, excluding highly exchangeable protons (N-terminal, Lys, amino and Arg guanido groups, hydroxyls of Ser, Thr, and Tyr), carboxyls of Asp and Glu, non-protonated aromatic carbons, and the C-terminal His6 tag.
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‡ Calculated using PSVS version 1.5 (Bhattacharya et al., 2007). Average distance violations were calculated using the sum over r−6.
§ Based on ordered residue ranges [S(φ) + S(ψ)>1.8].
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Values in (brackets) refer to the core structured region.
Table 3
Interdomain NOE restraints identified by Cyana during automated NOE assignment and structure calculation.
| Proton pair | Intensity | Distance (Å) |
|---|---|---|
| TYR 75 HD1 - THR 188 HA | Weak | 5.5 |
| TYR 75 HE1 - GLY 160 HA2 | Weak | 5.4 |
| TYR 108 HE1 - ALA 186 HA | Weak | 5.5 |
| TYR 108 HE2 - ALA 186 HA | Weak | 5.5 |
| TYR 108 HE1 - ALA 186 HB | Weak | 5.1 |
| TYR 75 HD1 - ALA 186 HB | Weak | 5.2 |
| TYR 75 HE1 - LEU 161 HA | Weak | 5.2 |
| TYR 75 HE1 - LEU 161 HB3 | Weak | 5.4 |
| TYR 75 HE1 - LEU 161 HG | Weak | 5.5 |
| TYR 75 HE1 - LEU 161 HD1 | Weak | 4.9 |
| TYR 75 HE1 - LEU 161 HD2 | Weak | 4.9 |
| THR 73 HG2 - ALA 186 HB | Weak | 5.5 |
| LYS 78 HD2 - PHE 187 hr | Weak | 5.5 |
| LYS 78 HD3 - PHE 187 hr | Weak | 5.5 |
| TYR 75 HD1 - HET 159 HA | Weak | 5.5 |
| TYR 108 HD1 - ALA 186 HB | Weak | 5.5 |
| GLN 76 HE22 - LEU 161 HB2 | Weak | 5.2 |
| GLN 76 HE22 - LEU 161 HG | Weak | 5.1 |
| GLN 76 HE22 - LEU 161 HD1 | Weak | 4.5 |
| GLN 76 HE22 - LEU 161 HD2 | Weak | 4.5 |
| TYR 75 HD1 - THR 188 HG2 | Weak | 4.2 |
| TYR 75 HE1 - LEU 161 hr | Weak | 4.3 |
| TYR 75 HE1 - VAL 162 hr | Weak | 5.5 |
| TYR 75 HE1 - LEU 161 HB2 | Weak | 4.1 |
| TYR 75 HE1 - THR 188 HG2 | Weak | 4.1 |
| TYR 75 HE1 - THR 188 hr | Weak | 5.5 |
| TYR 75 HE1 - GLY 160 hr | Weak | 4.8 |
| TYR 75 HD1 - GLY 160 hr | Weak | 4.7 |
| THR 73 HG2 - HET 159 HG | Weak | 4.4 |
| TYR 75 HE1 - LEU 161 HD | Weak | 4.0 |
| TYR 75 HE2 - LEU 161 HD | Weak | 5.1 |
| GLN 76 HE21 - LEU 161 HD | Medium | 3.7 |
| GLN 76 HE22 - LEU 161 HD | Medium | 3.7 |
| LYS 78 HG - PHE 187 hr | Weak | 4.9 |
| LYS 78 HD - ALA 186 HB | Weak | 5.1 |
| LYS 78 HD - PHE 187 hr | Weak | 4.7 |
| LYS 78 HE - PHE 187 hr | Weak | 5.3 |
| ARG 112 HA - ARG 182 HB | Weak | 5.3 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | BL21(DE3) | Invitrogen | T7 express, protein expression strain | |
| Strain, strain background (Escherichia coli) | BW25113 | Datsenko and Wanner, 2000 | rrnB3 ΔlacZ4787 ΔphoBR580 hsdR514 Δ(araBAD)567 Δ(rhaBAD)568 galU95 ΔendA9::FRT ΔuidA3::pir(wt) recA1 rph-1 | |
| Strain, strain background (Escherichia coli) | BW25113 △dolP | This paper | BW25113 with dolP deleted | |
| Strain, strain background (Escherichia coli) | BW25113 △lpp,△rcsF | This paper | BW25113 with lpp and rcsF deleted | |
| Strain, strain background (Escherichia coli) | BW25113 △lpp,△rcsF,△pgsA | This paper | BW25113 with lpp, rcsF and pgsA genes deleted | |
| Strain, strain background (Escherichia coli) | BW25113 △clsA,△clsB,△clsC | This paper | BW25113 with clsA, clsB and clsC genes deleted | |
| genetic reagent (E. coli) | KEIO library | Datsenko and Wanner, 2000 | Nonessential genes disrupted in E. coli BW25113 | |
| Recombinant DNA reagent | pKD4 | Datsenko and Wanner, 2000 | Plasmid | Template for the amplification of a kanamycin resistance cassette flanked by FRT sites. |
| Recombinant DNA reagent | pKD46 | Datsenko and Wanner, 2000 | Plasmid | Temperature sensitive, low copy number plasmid encoding the Lambda RED recombinase genes under the control of an arabinose inducible promoter |
| Recombinant DNA reagent | pCP20 | Datsenko and Wanner, 2000 | Plasmid | Temperature sensitive plasmid encoding the FLP recombinase gene |
| Recombinant DNA reagent | pET17b | Novagen | Plasmid | T7 expression vector, AmpR |
| Recombinant DNA reagent | pET17b dolP | This paper | Plasmid | pET17b with dolP cloned between NdeI and EcoRI |
| Recombinant DNA reagent | pET17b dolP TM | This paper | Plasmid | As described above with the dolP gene randomly disrupted by Transposon mutations |
| Recombinant DNA reagent | pET17b dolP STm | This paper | Plasmid | pET17b with the S. typhimurium dolP gene cloned between NdeI and HindIII |
| Recombinant DNA reagent | pET17b dolP H.i | This paper | Plasmid | pET17b encoding a codon optimised Haemophilus influenza dolP homolog |
