1. Biochemistry and Chemical Biology
  2. Microbiology and Infectious Disease
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Structure of dual BON-domain protein DolP identifies phospholipid binding as a new mechanism for protein localisation

  1. Jack Alfred Bryant
  2. Faye C Morris
  3. Timothy J Knowles
  4. Riyaz Maderbocus
  5. Eva Heinz
  6. Gabriela Boelter
  7. Dema Alodaini
  8. Adam Colyer
  9. Peter J Wotherspoon
  10. Kara A Staunton
  11. Mark Jeeves
  12. Douglas F Browning
  13. Yanina R Sevastsyanovich
  14. Timothy J Wells
  15. Amanda E Rossiter
  16. Vassiliy N Bavro
  17. Pooja Sridhar
  18. Douglas G Ward
  19. Zhi-Soon Chong
  20. Emily CA Goodall
  21. Christopher Icke
  22. Alvin CK Teo
  23. Shu-Sin Chng
  24. David I Roper
  25. Trevor Lithgow
  26. Adam F Cunningham
  27. Manuel Banzhaf
  28. Michael Overduin  Is a corresponding author
  29. Ian R Henderson  Is a corresponding author
  1. Institute of Microbiology and Infection, University of Birmingham, United Kingdom
  2. School of Biosciences, University of Birmingham, United Kingdom
  3. Institute for Cancer and Genomic Sciences, University of Birmingham, United Kingdom
  4. Infection & Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Australia
  5. Department of Chemistry, National University of Singapore, Singapore
  6. Institute for Molecular Bioscience, University of Queensland, Australia
  7. School of Life Sciences, The University of Warwick, United Kingdom
  8. Institute of Inflammation and Immunotherapy, University of Birmingham, United Kingdom
  9. Department of Biochemistry, University of Alberta, Canada
Research Article
Cite this article as: eLife 2020;9:e62614 doi: 10.7554/eLife.62614
5 figures, 6 tables, 2 data sets and 3 additional files

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 position 19 acylated by Lgt and Lnt and finally the protein is targeted to the OM by the Lol system (Figure 1—figure supplement 1). E. coli contains three BON-domain proteins. DolP shares a similar domain organisation with OsmY, which encodes a periplasmic protein that possesses a signal sequence (green) which is recognised and cleaved by the signal peptidase LepB. Kbp is more divergent from DolP and OsmY, has no predictable signal sequence and is composed of BON and LysM domains (Figure 1—figure supplement 2). (B) DolP, OsmY and Kbp are widespread amongst proteobacteria, and cluster into three distinct groups based on the program CLANS (Frickey and Lupas, 2004) with connections shown for a P value cut-off of <10−2 (Table 4). (C) Growth phenotypes for mutant isolates lacking DolP (ΔdolP), wild-type strain (WT) or the complemented mutant (COMP). Strains were grown on LB agar containing vancomycin (100 μg/ml) or sodium dodecyl sulphate (SDS; 4.8%). (D) DolP from diverse proteobacterial species expressed in an E. coli ΔdolP strain restores growth in the presence of vancomycin as assessed by a serial dilution plate growth assay. Plasmids expressing OsmY do not complement the defect.

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 BamC and BamE. DolP is not detected in the mutant but like BamC and BamE is found with the membrane fraction. (B) Western immunoblotting of OM fractions from E. colidolP complemented with a plasmid (pDolP-C19A) encoding DolP with a point mutation at position C19. (C) E. coli cells treated with protease in the presence (+) or absence (-) of polymyxin B, which permeablises the OM, allowing the protease access to the periplasm. Antibodies to the cytoplasmic RNA polymerase (RNAP) and the periplasmic chaperone SurA were used as controls. (D) Immunofluorescence photomicrographs of E. coli BW25113, an isogenic ∆dolP mutant and the complemented mutant. Cells were probed with anti-DolP before and after permeabilisation. Anti-SurA was used as a control.

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 predominant architecture is that observed for DolP and OsmY where the protein possesses a signal sequence and one or more BON domains. The second major architecture is that observed for Kbp, where proteins possess one or more BON domains and a LysM domain. The other major architectures include associations with Secretin (Pfam: PF00263), CBS (Pfam: PF00571), OmpA (Pfam: PF00691), MS_channel (Pfam: PF00924), FHA (Pfam: PF00498) or cytidylate kinase (Pfam: PF13189) domains. Due to their functions, many of these domains would place their associated BON domains in proximity to cell membranes.

