The structure of complement C3b provides insights into complement activation and regulation.

The human complement system is an important component of innate immunity. Complement-derived products mediate functions contributing to pathogen killing and elimination. However, inappropriate activation of the system contributes to the pathogenesis of immunological and inflammatory diseases. Complement component 3 (C3) occupies a central position because of the manifold biological activities of its activation fragments, including the major fragment, C3b, which anchors the assembly of convertases effecting C3 and C5 activation. C3 is converted to C3b by proteolysis of its anaphylatoxin domain, by either of two C3 convertases. This activates a stable thioester bond, leading to the covalent attachment of C3b to cell-surface or protein-surface hydroxyl groups through transesterification. The cleavage and activation of C3 exposes binding sites for factors B, H and I, properdin, decay accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46), complement receptor 1 (CR1, CD35) and viral molecules such as vaccinia virus complement-control protein. C3b associates with these molecules in different configurations and forms complexes mediating the activation, amplification and regulation of the complement response. Structures of C3 and C3c, a fragment derived from the proteolysis of C3b, have revealed a domain configuration, including six macroglobulin domains (MG1-MG6; nomenclature follows ref. 5) arranged in a ring, termed the beta-ring. However, because neither C3 nor C3c is active in complement activation and regulation, questions about function can be answered only through direct observations on C3b. Here we present a structure of C3b that reveals a marked loss of secondary structure in the CUB (for 'complement C1r/C1s, Uegf, Bmp1') domain, which together with the resulting translocation of the thioester domain provides a molecular basis for conformational changes accompanying the conversion of C3 to C3b. The total conformational changes make many proposed ligand-binding sites more accessible and create a cavity that shields target peptide bonds from access by factor I. A covalently bound N-acetyl-l-threonine residue demonstrates the geometry of C3b attachment to surface hydroxyl groups.

Crystal structure of human apolipoprotein A-I: insights into its protective effect against cardiovascular diseases.

Despite three decades of extensive studies on human apolipoprotein A-I (apoA-I), the major protein component in high-density lipoproteins, the molecular basis for its antiatherogenic function is elusive, in part because of lack of a structure of the full-length protein. We describe here the crystal structure of lipid-free apoA-I at 2.4 A. The structure shows that apoA-I is comprised of an N-terminal four-helix bundle and two C-terminal helices. The N-terminal domain plays a prominent role in maintaining its lipid-free conformation, indicating that mutants with truncations in this region form inadequate models for explaining functional properties of apoA-I. A model for transformation of the lipid-free conformation to the high-density lipoprotein-bound form follows from an analysis of solvent-accessible hydrophobic patches on the surface of the structure and their proximity to the hydrophobic core of the four-helix bundle. The crystal structure of human apoA-I displays a hitherto-unobserved array of positively and negatively charged areas on the surface. Positioning of the charged surface patches relative to hydrophobic regions near the C terminus of the protein offers insights into its interaction with cell-surface components of the reverse cholesterol transport pathway and antiatherogenic properties of this protein. This structure provides a much-needed structural template for exploration of molecular mechanisms by which human apoA-I ameliorates atherosclerosis and inflammatory diseases.

Structures of apolipoprotein A-II and a lipid-surrogate complex provide insights into apolipoprotein-lipid interactions.

Apolipoproteins A-I and A-II form the major protein constituents of high-density lipid particles (HDL), the concentration of which is inversely correlated with the frequency of heart disease in humans. Although the physiological role of apolipoprotein A-II is unclear, evidence for its involvement in free fatty acid metabolism in mice has recently been obtained. Currently, the best characterized activity of apolipoprotein A-II is its potent antagonism of the anti-atherogenic and anti-inflammatory activities of apolipoprotein A-I, probably due to its competition with the latter for lipid acyl side chains in HDL. Many interactions of apolipoprotein A-I with enzymes and proteins involved in reverse cholesterol transport and HDL maturation are mediated by lipid-bound protein. The structural bases of interaction with lipids are expected to be common to exchangeable apolipoproteins and attributable to amphipathic alpha-helices present in each of them. Thus, characterization of apolipoprotein-lipid interactions in any apolipoprotein is likely to provide information that is applicable to the entire class. We report structures of human apolipoprotein A-II and its complex with beta-octyl glucoside, a widely used lipid surrogate. The former shows that disulfide-linked dimers of apolipoprotein A-II form amphipathic alpha-helices which aggregate into tetramers. Dramatic changes, observed in the presence of beta-octyl glucoside, might provide clues to the structural basis for its antagonism of apolipoprotein A-I. Additionally, excursions of individual molecules of apolipoprotein A-II from a common helical architecture in both structures indicate that lipid-bound apolipoproteins are likely to have an ensemble of related conformations. These structures provide the first experimental paradigm for description of apolipoprotein-lipid interactions at the atomic level.