The unique structure of Haemophilus influenzae protein E reveals multiple binding sites for host factors

Abstract

Haemophilus influenzae protein E (PE) is a multifunctional adhesin involved in direct interactions with lung epithelial cells and host proteins, including plasminogen and the extracellular matrix proteins vitronectin and laminin. We recently crystallized PE and successfully collected X-ray diffraction data at 1.8 Å. Here, we solved the structure of a recombinant version of PE and analyzed different functional regions. It is a dimer in solution and in the asymmetric unit of the crystals. The dimer has a structure that resembles a flattened β-barrel. It is, however, not a true β-barrel, as there are differences in both the hydrogen-bonding pattern and the shape. Each monomer consisted of a 6-stranded antiparallel β-sheet with a rigid α-helix at the C terminus tethered to the concave side of the sheet by a disulfide bridge. The laminin/plasminogen binding region (residues 41 to 68) is exposed, while the vitronectin binding region (residues 84 to 108) is partially accessible in the dimer. The dimerized PE explains the simultaneous interaction with laminin and vitronectin. In addition, we found this unique adhesin to be present in many bacterial genera of the family Pasteurellaceae and also orthologues in other, unrelated species (Enterobacter cloacae and Listeria monocytogenes). Peptides corresponding to the surface-exposed regions PE 24 to 37, PE 74 to 89, and PE 134 to 156 were immunogenic in the mouse. Importantly, these peptide-based antibodies also recognized PE at the bacterial surface. Taken together, our detailed structure of PE explains how this important virulence factor of H. influenzae simultaneously interacts with host vitronectin, laminin, or plasminogen, promoting bacterial pathogenesis.

Fig 1

Purification of recombinant PE. (A) The gel filtration profile of the recombinant variant PE (approximately 2 mg protein purified by anion exchange as described in Materials and Methods) was injected into an equilibrated Superdex 200 column. The separation chromatogram of PE is shown, along with a standard molecular weight filtration marker. mAu, milliabsorbance unit. (B) Fractions collected from panel A were separated using 12% SDS-PAGE, followed by staining with Coomassie blue R250. A similar gel filtration profile and purity pattern were also observed with SeMet PE (data not shown). (C) DLS pattern of the recombinant variant PE purified by gel filtration. (D) Transmission electron microscopy showing PE molecules (arrowheads). Bar, 100 nM.

Fig 2

Secondary-structure elements and the PE monomer. (A) PE 28 to 159 amino acids showing the secondary structure. In total, 6 β-strands, 8 loops, and one C-terminal helix exist. (B) In the monomer, 6 antiparallel β-strands form the β-sheet. A longer α-helix packs on the concave face of the sheet, where strands β1, β2, and β3 are curved around it. It is tethered to the β-sheet through a conserved disulfide bond between cysteines 99 and 148.

Fig 3

The PE dimer. (A) Cartoon representation of the PE dimer. (B) Surface of the PE dimer shown from the top and bottom cavities, in addition to the charge distribution of the molecule. The top surface of the molecule is neutral in charge, whereas the bottom side of the dimer is basic. The positive and negative charges are shown in blue and red, respectively.

Fig 4

Dimer interface and PE internal cavities. (A) Stereoview showing side chains in the loop from residues 52 to 61 from one monomer (green A) to main-chain atoms in the strand β6b in the other monomer (red B). Two central residues are AsnA60 and TrpA57. The Nδ2 of AsnA60 forms a hydrogen bond with the backbone oxygen of ThrB131, and Oδ1 makes a hydrogen bond with the backbone nitrogen from the same ThrB131. The side chain of TrpA57 occupies a hydrophobic pocket formed by the aliphatic part of the side chain of Lys B129, the methyl moiety of Thr B105, and the side chain of His A67. The Nε1 of TrpA57 makes a hydrogen bond with the backbone oxygen of LysB129. Other interactions are formed between AspA59 and ThrB131, Gln A61, and Ser133. Tyr A94 from strand β4 completes the main interactions through a hydrogen bond to the Nz of Lys B129. (B) Top and bottom pockets of the dimer (red and blue, respectively). The side chains of Arg108 and Arg121 in each monomer are shown. (C) Schematic drawing of the residues lining the walls of the top pocket of the dimer. The colored halves represent the two monomers, and the colored residues inside the pocket are located at the bottom wall of the pocket.