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; Lennon et al., 2015) (A) Investigation of osmY and kbp null mutants of E. coli revealed neither was sensitive to vancomycin or SDS. Growth phenotypes for mutant isolates lacking BON-domain proteins, wild-type strains (WT) or complemented mutants (COMP). Strains were grown on LB agar containing vancomycin (100 μg/ml) or sodium dodecyl sulphate (SDS; 4.8%). (B) A plasmid encoding a DolP-OsmY chimeric protein composed of the lipoprotein targeting sequence of DolP and the BON domains of OsmY failed to complement the OM defect associated with loss of dolP. (C) E. coli BW25113 ΔdolP is not more susceptible to osmotic stress induced by NaCl than the parent strain as assessed by a serial dilution plate assay. Interestingly, our investigations did not reveal a role for either kbp or osmY in survival of osmotic stress as the E. coli BW25113 parent strain and isogenic osmY::aph and kbp::aph mutants survived equally well.

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 electron microscopy reveals parental and E. colidolP cells have no discernible differences in cellular morphology.

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 sequence of dolP was also swapped for that of pelB in order to create the construct pET17b-dolPpelB in order to target DolP to the periplasmic space with no modification. Fluorescence microscopy of ΔdolP cells expressing either DolPWT-mCherry or DolPIM-mCherry or DolPpelB-mCherry from the pET17b plasmid after growth to mid-exponential phase (OD600 ~0.4–0.8). Scale bars represent 2 μM and both phase contrast and the mCherry channel are shown in greyscale and red, respectively. The capacity of DolPWT, DolPIM, DolPWT-mCherry or DolPIM-mCherry to complement the ΔdolP mutant sensitivity phenotype was screened by dilution assay on 4.8% SDS. The expression of each construct was checked by Western blotting of total protein extracts with anti-DolP antiserum.

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 highlighted in red whilst the flexible N-terminal is shown in grey. The dynamic nature of the linker was demonstrated from S2 order parameter analysis calculated from chemical shift assignments using TALOS+. (C) Small-angle X-ray scattering curve of DolP with corresponding best fit of the solution structure of DolP. Best fit calculated based on the core DolP solution structure with flexibility accommodated in residues 20–46, 112–118, and 189–195. The corresponding ab-initio bead model is shown calculated using Dammif (Franke and Svergun, 2009) based solely on the scattering data. (D) Western blots of total protein extracts show plasmid-mediated expression of DolP in E. coli ΔdolP after site-directed mutation of conserved residues. The empty vector (EV) control is labelled and WT represents wild-type DolP. The presence of the OM lipoprotein BamB was used as a control. Colony growth assays by serial dilution of mutants on 4.8% SDS reveal which residues are critical for the maintenance of the OM barrier function. (E) Structure of DolP showing position of transposon-mediated insertions. Western blots of total protein extracts show plasmid-mediated expression of mutant versions of DolP in E. coli ΔdolP. The empty vector (EV) control is labelled and WT represents wild-type DolP. Colony growth assays by serial dilution of mutants on 4.8% SDS reveal which insertions abolish DolP function. Blue labels represent position of non-functional insertions. Orange labels represent position of tolerated insertions. The presence of the OM lipoprotein BamB was used as a control.

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
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) Column chromatography of purified DolP revealed that it had an elution profile consistent with a single monomeric species.

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 degrees of freedom available to each domain. (B) Dalilite superposition of DolP BON domains 1 (Red; residues 46–114) and 2 (Blue; residues 117–189). The BON domains are similar except for the double turn extension of the BON2:α1 helix and the presence of the α1’ helix present in BON1 that is absent in BON2. The pairwise RMSD for backbone heavy atoms is 1.8 Å and dalilite Z-score is 8.4. (C) Superposition of DolP BON2 (Blue) on to the BON subdomain of Rv0899 (OmpATb) (Green; accession code – 2KSM; residues 136–196). For BON2 the pairwise RMSD for backbone heavy atoms was 2.7 Å and the dalilite Z-score was 4.9. Similarly, for BON1 the pairwise RMSD was 2.6 Å and the dalilite Z-score was 5.3.

