Structure of the haptoglobin–haemoglobin complex

Abstract

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 β-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 β-subunits of haemoglobin, explaining the tight binding between haptoglobin and haemoglobin. The haemoglobin-interacting region in the αβ dimer is highly overlapping with the interface between the two αβ 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).

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

“…The Hp-Hb-HpHbR structure also for the first time reveals the structure of the human Hp-Hb complex that has previously failed to form high-resolution diffracting crystals 32 . The human Hp-Hb is essentially similar to the recently determined porcine Hp-Hb complex 30 with two CCP domains merging into a CCP fusion domain via b-strand swapping (Fig. 1a).…”

“…The gene encoding Hpr has arisen by a duplication of the Hp gene in primate evolution 38 . The Hpr amino-acid sequence is 91% identical to Hp, and the two proteins are predicted to have nearly identical three-dimensional structures 30 . Despite their structural similarity and common ability to bind Hb, Hp and Hpr serve quite different biological functions.…”

“…Hp (isoform 1) is a bivalent molecule owing to the association of its complement control protein (CCP) domains 29 . Each Hp moiety forms a tight interaction with an abHb-dimer via its serine protease-like (SP) domain and the Hp-Hb complex has a barbell-like structure with abHb loaded at the ends 30,31 . Our structure reveals that the dimeric Hp-Hb is recognized by two HpHbR receptors interacting with both the Hp SP domains and the b-subunits of Hb (Fig.…”

Hemolysis and free hemoglobin revisited: exploring hemoglobin and hemin scavengers as a novel class of therapeutic proteins

“…30,31 The recently resolved crystal structure of the porcine Hb:Hp complex provided some structural basis for the protection of critical amino acids that are primary targets of globin oxidation. 32 As a result of this protection, globin oxidation with subsequent protein degradation does not occur when Hb is sequestered in the Hb:Hp complex. 33 The structural stability of the complex may prevent accumulation of proinflammatory Hbdegradation products that can evade clearance by scavenger receptors.…”

Efficient Covalent Bond Formation in Gas-Phase Peptide–Peptide Ion Complexes with the Photoleucine Stapler

“…An entirely different approach to studying noncovalent protein-ligand and protein-protein complexes relies on mass spectrometry that has been used to study their stoichiometry [16,17], stability [18][19][20], and thermochemistry in the gas phase [21,22]. Formation of covalent bonds in gas-phase ion complexes has been reported for several systems that relied on collision-induced chemical reactions between an anion and a cation in a complex that mimicked chemical methods used in solution [23,24].…”