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.2017 Aug 3;12(8):e0182016.
doi: 10.1371/journal.pone.0182016. eCollection 2017.

An evolutionarily conserved glycine-tyrosine motif forms a folding core in outer membrane proteins

Affiliations

An evolutionarily conserved glycine-tyrosine motif forms a folding core in outer membrane proteins

Marcin Michalik et al. PLoS One..

Abstract

An intimate interaction between a pair of amino acids, a tyrosine and glycine on neighboring β-strands, has been previously reported to be important for the structural stability of autotransporters. Here, we show that the conservation of this interacting pair extends to nearly all major families of outer membrane β-barrel proteins, which are thought to have originated through duplication events involving an ancestral ββ hairpin. We analyzed the function of this motif using the prototypical outer membrane protein OmpX. Stopped-flow fluorescence shows that two folding processes occur in the millisecond time regime, the rates of which are reduced in the tyrosine mutant. Folding assays further demonstrate a reduction in the yield of folded protein for the mutant compared to the wild-type, as well as a reduction in thermal stability. Taken together, our data support the idea of an evolutionarily conserved 'folding core' that affects the folding, membrane insertion, and thermal stability of outer membrane protein β-barrels.

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Conflict of interest statement

Competing Interests:The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. OmpX structure.
(A) The topology model of OmpX with the highlighted mortise-tenon motif in red (Y80) and blue (G112) on the 5th and 6th β-strands. Residues forming the β-strands are represented as squares, while loop residues are shown as circles (adapted from [24]).(B) The OmpX solution NMR structure in 3D representation using PyMOL [PDB code 2MNH].(C) The mortise-tenon motif involves residues Y80 (red) and G112 (blue). The tyrosine’s aromatic side chain is positioned in a planar manner over the glycine in the neighboring strand (G112).
Fig 2
Fig 2
(A) Alignments of the mortise-tenon regions in 8-stranded OMP β-barrels. There is 100% conservation of the glycine (dark blue, strand 6th) and very high conservation of the tyrosine (red, strand 5th) in the mortise-tenon motif. Alignments were prepared using PROMALS3D web server; the predicted β-strands are colored in blue, and the consensus secondary structure prediction is given below the alignment.OmpX—APC51088.1 Outer membrane protein X [Escherichia coli str. K-12 substr. W3110];HypoProt_1—WP_039105847.1 hypothetical protein [Frischella perrara];HypoProt_2—WP_044834085.1 hypothetical protein [Thalassomonas actiniarum];OmpA—BAA35715.1 Outer membrane protein A [Escherichia coli str. K-12 substr. W3110];OmpW—CDO14140.1 Outer membrane protein W [Klebsiella pneumoniae];PagL_1—BAR70092.1 PagL [Pseudomonas aeruginosa];PagL_2—AGW82166.1 PagL [Bordetella avium];Ail—AAB36601.1 Ail [Yersinia pseudotuberculosis];NspA—CAX50548.1 NspA [Neisseria meningitidis 8013];OprF—AFM37279.1 OprF [Pseudomonas aeruginosa];OmpP5—AAA03346.1 outer membrane protein P5 [Haemophilus influenzae];MotB—WP_012154185.1 flagellar motor protein MotB [Shewanella pealeana];OmpF—WP_013260126.1 Outer membrane porin F [gamma proteobacterium HdN1];OmpA-Like—WP_011053841.1 OmpA-like protein [Buchnera aphidicola];PagC—WP_067708083.1 PagC [Erwinia sp. ErVv1];TailProt—CSH02213.1 Tail assembly protein [Shigella sonnei];VirProt—WP_038626525.1 virulence protein [Pantoea sp. PSNIH2];InvProt—SDG65145.1 attachment invasion locus protein [Vibrio xiamenensis].(B) Structures of different 8-stranded β barrel proteins. The 5th and 6th β-strands are highlighted in yellow and the mortise-tenon motif is highlighted in blue and red accordingly. PDB codes: NspA [1P4T]; OmpA[1QJP]; OmpX[2MNH]; OprF[4RLC]; PagL[2ERV].
Fig 3
Fig 3