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 the experimentally derived secondary structure for DolP BON1 and BON2 and OmpATb are depicted below the sequence alignment. (B) Alignments of the amino acid sequences of DolP and OsmY from various Gram-negative bacteria. The positions of the experimentally-derived secondary structural elements of E. coli DolP are depicted below the sequence alignment. The signal sequence is depicted by the red box. The Lipobox associated with recognition by LspA and acylation is highlighted in purple. The conserved glycine residues are highlighted in blue and the tyrosine residue associated with interdomain interactions is highlighted in green. Residues showing CSPs are highlighted in pink.

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 DolP solution structure and the small-angle X-ray scattering curve highlighting the closeness of fit.

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 resulting in 83 NOEs being displayed.

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 P(r), calculated from the scattering curve of DolP, calculated using gnom arbitrary units (a.u.).

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 signal when DHPG (40 mM) was added to DolP (300 µM). Examples of significant CSPs are shown. (B) Histogram showing intensity reductions of HN signals of DolP induced by adding 5-doxyl PC and DMPG into DPC/CHAPs micelles and the corresponding structure of a representative DolP-micelle complex calculated using CSPs and doxyl restraints using the program HADDOCK. Only the BON2:α1 helix is observed making contact with the micelle surface. No corresponding interaction of the BON1:α1 helix is observed. Zoom panels show burial of BON2:α1 into the micelle. The side chains of DolP residues that intercalate between the acyl chains (G120, S123, W127, T130, and S134) are coloured red. The side chains of residues that buttress the interface (E121, N124, T126, I128, K131, R133, and Q135) are coloured yellow. DolP is shown in blue and the phospholipid micelle is shown in tan.

Figure 3—source data 1

Chemical shift perturbations for lipid titration results.

https://cdn.elifesciences.org/articles/62614/elife-62614-fig3-data1-v2.xlsx
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 interaction plates were incubated for 12 hr at 37°C and imaged. An example of a 384‐well plate is shown above the graph. Each plate contained a total of 384 colonies consisting of 96 wildtype, single, and double mutant clones. Fitness was measured by quantifying colony size and integral opacity, which represents colony density, using the image analysis software Iris (Kritikos et al., 2017). Bar plots show the averaged values 96 technical replicates. The error bars represent the 95% confidence interval. (B) Phase contrast microscopy of WT, ΔdolP, ΔbamB, ΔbamC, ΔbamE, ΔbamBΔdolP, ΔbamCΔdolP and ΔbamEΔdolP cells after growth to mid-exponential phase (OD600 ~0.4–0.8). Scale bars represent 2 μM. Phase light cells can be observed for the ΔbamBΔdolP and ΔbamEΔdolP cells.( C) DolP immunoprecipitation. Whole cell triton X-100 solubilised lysates of E. coli BW25113 pDolPpelB, pBamA-His, and ΔdolP, were purified by Ni-NTA affinity chromatography then detected by western blot using anti-DolP and BamA-E antibodies. (D) Purified OM samples from E. coli BW25113 parent (WT) or ΔdolP cells were separated by SDS-PAGE, with (d) and without (n) boiling before being visualised by staining with coomassie.

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) Analysis of phospholipid profiles from purified ∆dolP cell envelopes. Phospholipids were extracted by the Bligh-Dyer method from E. coli IM or OM samples purified by sucrose density gradient centrifugation. Phospholipids were visualised by staining with phosphomolybdic acid and charring after being separated by thin-layer chromatography with the following mobile phase: Chloroform:methanol:acetic acid (65:25:10). Phospholipid profiles were also analysed by LC/MS-MS following separation on the Luna C8(2) column under a THF/MeOH/H2O gradient. Phospholipid compositions are shown as sum for each of the four major classes observed: lyso-phophatidylethanolamines (LysoPE), phosphatidylethanolamines (PE), phosphatidylglycerols (PG) and cardiolipins (CL). Each data set is from three biological replicates generated from three separately purified membranes. Error bars represent ±S.D. (C) PagP-mediated Lipid A palmitoylation assay. PagP transfers an acyl chain from surface exposed phospholipid to hexa-acylated Lipid A to form hepta-acylated Lipid A. [32P]-labelled Lipid A was purified from cells grown to mid-exponential phase in LB broth with aeration. An equal amount of radioactive material (cpm/lane) was loaded on each spot and separated by thin-layer chromatography before quantification. As a positive control, cells were exposed to 25 mM EDTA for 10 min prior to Lipid A extraction in order to chelate Mg2+ ions and destabilise the LPS layer, leading to high levels of Lipid A palmitoylation. Hepta-acylated and hexa-acylated lipid A was quantified and hepta-acylated Lipid A represented as a percentage of total. Triplicate experiments were utilised to calculate averages and standard deviations with students t-tests used to assess significance. Student’s t-tests: NS* p>0.1 compared with Parent EV. (D) E. coli BW25113 cells were grown overnight in LB (~16 hr) before being harvested by centrifugation and washed three times in PBS. Membrane fluidity was measured for each strain in triplicate and error bars represent standard deviation. Membrane fluidity is expressed as relative to E. coli BW25113 parent cells (WT).

