Structural basis for iron piracy by pathogenic Neisseria

Abstract

Neisseria are obligate human pathogens causing bacterial meningitis, septicaemia and gonorrhoea. Neisseria require iron for survival and can extract it directly from human transferrin for transport across the outer membrane. The transport system consists of TbpA, an integral outer membrane protein, and TbpB, a co-receptor attached to the cell surface; both proteins are potentially important vaccine and therapeutic targets. Two key questions driving Neisseria research are how human transferrin is specifically targeted, and how the bacteria liberate iron from transferrin at neutral pH. To address these questions, we solved crystal structures of the TbpA-transferrin complex and of the corresponding co-receptor TbpB. We characterized the TbpB-transferrin complex by small-angle X-ray scattering and the TbpA-TbpB-transferrin complex by electron microscopy. Our studies provide a rational basis for the specificity of TbpA for human transferrin, show how TbpA promotes iron release from transferrin, and elucidate how TbpB facilitates this process.

Figure 1. Crystal structure of the TbpA-(apo)hTF complex

a, The TbpA beta barrel is lime, the plug is red, the helix finger is magenta. For hTf, the C-lobe is gold and the N-lobe is salmon. A ferric ion has been modeled into the C-lobe as a red sphere. b, Electrostatic potential of TbpA viewed from the extracellular surface with hTf shown in gold ribbon. c, Residues of TbpA that bind hTf: gold, hydrophobic interactions; green, hydrogen bonds; red, salt bridges (residues labeled). d, Electrostatic potential of hTf viewed from the extracellular surface with TbpA shown in green ribbon. e, Surface representation of hTf showing regions that bind TbpA.

Figure 2. TbpA distorts the iron coordination site in the hTf C-lobe by inserting a helix from extracellular loop three

a, TbpA (green) inserts a helix finger from extracellular loop 3 (magenta) into the cleft of the hTf C-lobe (gold). b, The helix finger interacts with the hTf C-lobe residues through mainchain and sidechain interactions. c, The long TbpA plug loop (pink) interacts with residues from the C1 subdomain. d, Comparison of C-lobe conformations for holo (green), apo (gray, PDB code 2HAU), and TbpA-bound Tf (gold). e, Superposition of residues coordinating iron in diferric hTf (gray) with the same residues in hTf when bound to TbpA (gold). Distances for the residues coordinating iron are shown for the TbpA-bound state.

Figure 3. SAXS analysis of the N. meningitidis TbpB-(holo)hTf complex

a, Superposition of TbpB from N. meningitidis with three TbpB structures from porcine pathogens. While C-lobes align closely, the N-lobes show sequence and structural variability. Residues that diminish hTf binding when mutated are shown as spheres. Position 206 (proline in N. meningitidis) is critical for interaction with hTf. b, Competition ELISA showing relative binding affinities of TbpA and TbpB for apo hTf, holo hTf, hTf-Fe_N, and hTf-Fe_C. c, Fitting of the TbpB-(holo)hTf complex model into the averaged ab initio envelope was performed using Chimera. TbpB is shown in cyan, hTf is shown in salmon (N-lobe) and gold (C-lobe).

Figure 4. Analysis of the TbpA-TbpB-(holo)hTf triple complex by negative stain electron microscopy

a, Model of the triple complex. TbpA is green, TbpB is light blue, and hTf is gold. b, Purification of the triple complex by size exclusion chromatography. The Coomassie-stained SDS gel shows purified TbpA in lane 1, the TbpB-(holo)hTf complex in lane 2, and the TbpA-TbpB-(holo)hTf complex in lane 3. c, Row 1: set of seven non-redundant class averages of negatively stained complexes. Row 2: examples of individual images assigned to the respective classes. Row 3: reprojections of the model in the corresponding orientations, band-limited to 15Å resolution; Row 4: surface renderings of the band-limited model in the corresponding orientations.

Figure 5. Mechanism for iron import

a, Binding surfaces of TbpA (green) and TbpB (cyan) mapped onto the hTF C-lobe. b, Enclosed chamber formed by TbpA-TbpB-(holo)hTF (left, magenta sphere). A cutaway view (right) from inside the chamber illustrates the proximity of the iron (red). c, Model for iron release. Conserved K359 in the L3 helix finger is positioned to interact with residues that regulate iron release in eukaryotic iron uptake. d, Import of iron through TbpA. (I) An electrostatic surface depicts cavities between the TbpA barrel and plug domain. (II) Plug domain constrictions close the tunnel. (III) MD simulations show removal of constrictions upon interaction with TonB.