(A) Alignments of the mortise-tenon motifs in 16-stranded OMP β-barrels. Three conserved motifs are present on the 1st and 2nd β-strands, 3rd and 4th β-strands and on the 15th and 16th β-strands. The color scheme is the same as in Fig 2.OmpF—AJW81160.1 OmpF [Yersinia ruckeri];OmpD—GAL44027.1 outer membrane porin OmpD [Citrobacter werkmanii NBRC 105721];MProt_1—WP_052899673.1 membrane protein [Erwinia iniecta];MProt_2—WP_045859316.1 membrane protein [Raoultella terrigena];MProt_3—WP_034791683.1 membrane protein [Ewingella americana];MProt_4—WP_024486820.1 membrane protein [Serratia fonticola];MProt_5—WP_028724068.1 membrane protein [Pantoea ananatis];Porin_1—SCX53975.1 Outer membrane protein (porin) [Variovorax sp. EL159];Porin_2—WP_005794534.1 porin [Acidovorax delafieldii];Porin_3—WP_003058791.1 porin [Comamonas testosteroni];Porin_4—WP_011146030.1 porin [Photorhabdus luminescens];Porin_5—WP_035609637.1 porin [Edwardsiella ictaluri];Porin_6—WP_034397082.1 porin [Delftia acidovorans];OmpC_1—WP_019081949.1 porin OmpC [Yersinia enterocolitica];OmpC_3—WP_058683256.1 porin OmpC [Enterobacter cloacae];PhoE_1—WP_002439127.1 phosphoporin PhoE [Shimwellia blattae];PhoE_2—WP_017456205.1 phosphoporin PhoE [Kosakonia sacchari];PhoE_3—WP_072077160.1 phosphoporin PhoE [Salmonella enterica];PhoE_4—WP_032085013.1 phosphoporin PhoE [Escherichia coli].(B) Structures of different 16-stranded β barrel proteins. The 1st-2nd, 3rd-4th and 15th-16th β-strands are highlighted in yellow and the mortise-tenon motifs are highlighted in blue and red accordingly. The amino-acid pairs forming the motif are given under the names of the proteins. PDB codes: OmpK36 [1OSM]; PhoE [1PHO]; Omp32 [2FGQ]; OmpC [2J1N]; OmpF [3NSG].
Fig 4
Fig 4
(A) Alignments of the mortise-tenon motifs in 22-stranded OMP β-barrels. Two conserved motifs are present on the 9th and 10th β-strands and on the 15th and 16th β-strands. Compared to 8-stranded proteins, the glycine in the motif is also completely conserved, while the tyrosine in the motif is less conserved, with the aromatic residues phenylalanine and tryptophan also present at a higher frequency. The color scheme is the same as in Fig 2.ShuA—AIN19718.1 outer membrane heme receptor ShuA [Yersinia kristensenii];FetA—AHW74795.1 FetA [Neisseria meningitidis];CirA—CDO15561.1 cirA [Klebsiella pneumoniae];PdtK—ABC68350.1 PdtK [Pseudomonas putida];TonB-dep—WP_043878013.1 TonB-dependent receptor [Pectobacterium atrosepticum];FauA—AAD26430.1 ferric alcaligin siderophore receptor FauA [Bordetella pertussis];FpvA—AOX26958.1 FpvA [Pseudomonas aeruginosa];TbpA—AAF81744.1 transferrin-binding protein A [Neisseria meningitidis];HasR_1—CAE46936.1 hasR [Serratia marcescens];HasR_2—AIN17416.1 hasR protein [Yersinia kristensenii];PirA—CFU88752.1 ferric enterobactin receptor PirA [Pseudomonas aeruginosa];PiuA—SCM64637.1 PiuA putative outer membrane ferric siderophore receptor [Pseudomonas aeruginosa];FecA—AAL08456.2 FecA [Shigella flexneri 2a];FhuA_1—CDO15921.1 fhuA [Klebsiella pneumoniae];FhuA_2—ANK05474.1 fhuA [Escherichia coli O25b:H4];FepA—ADB98042.1 FepA [Escherichia coli];BtuB_1—CDO16333.1 btuB [Klebsiella pneumoniae];BtuB_2—AEV65209.1 BtuB [Pseudomonas fluorescens F113];BtuB_3—EFE98543.1 btuB [Escherichia coli FVEC1412].(B) Structures of different 22-stranded OMP β-barrels. The 9th-10th and 15th-16th β-strands are highlighted in yellow and the mortise-tenon motifs are highlighted in blue and red accordingly. The amino acid pairs forming the motif are given under the names of the proteins. Observed mortise-tenon pairs that were at non-conserved positions are marked with an astrix. PDB codes: BtuB [1NQE]; FpvA [2O5P]: FauA [3EFM]; PirA [5FR8]; HasR [3CSN]; ShuA [3FHH].
Fig 5
Fig 5
Raw stopped-flow data and exponential fits for wt (panel A) and Y80G OmpX (panel B). The raw data is shown in black, the single-exponential fit in blue, and the double-exponential fit in red. Clearly, a single exponential fit is not sufficient to describe the data. Panel C shows the difference between wt and Y80G OmpX double exponential fits (see alsoTable 2).
Fig 6
Fig 6.In vitro folding experiments for wt (WT) and Y80G OmpX (MT) at different temperatures.
Protein samples in urea were diluted 1:20 into detergent-containing buffer, and incubated for 2h on ice, and at 20, 30, 40 and 50°C. The negative controls (boiled) in the gels are detergent-refolded samples which were boiled excessively at 100°C, while heated refolded samples (boil+20°C) are samples that after heating to 100°C for 1h to denature were shifted to 20°C for another hour to test for reversible refolding. The folded fraction (F) is represented by the upper band, while the unfolded fraction (U) is the lower band.