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
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
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
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 perturbations observed on DHPG binding. (B) Histograms showing the normalised CSP values observed in 15N-labelled DolP (300 μM) amide signals in the presence of 5 mM cardiolipin, 20 mM 1,2,-dihexanoyl-sn-glycero-3-phosphethanolamine and 20 and 40 mM 1,2-dihexanoyl-sn-glycero-3-phospho-(1'-rac-glycerol).

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, W127, and T138 are shown with corresponding estimations for Bmax, the maximum Δδppm, and Kd highlighted.

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 1,2-dihexanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) and 5 mM cardiolipin.( B) Mutagenesis of the BON2:α1 helix residues identified by CSPs. The positions of W127 and L137 are indicated as sticks. Western blots of total protein extracts show plasmid-mediated expression of DolP in E. coli ΔdolP after site-directed mutation of amino acid residues. The empty vector (EV) control is labelled and WT represents wild-type DolP. Colony growth assays of E. coli ΔdolP complemented with DolP mutants reveal which residues are critical for the maintenance of OM barrier function. The presence of the protein PqiB was used as a control. (C) Histograms showing the normalised CSP values observed in 15N-labelled DolPWT or DolPW127E mutant (300 μM) amide signals in the presence of 40 mM 1,2-dihexanoyl-sn-glycero-3-phospho-(1'-rac-glycerol).

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 surface is shown in red and the +3kT/e surface is shown in blue. A formal charge library was used, with a dielectric of 2 assigned to the protein interior and a dielectric of 80 assigned to the exterior. Cartoon representations of the BON structures are shown to the right of each surface to more clearly highlight the orientations of the protein. The BON1:α1 and BON2:α1 helices show clear differences, with BON1:α1 being predominantly neutral with an electronegative patch towards its N-terminus, whilst BON2:α2 shows no electronegatively at all, but rather has a large electropositive patch towards the centre of this helix presumably explaining its specificity for the electropositive surface of phosphatidylglycerol. (B) Hydrophobic surface map of DolP BON domains 1 and 2, hydrophobic residues (A, G, V, I, L, F, M) are shown in cyan, W127 (Red) is shown exposed on the surface of the BON2:α1 helix. Cartoon representations of the BON structures are shown to the right of each surface to more clearly highlight the orientations of the protein.

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 vancomycin (100 μg/ml) or SDS (4.8% wt/vol), and growth was observed after overnight incubation. The W127E and L137E mutants failed to grow. (B) Western immunoblotting of whole cell lysates derived from overnight cultures of mutants highlighted in the top panel. Blots were probed with antibodies to the outer-membrane lipoprotein BamB and to DolP.

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 represent 2 μM and both phase contrast and the mCherry channel are shown in greyscale and red respectively. White arrows highlight division site localisation of DolPWT-mCherry. Demographic representations of the DolPWT-mCherry or DolPW127E-mCherry fluorescence intensities measure along the medial axis of the cells. Images of >500 cells were analysed using the MicrobeJ software and sorted according to length where the y-axis represents relative cellular position with 0 being mid-cell and 3 or −3 being the cell poles (Ducret et al., 2016). (B) Thin layer chromatography of phospholipids extracted from either E. coli BW25113 (WT), ΔrcsFΔlpp, ΔrcsFΔlppΔpgsA (referred to as ΔpgsA) or ΔclsAΔclsBΔclsC (referred to as ΔclsABC) strains. The rcsF and lpp genes must be removed in order to prevent toxic build-up of Lpp on the IM in the pgsA mutant. Phospholipids were separated using chloroform:methanol:acetic acid (65:25:10) as the mobile phase before staining with phophomolybdic acid and charring.( C) Fluorescence microscopy of ΔpgsA or ΔclsABC cells expressing DolPWTmCherry from the pET17b plasmid after growth to mid-exponential phase (OD600 ~0.4–0.8). White arrows highlight DolP-mCherry mislocalisation.

