Structural basis for trypanosomal haem acquisition and susceptibility to the host innate immune system.

Sleeping sickness is caused by trypanosome parasites, which infect humans and livestock in Sub-Saharan Africa. Haem is an important growth factor for the parasites and is acquired from the host by receptor-mediated uptake of haptoglobin (Hp)-haemoglobin (Hb) complexes. The parasite Hp-Hb receptor (HpHbR) is also a target for a specialized innate immune defence executed by trypanosome-killing lipoprotein particles containing an Hp-related protein in complex with Hb. Here we report the structure of the multimeric complex between human Hp-Hb and Trypanosoma brucei brucei HpHbR. Two receptors forming kinked three-helical rods with small head regions bind to Hp and the ß-subunits of Hb (ßHb), with one receptor at each end of the dimeric Hp-Hb complex. The Hb ß-subunit haem group directly associates with the receptors, which allows for sensing of haem-containing Hp-Hb. The HpHbR-binding region of Hp is conserved in Hp-related protein, indicating an identical recognition of Hp-Hb and trypanolytic particles by HpHbR in human plasma.

Structure of the haptoglobin-haemoglobin complex.

Red cell haemoglobin is the fundamental oxygen-transporting molecule in blood, but also a potentially tissue-damaging compound owing to its highly reactive haem groups. During intravascular haemolysis, such as in malaria and haemoglobinopathies, haemoglobin is released into the plasma, where it is captured by the protective acute-phase protein haptoglobin. This leads to formation of the haptoglobin-haemoglobin complex, which represents a virtually irreversible non-covalent protein-protein interaction. Here we present the crystal structure of the dimeric porcine haptoglobin-haemoglobin complex determined at 2.9 Å resolution. This structure reveals that haptoglobin molecules dimerize through an unexpected ß-strand swap between two complement control protein (CCP) domains, defining a new fusion CCP domain structure. The haptoglobin serine protease domain forms extensive interactions with both the α- and ß-subunits of haemoglobin, explaining the tight binding between haptoglobin and haemoglobin. The haemoglobin-interacting region in the αß dimer is highly overlapping with the interface between the two αß dimers that constitute the native haemoglobin tetramer. Several haemoglobin residues prone to oxidative modification after exposure to haem-induced reactive oxygen species are buried in the haptoglobin-haemoglobin interface, thus showing a direct protective role of haptoglobin. The haptoglobin loop previously shown to be essential for binding of haptoglobin-haemoglobin to the macrophage scavenger receptor CD163 (ref. 3) protrudes from the surface of the distal end of the complex, adjacent to the associated haemoglobin α-subunit. Small-angle X-ray scattering measurements of human haptoglobin-haemoglobin bound to the ligand-binding fragment of CD163 confirm receptor binding in this area, and show that the rigid dimeric complex can bind two receptors. Such receptor cross-linkage may facilitate scavenging and explain the increased functional affinity of multimeric haptoglobin-haemoglobin for CD163 (ref. 4).