Fig 5

Binding sites of the various host factors on the PE dimer. (A) Ribbon diagram of a PE dimer that shows the Vn binding region. The two separate monomers are shown in green and light blue, whereas the amino acid region 84 to 108 (based on peptide mapping) is shown in dark magenta and dark blue in the two monomers. (B) Surface of the PE dimer with exposed K85 and R86 side chains, along with other residues. (C) The Ln/plasminogen binding region of PE. The ribbon model shows the Ln/plasminogen binding region in red. The amino acids of the binding region are shown as stick models. For ease of following the chain, several residues along this fragment are numbered. (D) Surface of PE with the Ln and plasminogen binding region indicated in red.

Fig 6

PE homologues also exist in other pathogens. (A) BLAST results revealed that several other pathogens have PE homologues. Retrieved sequences were aligned using the MultAlin online tool (42). Highly conserved amino acids are shown in red, and partially conserved amino acids are in blue. Secondary structures are shown at the top, with beta strands as arrows and helices as cylinders. (B) Identity and similarity matrix of protein sequences analyzed by using the pairwise alignment tool Needle (http://www.ebi.ac.uk). (C) Rooted phylogenetic tree showing 5 different clusters. Haemophilus spp. formed a single cluster, and distantly related sequences of Pasteurella spp. and E. cloacae and L. monocytogenes formed two separate clusters. The sequences were analyzed for phylogenetic relations by using MEGA5 software (43).

Fig 7

PE has a monellin-like fold. Shown are the superimposed structures of monellin (mon) (PDB code 2O9U) chain A (blue) and PE monomer (green). The beta sheet arrangements in the protein backbone are very similar. However, helices are present at the C terminus in PE and at the N terminus in monellin.

Fig 8

Mapping of surface-exposed immunogenic regions using mouse anti-PE peptide Abs. (A) Ribbon diagram showing PE amino acid regions (indicated with different colors) that were selected for immunization of a series of mice. (B) Surface structure of selected regions as shown in panel A. (C) Results from ELISA that demonstrate Ab recognition of peptides with which microtiter plates were coated. (D) Recognition of full-length recombinant PE 22 to 160 by peptide Abs shown in ELISA. (E) Flow cytometry profiles of NTHI showing surface recognition of PE at the surfaces of bacteria using anti-PE peptide Abs.

Fig 9

Schematic representation of the multiple functions of PE. (Left) H. influenzae PE interacts with the host epithelium. The PE molecule mediates binding to the epithelial surface utilizing a hitherto unknown receptor. This interaction contributes to bacterial adhesion and induction of a proinflammatory response by the epithelial cells. PE binds to Ln, which contributes to adhesion of H. influenzae to the basement membrane and the host ECM. In addition to Ln, H. influenzae binds plasminogen by using PE. When plasminogen is bound to PE, it is converted into active plasmin by host urokinase plasminogen activator (uPA) or tissue plasminogen activator (tPA). Active plasmin may help in bacterial invasion and degradation of the ECM. (Right) PE inhibits the MAC at the surface of H. influenzae, which is accomplished by the binding of Vn. Vitronectin is a well-known complement regulator that inhibits the terminal complement pathway by interacting with the C5b-7 complex assembly and also inhibits C9 polymerization during formation of a lytic pore. PE is able to bind both Ln and Vn at the same time, and the two ligands do not interfere with each other; thus, adhesion and MAC inhibition can be carried out simultaneously.