Fig 7
Fig 7. Heat stability for wt and Y80G OmpX monitored by gel shifts.
Proteins were refoldedin vitro prior to incubation at 85°C for the indicated time. The denatured OmpX appears as the lower band (U) on the gel, while folded OmpX is shifted to the upper band (F). An estimation of folded/unfolded yields of proteins can be made by comparing the intensities of the two bands. In the second lane of both gels OmpX samples were boiled at 100°C for 30 min before loading as a control.
Fig 8
Fig 8. Stability of wt and Y80G OmpX as a function of urea concentration.
As OmpX unfolds, the 2 tryptophan residues are exposed to a hydrophilic environment, resulting in a shift of the fluorescence maximum to higher wavelengths (336 nm to 356 nm), indicating that the protein has unfolded. Three technical replicates were used, and the averaged results are shown with standard deviation.
Fig 9
Fig 9. The folding of OmpXin vivo andin vitro.
Highlighted in white spheres in both folding models are where similarities between the two folding processes may be present.In vivo: The BAM and Skp-assisted folding of OmpX into the OM.(A) OmpX is held in a flexible, relatively packed state by the Skp complex.(B) Recognition of the β-signal by the BAM complex leads to its release from Skp, and delivery to the BAM Complex. Possible interactions between the periplasmic leaflet and the β -signal and region 73–82 may also occur.(C) The BAM-assisted folding and insertion of OmpX into the OM is stabilized by the G112-Y80 motif by aligning strands 5 and 6.(D) OmpX is released from the BAM complex, and the fully-folded protein is thermally stabilized by the G112-Y80 motif.In vitro:(B) OmpX is expected to be extended in 8M urea compared to the Skp-bound form. Residual secondary structure and micelle interactions are present in the β-signal and region 73–82.(C) Upon urea dilution, OmpX will collapse and simultaneously fold into the DHPC micelle. This process is mediated by Y80G-Y112 motif, which kinetically increases the speed of folding by aligning strands 5 and 6. Strands 1 and 8 come together after folding of the protein core, followed by loop formation and a stable hydrogen bond network.(D) The final structure is thermally stabilized by the G112-Y80 motif.
Fig 10
Fig 10
(A) Alignments of the mortise-tenon motif in VDAC proteins. The conserved motif is located on the 13th and 14th β-strands, and most commonly consists of a threonine and a tyrosine. The color scheme is the same as in Fig 2.Mouse_VDAC—AAB47777.1 isoform 1 [Mus musculus];Human_VDAC_1—AAA61272 isoform 1 [Homo sapiens];Human_VDAC_2—AAB59457.1 isoform 2 [Homo sapiens];Human VDAC_3—AAB93872.1 isoform 3 [Homo sapiens];Elephant_VDAC—XP_010588919.1 isoform 1 [Loxodonta africana]; Platypus_VDAC—XM_007668866.2 isoform 1 [Ornithorhynchus anatinus]; Croc_VDAC—XP_019390612.1 isoform 1 [Crocodylus porosus];Gecko_VDAC—AAW79047.1. isoform 1 [Gekko japonicus];Mallard_VDAC—XP_005011296.1 isoform 1 [Anas platyrhynchos];Zebrafish_VDAC—AAH42329.1 isoform 2 [Danio rerio];Yeast_VDAC—AAA35208.1 isoform 1 [Saccharomyces cerevisiae (strain ATCC 204508 / S288c)];Cobra_VDAC—ETE66681.1 isoform 3 [Ophiophagus hannah];Nematode_VDAC—CDP92897.1 isoform 1 [Brugia malayi];Rice_VDAC—CAB82853.1—isoform 1 [Oryza sativa subsp. japonica].(B) Available VDAC structures. The 13th and 14th β-strands containing the motif are highlighted in yellow with the marked mortise-tenon pair in blue and red respectively. The amino acid pair forming the motif is given under the names of the proteins. PDB codes: Mouse VDAC1 [3EMN]; Human VDAC1 [2JK4]; Zebrafish VDAC2 [4BUM].
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Funding to DL from the German Science Foundation (DFG Li 1673/4-1) and from the Department of Biosciences, University of Oslo, and funding to MOR from the Norwegian Research Council (NFR 240909), is gratefully acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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