| Recombinant DNA reagent | pET17b dolP P.m | This paper | Plasmid | pET17b encoding a codon optimised Pasteurella multocida dolP homolog |
| Recombinant DNA reagent | pET17b dolP N.m | This paper | Plasmid | pET17b encoding a codon optimised Neisseria meningitidis dolP homolog |
| Recombinant DNA reagent | pET17b dolP V.c | This paper | Plasmid | pET17b encoding a codon optimised Vibrio cholera dolP homolog |
| Recombinant DNA reagent | pET17b osmY | This paper | Plasmid | pET17b encoding a codon optimised E. coli K12 osmY |
| Recombinant DNA reagent | p(OM)OsmY | This paper | Plasmid | pET17b encoding a codon optimised E. coli K12 osmY synthesised with the dolP signal sequence and acylation site in place of the osmY signal sequence |
| Recombinant DNA reagent | pET20b | Novagen | Plasmid | T7 expression vector, AmpR |
| Recombinant DNA reagent | pET20b dolP | This paper | Plasmid | pET20b with dolP cloned between NdeI and EcoRI |
| Recombinant DNA reagent | pET20b dolP PM | This paper | Plasmid | pET20b with dolP cloned between NdeI and EcoRI with site-directed point mutations at various sites |
| Recombinant DNA reagent | pET20b wbbL | This paper | Plasmid | pET20b with wbbL gene cloned between NdeI and HindIII |
| Recombinant DNA reagent | pET20b dolP::mCherry | This paper | Plasmid | pET20b encoding dolP fused to a codon optimised mCherry gene via a C-terminal 11-codon flexible linker (GGSSLVPSSDP) |
| Recombinant DNA reagent | pET26b dolPpelB::mCherry | This paper | Plasmid | pET26b dolP::mCherry with the dolP signal sequence replaced with that of pelB |
| Recombinant DNA reagent | pET20b dolPIM::mCherry | This paper | Plasmid | pET20b dolP::mCherry with codon 20 and 22 of dolP each mutated to aspartic acid |
| Recombinant DNA reagent | pET20b dolPW127E::mCherry | This paper | Plasmid | pET20b dolP::mCherry with codon 127 mutated to glutamic acid |
Table 4
Accession numbers for the sequences used for CLANS clustering shown in Figure 1.
| Organism | OsmY | DolP | Kbp |
|---|---|---|---|
| Escherichia coli K12 | P0AFH8 | P64596 | P0ADE6 |
| Klebsiella pneumoniae MGH 78578 | A6THZ1 | A6TEG9 | A6T985 |
| Enterobacter cloacae ENHKU01 | J7G7C8 | J7GHD1 | J7GFT3 |
| Salmonella enterica Typhimurium | Q7CP68 | Q7CPQ6 | Q8ZML9 |
| Erwinia billingiae Eb661 | D8MMS8 | D8MME2 | D8MNV6 |
| Serratia proteamaculans 568 | A8G9G9 | A8GJZ3 | A8GFP7 |
| Cronobacter sakazakii ATCC BAA-894 | A7MGB6 | A7MIQ1 | A7MEA9 |
| Pantoea sp. Sc1 | H8DPK0 | H8DQ90 | H8DIH9 |
| Hafnia alvei ATCC 51873 | G9Y3J7 | G9Y4J4 | G9YAM4 |
| Citrobacter rodentium ICC168 | D2TRY4 | D2TQ24 | D2TM58 |
| Shigella flexneri 1235–66 | I6F1Q5 | I6GLP1 | I6HD15 |
| Yersinia enterocolitica 8081 | A1JJ93 | A1JR75 | |
| Yersinia pestis KIM10+ | Q7CG58 | Q8D1R6 | |
| Dickeya dadantii 3937 | E0SJX0 | E0SHF6 |
Table 5
HADDOCK docking statistics for ensemble 20 lowest-energy DolP-DPC micelle solution structures calculated.
| Experimental parameters* | |
|---|---|
| Ambiguous distance restraints | 19 including NH of I20, G120-T130, V132-Q135, T138, S139, and NHε of W127 |
| Number of flexible residues† | 50 (I20-V45 (flexible linker as ascertained by NMR), A74, G120-I128, K131-R133, Q135-L137, V142-S145, I173,S178-V180) |
| Atomic pairwise RMSD (Å) | |
| All backbone | |
| Flexible interface backbone | |
| Intermolecular energies (kcal.mol−1) | |
| Evdw | −100.81 ± 7.74 |
| Eelec | −231.67 ± 64.14 |
| Erestraints | 22.30 ± 4.29 |
| Buried surface area (Å2) | 2186.78 ± 133.277 |
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* deduced from intensity reductions observed in presence of 5-doxl derivative.
† according to their surface accessibility and the chemical shift perturbation in presence of DPC/CHAPS.
Additional files
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Supplementary file 1
UniProt accession numbers for the proteins in the respective clusters as shown in Table 1.
- https://cdn.elifesciences.org/articles/62614/elife-62614-supp1-v2.xlsx
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Supplementary file 2
Mass spectrometry of outer membrane fractions to assess presence of protein.
- https://cdn.elifesciences.org/articles/62614/elife-62614-supp2-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/62614/elife-62614-transrepform-v2.docx