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§αβγδεζAci††Act††Bac††Chl††Chl††Chl††Cya††Dei††Fib††Fir††Gem††Nit††Pla††Spi††Syn††The††The††The††Ver††
112802723OsmY-like and 1 x BON41 (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)
28332395DolP-like97 (103)330 (335)1892 (1919)15 (17)2 (2)1 (1)1 (2)1 (1)
3579690three x BON + 1 x BON95 (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)
4476537BON + secretin207 (276)77 (80)70 (117)32 (34)4 (4)1 (1)3 (3)10 (11)1 (1)7 (7)1 (1)
54091570Kbp-like66 (66)131 (132)1323 (1328)1 (1)1 (1)31 (31)5 (5)1 (1)1 (1)
6282300CBS + CBS + BON82 (136)17 (29)4 (4)53 (127)4 (4)
7220318BON + BON + OmpA157 (161)55 (57)9 (11)62 (64)1 (1)19 (23)1 (1)
87075BON + Mschannel31 (32)1 (1)24 (25)2 (3)1 (1)8 (13)
95252one x BON1 (1)42 (51)
104380one x BON and 1 x DUF22041 (1)1 (1)77 (77)1 (1)
1133871–2 X Forkhead + BON2 (2)4 (4)2 (2)78 (79)
123033one x BON26 (27)3 (3)1 (1)1 (1)1 (1)
smaller cluster/unclustered:
8310922 (29)19 (19)25 (25)9 (9)1 (1)4 (12)2 (2)1 (1)
  1. * 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**.

  2. †† 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 completeness74.14%
Backbone completeness98.42%
Sidechain completeness84.84%
Unambiguous CH2 completeness100%
Unambiguous CH3 completeness100%
Unambiguous sidechain NH2 completeness100%
Conformationally restricting restraints‡
Distance restraints
Total NOEs2930 (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)
Interdomain38
Dihedral angle restraints258
Hydrogen bond restraints128
No. of restraints per residue16.6 (20.9)
No. of long range restraints per residue5.0 (6.5)
Residual restraint violations‡
Average No. of distance violations per structure
0.2 Å-0.5 Å3.55
>0.5 Å0
Average No. of dihedral angle violations per structure
>5o0 (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 3D0.38 /- 1.28
Prosall0.52 /- 0.54
Procheck (phi-psi)d−0.28 /- 0.79
Procheck (all)d−0.75 /- 4.44
Molprobity clash score47.99 /- 6.71
Model Contents
Ordered residue ranges§45–193
Total number of residues178
BMRB accession number19760
PDB ID code7A2D
  1. * 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.

  2. ‡ 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].

