1 Nature 2012 Vol: 489(7416):456-459. DOI: 10.1038/nature11369

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[thinsp]A resolution. This structure reveals that haptoglobin molecules dimerize through an unexpected [bgr]-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 [agr]- and [bgr]-subunits of haemoglobin, explaining the tight binding between haptoglobin and haemoglobin. The haemoglobin-interacting region in the [agr][bgr] dimer is highly overlapping with the interface between the two [agr][bgr] 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 [agr]-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).

Editor's summary

How haptoglobin neutralizes free haemoglobin The release of extracellular haemoglobin into the plasma is potentially hazardous because the exposed haem group is highly reactive and therefore toxic. The circulating protein haptoglobin counters this by soaking up free haemoglobin in a stable complex that is cleared from the blood through its binding to the macrophage scavenger receptor CD163. This paper presents the 2.9-ångström crystal structure of the dimeric porcine haptoglobin–haemoglobin complex. The structure provides a mechanism for haptoglobin-mediated recognition of haemoglobin.

Mentions
Figures
Figure 1: Crystal structure of porcine Hp–Hb. a, Structure of porcine Hp–Hb. Hp is coloured blue and cyan, αHb and βHb are orange. Haem groups are shown as dark grey sticks. Red spheres represent Fe ions. Glycosylations are shown as light grey sticks and disulphide bridges as yellow sticks. b, Stick representation of the B1/B2-strands (blue and cyan) of Hp CCP domains. c, Interface between Hp CCP and the Hp serine protease (SP) domain. Hp SP is coloured dark blue and Hp CCP light blue. The opposing Hp CCP domain is coloured cyan. Dashed lines represent hydrogen bonds and numbers indicate bond lengths (Å). d, Structure of the Hp serine protease domain loop 3 region. Residues important for interaction with the CD163 scavenger receptor3 are shown as sticks. Figure 2: The Hp-bound Hb dimer is in its oxy-state. a, Ultraviolet–visible spectra of a porcine Hp–Hb crystal (black line), porcine Hp–Hb in solution (red lines) and porcine Hb in solution (blue lines). b, c, Superimposition of porcine Hp-bound αHb (b) or βHb (c) (orange), oxygenated human αHb (b) or βHb (c) (blue, PDB accession 2DN1), and deoxygenated αHb (b) or βHb (c) (light blue, PDB accession 2DN2). Haem groups, oxygen molecules, proximal histidines (His 87 in αHb and His 92 in βHb) and distal histidines (His 58 in αHb and His 63 in βHb) are shown as sticks. Fe ions are shown as spheres. Figure 3: The Hb contact area overlaps with the Hb dimer contact area in Hb tetramers. a, Surface representation of Hb in the Hp–Hb complex. Residues within 3.8 Å of the Hp serine protease domain are coloured blue. b, Surface representation of human α1β1Hb in oxygenated tetrameric Hb (PDB accession 2DN1). α1Hb residues within 3.8 Å of β2Hb, and β1Hb residues within 3.8 Å of α2Hb, are coloured red. c–f, Selected interactions between αHb (c, d) or βHb (e, f) and the Hp serine protease domain. Residues from the Hp serine protease domain are shown as light blue sticks, and Hb residues as light orange sticks. Dashed lines represent electrostatic interactions or hydrogen bonds. Figure 4: Hb residues prone to oxidative modifications and SAXS. a, Human Hb residues prone to oxidative modifications indicated on the structure of porcine Hp–Hb (green spheres, corresponding porcine residues in parentheses). b, SAXS curves of porcine Hp–Hb (pHp–Hb), human Hp–Hb (hHp–Hb) and CD163 SRCR 1–5 bound to hHp–Hb. Circles represent experimental data and lines theoretical intensities. The structure of pHp–Hb with modelled glycosylations gives the best fit to the experimental data. AU, arbitrary units. c, Ab initio modelling of hHp–Hb bound to CD163 SRCR 1–5. The modelling on the basis of imposed P2 symmetry (a, Supplementary 1) was performed using the structure of pHp–Hb with modelled glycosylations and the structure of M2BP (PDB accession 1BY2).
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References
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