Properties and Phylogeny of 76 Families of Bacterial and Eukaryotic Organellar Outer Membrane Pore-Forming Proteins

Abstract

We here report statistical analyses of 76 families of integral outer membrane pore-forming proteins (OMPPs) found in bacteria and eukaryotic organelles. 47 of these families fall into one superfamily (SFI) which segregate into fifteen phylogenetic clusters. Families with members of the same protein size, topology and substrate specificities often cluster together. Virtually all OMPP families include only proteins that form transmembrane pores. Nine such families, all of which cluster together in the SFI phylogenetic tree, contain both α- and β-structures, are multi domain, multi subunit systems, and transport macromolecules. Most other SFI OMPPs transport small molecules. SFII and SFV homologues derive from Actinobacteria while SFIII and SFIV proteins derive from chloroplasts. Three families of actinobacterial OMPPs and two families of eukaryotic OMPPs apparently consist primarily of α-helices (α-TMSs). Of the 71 families of (putative) β-barrel OMPPs, only twenty could not be assigned to a superfamily, and these derived primarily from Actinobacteria (1), chloroplasts (1), spirochaetes (8), and proteobacteria (10). Proteins were identified in which two or three full length OMPPs are fused together. Family characteristic are described and evidence agrees with a previous proposal suggesting that many arose by adjacent β-hairpin structural unit duplications.

Fig 1. Binary alignment of a member of the BRP family (1.B.4) with a member of the CPP1 family (1.B.43).

The alignment was generated with the GSAT program and shows the full sequences of both proteins. Residue numbers are indicated at the beginning and end of each line. A vertical line indicates an identity, and a colon indicates a similarity. The comparison score is provided in Table 4.

Fig 2. Illustration of the use of the Superfamily Principle to establish homology between two proteins in different families that have shown insufficient sequence similarity to allow demonstration of homology by direct comparison.

A, B, and C show alignments of proteins A with B, B with C and C with D, respectively. Protein A, 1.B.24.1.2; Protein B, Req1, obtained with Protocol 1 with 1.B.24.1.2 as the query sequence. Protein C, Nbr4, obtained with Protocol 1 with 1.B.58.1.2 as the query sequence, Protein D; 1.B.58.1.2. Comparison scores are provided in Table 4.

Fig 3. Topological comparison between proteins in Superfamily I ([] ) and those not in a superfamily (| = |).

A single established or predicted topology is included for each family, although it is possible that some families include members with more than one topology (see Table 1).

Fig 4. AveHAS plots of representative OMPP families showing upper light line, average amphipathicity; upper dark line, average hydropathy, and lower light line, average sequence similarity.

Numbers above the hydropathy plot indicate the known positions of the β-strands. (A) the OOP Family (TC #1.B.6.1); (B) the GBP Family (1.B.1.1); (C) the SP Family (1.B.3.1); and (D) the KdgM Family, (TC #1.B.35.1). β-TMS positions are based on high resolution X-ray crystallographic structures of representative family members. The alignments upon which these plots were based included all proteins within the indicated subfamily in TCDB. Note the correlation between the peaks of hydropathy (middle plots), and the peaks of sequence similarity (lower plots).

Fig 5. Phylogenetic tree based on the SuperfamilyTree programs (SFT1 and SFT2) for Superfamily I, showing the estimated family relationship based on tens of thousands of BLAST bit scores and the consensus of 100 trees.

Fig 6

Phylogenetic trees based on the SuperfamilyTree I program for Superfamily II (A) and Superfamily III (B). The TC numbers of the proteins in TC subclass 1.B are provided.

Fig 7. Putative β-hairpin repeats in the β-barrel OMPP, 1.B.1.2.1.

Repeats were identified using the HHRepID program and aligned using the MEME program. Residue position is indicated on the left. Positions with conservation are shaded as dictated by the program.