  3. 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 pairIntensityDistance (Å)
TYR 75 HD1 - THR 188 HAWeak5.5
TYR 75 HE1 - GLY 160 HA2Weak5.4
TYR 108 HE1 - ALA 186 HAWeak5.5
TYR 108 HE2 - ALA 186 HAWeak5.5
TYR 108 HE1 - ALA 186 HBWeak5.1
TYR 75 HD1 - ALA 186 HBWeak5.2
TYR 75 HE1 - LEU 161 HAWeak5.2
TYR 75 HE1 - LEU 161 HB3Weak5.4
TYR 75 HE1 - LEU 161 HGWeak5.5
TYR 75 HE1 - LEU 161 HD1Weak4.9
TYR 75 HE1 - LEU 161 HD2Weak4.9
THR 73 HG2 - ALA 186 HBWeak5.5
LYS 78 HD2 - PHE 187 hrWeak5.5
LYS 78 HD3 - PHE 187 hrWeak5.5
TYR 75 HD1 - HET 159 HAWeak5.5
TYR 108 HD1 - ALA 186 HBWeak5.5
GLN 76 HE22 - LEU 161 HB2Weak5.2
GLN 76 HE22 - LEU 161 HGWeak5.1
GLN 76 HE22 - LEU 161 HD1Weak4.5
GLN 76 HE22 - LEU 161 HD2Weak4.5
TYR 75 HD1 - THR 188 HG2Weak4.2
TYR 75 HE1 - LEU 161 hrWeak4.3
TYR 75 HE1 - VAL 162 hrWeak5.5
TYR 75 HE1 - LEU 161 HB2Weak4.1
TYR 75 HE1 - THR 188 HG2Weak4.1
TYR 75 HE1 - THR 188 hrWeak5.5
TYR 75 HE1 - GLY 160 hrWeak4.8
TYR 75 HD1 - GLY 160 hrWeak4.7
THR 73 HG2 - HET 159 HGWeak4.4
TYR 75 HE1 - LEU 161 HDWeak4.0
TYR 75 HE2 - LEU 161 HDWeak5.1
GLN 76 HE21 - LEU 161 HDMedium3.7
GLN 76 HE22 - LEU 161 HDMedium3.7
LYS 78 HG - PHE 187 hrWeak4.9
LYS 78 HD - ALA 186 HBWeak5.1
LYS 78 HD - PHE 187 hrWeak4.7
LYS 78 HE - PHE 187 hrWeak5.3
ARG 112 HA - ARG 182 HBWeak5.3
Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Escherichia coli)BL21(DE3)InvitrogenT7 express, protein expression strain
Strain, strain background (Escherichia coli)BW25113Datsenko and Wanner, 2000rrnB3 ΔlacZ4787 ΔphoBR580 hsdR514 Δ(araBAD)567 Δ(rhaBAD)568 galU95 ΔendA9::FRT ΔuidA3::pir(wt) recA1 rph-1
Strain, strain background (Escherichia coli)BW25113 △dolPThis paperBW25113 with dolP deleted
Strain, strain background (Escherichia coli)BW25113 △lpp,rcsFThis paperBW25113 with lpp and rcsF deleted
Strain, strain background (Escherichia coli)BW25113 △lpp,rcsF,pgsAThis paperBW25113 with lpp, rcsF and pgsA genes deleted
Strain, strain background (Escherichia coli)BW25113 △clsA,clsB,clsCThis paperBW25113 with clsA, clsB and clsC genes deleted
genetic reagent (E. coli)KEIO libraryDatsenko and Wanner, 2000Nonessential genes disrupted in E. coli BW25113
Recombinant DNA reagentpKD4Datsenko and Wanner, 2000PlasmidTemplate for the amplification of a kanamycin resistance cassette flanked by FRT sites.
Recombinant DNA reagentpKD46Datsenko and Wanner, 2000PlasmidTemperature sensitive, low copy number plasmid encoding the Lambda RED recombinase genes under the control of an arabinose inducible promoter
Recombinant DNA reagentpCP20Datsenko and Wanner, 2000PlasmidTemperature sensitive plasmid encoding the FLP recombinase gene
Recombinant DNA reagentpET17bNovagenPlasmidT7 expression vector, AmpR
Recombinant DNA reagentpET17b dolPThis paperPlasmidpET17b with dolP cloned between NdeI and EcoRI
Recombinant DNA reagentpET17b dolP TMThis paperPlasmidAs described above with the dolP gene randomly disrupted by Transposon mutations
Recombinant DNA reagentpET17b dolP STmThis paperPlasmidpET17b with the S. typhimurium dolP gene cloned between NdeI and HindIII
Recombinant DNA reagentpET17b dolP H.iThis paperPlasmidpET17b encoding a codon optimised Haemophilus influenza dolP homolog
Recombinant DNA reagentpET17b dolP P.mThis paperPlasmidpET17b encoding a codon optimised Pasteurella multocida dolP homolog
Recombinant DNA reagentpET17b dolP N.mThis paperPlasmidpET17b encoding a codon optimised Neisseria meningitidis dolP homolog
Recombinant DNA reagentpET17b dolP V.cThis paperPlasmidpET17b encoding a codon optimised Vibrio cholera dolP homolog
Recombinant DNA reagentpET17b osmYThis paperPlasmidpET17b encoding a codon optimised E. coli K12 osmY
Recombinant DNA reagentp(OM)OsmYThis paperPlasmidpET17b 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 reagentpET20bNovagenPlasmidT7 expression vector, AmpR
Recombinant DNA reagentpET20b dolPThis paperPlasmidpET20b with dolP cloned between NdeI and EcoRI
Recombinant DNA reagentpET20b dolP PMThis paperPlasmidpET20b with dolP cloned between NdeI and EcoRI with site-directed point mutations at various sites
Recombinant DNA reagentpET20b wbbLThis paperPlasmidpET20b with wbbL gene cloned between NdeI and HindIII
Recombinant DNA reagentpET20b dolP::mCherryThis paperPlasmidpET20b encoding dolP fused to a codon optimised mCherry gene via a C-terminal 11-codon flexible linker (GGSSLVPSSDP)
Recombinant DNA reagentpET26b dolPpelB::mCherryThis paperPlasmidpET26b dolP::mCherry with the dolP signal sequence replaced with that of pelB
Recombinant DNA reagentpET20b dolPIM::mCherryThis paperPlasmidpET20b dolP::mCherry with codon 20 and 22 of dolP each mutated to aspartic acid
Recombinant DNA reagentpET20b dolPW127E::mCherryThis paperPlasmidpET20b dolP::mCherry with codon 127 mutated to glutamic acid
Table 4
Accession numbers for the sequences used for CLANS clustering shown in Figure 1.
OrganismOsmYDolPKbp
Escherichia coli K12P0AFH8P64596P0ADE6
Klebsiella pneumoniae MGH 78578A6THZ1A6TEG9A6T985
Enterobacter cloacae ENHKU01J7G7C8J7GHD1J7GFT3
Salmonella enterica TyphimuriumQ7CP68Q7CPQ6Q8ZML9
Erwinia billingiae Eb661D8MMS8D8MME2D8MNV6
Serratia proteamaculans 568A8G9G9A8GJZ3A8GFP7
Cronobacter sakazakii ATCC BAA-894A7MGB6A7MIQ1A7MEA9
Pantoea sp. Sc1H8DPK0H8DQ90H8DIH9
Hafnia alvei ATCC 51873G9Y3J7G9Y4J4G9YAM4
Citrobacter rodentium ICC168D2TRY4D2TQ24D2TM58
Shigella flexneri 1235–66I6F1Q5I6GLP1I6HD15
Yersinia enterocolitica 8081A1JJ93A1JR75
Yersinia pestis KIM10+Q7CG58Q8D1R6
Dickeya dadantii 3937E0SJX0E0SHF6
Table 5
HADDOCK docking statistics for ensemble 20 lowest-energy DolP-DPC micelle solution structures calculated.
Experimental parameters*
Ambiguous distance restraints19 including NH of I20, G120-T130, V132-Q135, T138, S139, and NHε of W127
Number of flexible residues50 (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
Erestraints22.30 ± 4.29
Buried surface area (Å2)2186.78 ± 133.277
  1. * 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.

