Structural basis for haem piracy from host haemopexin by Haemophilus influenzae

Abstract

Haemophilus influenzae is an obligate human commensal/pathogen that requires haem for survival and can acquire it from several host haemoproteins, including haemopexin. The haem transport system from haem-haemopexin consists of HxuC, a haem receptor, and the two-partner-secretion system HxuB/HxuA. HxuA, which is exposed at the cell surface, is strictly required for haem acquisition from haemopexin. HxuA forms complexes with haem-haemopexin, leading to haem release and its capture by HxuC. The key question is how HxuA liberates haem from haemopexin. Here, we solve crystal structures of HxuA alone, and HxuA in complex with the N-terminal domain of haemopexin. A rational basis for the release of haem from haem-haemopexin is derived from both in vivo and in vitro studies. HxuA acts as a wedge that destabilizes the two-domains structure of haemopexin with a mobile loop on HxuA that favours haem ejection by redirecting key residues in the haem-binding pocket of haemopexin.

Figure 1. X-ray structure of the HxuA molecule.

(a) Cartoon representation of HxuA, a right-handed β-helix. Parallel β-sheet PB1 is coloured in magenta, PB2 in blue, PB3 in green and the extra-helix strand motifs in brown. The α-helix elements H1–H6 are shown in purple. (b) Enlarged view of α-helix H1, which splits PB1 into two parts. Together with the extra motifs β37/β38 and the swap of β40 and β41, this element induces a twist in the β-helix, highlighting the separation between the secretion domain and the functional domain. (c) Enlarged view of loop 706–731, in red, referred to as the M loop, which undergoes an important conformational change during complex formation.

Figure 2. Interaction of HxuA with haemopexin and its domains.

Interaction of HxuA with haemopexin (Hpx) and the N and C-terminal fragments of haemopexin (NtHpx and CtHpx, respectively), was measured by ITC. The upper part shows the heat signal for the titration. The lower part of each panel shows the binding isotherm derived from the heat signal, together with the fit calculated with Origin software.

Figure 3. TEM analysis of the HxuA-NtHpx and HxuA-Hpx complexes.

(a) Micrograph of the negatively stained HxuA-NtHpx complex. The arrows indicate the protein particles adsorbed on the carbon film. The right panel shows a representative gallery of class averages from 7,104 particle projections analysed using the EMAN2 software. (b) Micrograph of the negatively stained HxuA-Hpx complex. The arrows indicate the protein particles adsorbed on the carbon film. The right panel shows a representative gallery of class averages from 9,847 particle projections analysed using the EMAN2 software. In c, the arrow indicates the extra density clearly visible on the side views of the HxuA-Hpx complex (bottom) compared with the HxuA-NtHpx complex (top panel). The scale bar in a is 50 nm. The boxes are 18 nm in a and b and 20 nm in c.

Figure 4. X-ray structure of the HxuA-NtHpx complex.

(a) Cartoon representation of HxuA (silver) in complex with the N-terminal domain of haemopexin (NtHpx; orange). The secondary structure elements in HxuA that provide residues for the interaction with residues from blades 3 and 4 of NtHpx are coloured blue. The M loop is coloured red. (b) Surface representation of the complex between HxuA (blue) and NtHpx (orange). (c) Enlarged view of the interaction zone. The side chains of residues involved in hydrogen bonds or salt bridges formation is represented in sticks and coloured as in 4 A side chains of residues Glu713 and Asp726 are shown in green. (d) Superposition of full-length haem-haemopexin (1QHU, in grey) with the structure of the HxuA-NtHpx complex (in blue and orange), showing the potential steric clashes of the C-terminal domain of haemopexin with HxuA (pale blue) and of haem with the M loop of HxuA (red). (e,f) Enlarged view of the region of interaction involving the M loop together with the reorientation of side chains of Arg174 and Arg185 from haem-haemopexin. The dashes represent atoms located within hydrogen bonding distances.

Figure 5. Phenotypes of HxuA mutants.

(a) Amount of HxuA in the various strains, as detected by immunoblotting with anti-HxuA antibodies on whole-cell contents analysed by SDS-PAGE (control, WTHxuA, HxuA^Asp726Ala, HxuA^Glu713Ala and HxuA^DEL). The equivalent of 0.05 OD_600nm (10⁷ cells) was deposited in each lane. Known amounts of purified HxuA were run alongside. The scale on the left (m) represents molecular weight in kDa. (b) Detection of interaction of biotinylated haemopexin with HxuA and its mutants in whole cells by dot-blot analysis in vivo (control, wild-type, HxuA^Asp726Ala, HxuA^Glu713Ala, HxuA^DEL). Samples were spotted in duplicate. The equivalent of 0.05 OD_600nm (10⁷ cells) was deposited in each spot, in duplicate. Known amounts of purified biotinylated haemopexin were run alongside, in duplicate. (c) Analysis of the in vivo activities of HxuA, HxuA^DEL, HxuA^Glu713Ala and HxuA^Asp726Ala, in haem acquisition tests from haem and haem-haemopexin. Each well contained 100 μl of either 5 μM haem (He) or 5 μM haem-haemopexin (He-Hpx). The bottom panel shows the same activity test for haem (He), haem-haemopexin (He-Hpx) and the biotinylated haem-haemopexin (He-HpxB) for WT strain.