Not All Bacterial Outer-Membrane Proteins Are β-Barrels

Abstract

The discovery of Wza, an octomeric helical barrel integral bacterial outer-transmembrane protein, has challenged the widely held understanding that all integral outer-membrane proteins of Gram-negative bacteria are closed β-barrels composed of transmembrane β- strands. Wza is a member of the Outer-Membrane Polysaccharide Exporter family and our bioinformatics analysis suggests that other members of the family may also contain outer-membrane transmembrane segments that are helical. A review of the literature indicates that in addition to Wza, outer-membrane core complex proteins of the type IV secretion systems also contain transmembrane segments that are helical.

Figure 1. Alpha Helical Transmembrane Segments in Bacterial Outer-Membrane Proteins

(A) Representative structures of three classes of bacterial outer-membrane proteins. Structure of the porin OmpF (PDB ID: 2OMF monomer) where the transmembrane (TM) region is composed of a 16- stranded β-barrel formed from a single polypeptide chain. Structure of the Curli secretion channel CsgG (PDB ID: 4UV3) where the TM region is composed of TM β-strands from nine individual polypeptide chains combining to form a 36-stranded β-barrel. Structure of the group 1 capsular polysaccharide transporter Wza (PDB ID: 2J58) where the TM region is composed of α-helical TM segments from eight polypeptide chains combining to form an α-helical barrel. (B) Sequence identity (top row in each entry) and similarity (bottom row in each entry) of group A, B, C, D, E, and F OPX proteins. Group A: Wza (UniProt-P0A930), AmsH (Q46629), EpsA (Q45407); Group B: BexD (P22236), CtrA (P0A0V9), PssN (Q27SU9), VexA Q04976); Group C: GumB (Q456768), HfsD (Q(A5L5); Group D: ExoF (Q02728), AceH (Q8RR80); Group E: OntA (Q56653); Group F: KpsD (Q03961). OPX proteins from groups A, B, C, and D show high sequence similarity to each other (ranging from 40 to 85% ) and exhibit more than 25 % sequence similarity to Wza. Group E and F proteins are much larger than other OPX proteins and exhibit less similarity to Wza. (C) Predicted secondary structure distribution of thirteen OPX proteins representing the six OPX classes. β-strands are shown as yellow arrows while helices are shown as pink rectangles. Secondary structure predicted using PsiPred. All proteins except ExoF and AceH from group D indicate the presence of a C-terminal segment (*). MPEx and PsiPred also predict these regions to be possible TM segments. (D) C-terminal helical TM segments predicted in representative examples of OPX proteins. The transmembrane segment of Wza, the only OPX protein whose structure is known, is located at the C-terminus of the protein. We used PsiPred (MEMSAT SVM) and MPEx to identify any possible C-terminal TM segments present in OPX proteins. We also predicted the secondary structure distribution of these proteins using PsiPred and identified the most C-terminal helix. All of the proteins investigated, except the two from group D, contain a C-terminal helical region and these are predicted to be possible TM regions. (E) Structure of the outer-membrane core complex of T4SS of Xanthomonas citri (PDB ID 6GYB). Side (left) and top (right) view of the complex composed of VirB9 and VirB10 are shown in ribbon representation. The outer-membrane α-helical transmembrane segments of VirB10 are shown in magenta. The TM helices form two concentric rings that line the pore of the T4SS outer-membrane channel. VirB10 appears to be present in nearly all T4SS complexes (Sheedlo 2022) and therefore, the presence of helical outer-membrane segments may be a common feature of T4SSs. (F) Inner membrane insertion potential (ΔG _app ) of helical TM segments of outer-membrane proteins. Almost all of the TM segments bitopic inner membrane proteins (BIMP) inserted into the bacterial inner membrane (by the translocon) exhibit ΔG _app <0 (left, black circles) while the ΔG _app values of the α-helical TM segments of outer-membrane OPX (orange circles) and T4SS proteins (blue for helix 1 and pink, for helix 2, circles) have ΔG _app >0 suggesting that the helical TM segments from the outer-membrane proteins investigated here have low inner membrane insertion potential. (G) Hydrophobic moment (amphipathicity) of helical TM segments of outer-membrane proteins. Helical TM segments from the OPX proteins and TM helix 2 of the T4SSs have hydrophobic moments that are on average larger than those observed for the helical TM segments of bitopic inner membrane proteins. It has been suggested that the amphipathic nature of the helical TM segments facilitate membrane insertion (Dunstand 2015, Jeeves 2015). In F and G the bars represent the means of each dataset.