Data availability

Structural data have been deposited in PDB under the accession code 7A2D and the BMRB 19760. All data generated or analysed during this study are included in the manuscript and supporting files. We have supplied original images in the source data as appropriate. We have also supplied input data for Figure 1B in Supplementary file 1 and raw data for mass spectrometry results in Supplementary file 2.

The following data sets were generated
  1. 1
  2. 2
    Biological Magnetic Resonance Data Bank
    1. JA Bryant
    2. FC Morris
    3. TJ Knowles
    4. R Maderbocus
    5. E Heinz
    6. G Boelter
    7. D Alodaini
    8. A Colyer
    9. PJ Wotherspoon
    10. KA Staunton
    11. M Jeeves
    12. DF Browning
    13. YR Sevastsyanovich
    14. TJ Wells
    15. AE Rossiter
    16. VN Bavro
    17. P Sridhar
    18. DG Ward
    19. ZS Chong
    20. ECA Goodall
    21. C Icke
    22. ACK Teo
    23. SS Chng
    24. DI Roper
    25. T Lithgow
    26. AF Cunningham
    27. M Banzhaf
    28. M Overduin
    29. IR Henderson
    (2020)
    ID bmrbId=19760. Structure-function analyses of dual-BON domain protein DolP identifies phospholipid binding as a new mechanism for protein localisation to the cell division site.

Additional files

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
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
Transparent reporting form
https://cdn.elifesciences.org/articles/62614/elife-62614-transrepform-v2.docx

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