Structural mechanism of serum amyloid A-mediated inflammatory amyloidosis

Abstract

Serum amyloid A (SAA) represents an evolutionarily conserved family of inflammatory acute-phase proteins. It is also a major constituent of secondary amyloidosis. To understand its function and structural transition to amyloid, we determined a structure of human SAA1.1 in two crystal forms, representing a prototypic member of the family. Native SAA1.1 exists as a hexamer, with subunits displaying a unique four-helix bundle fold stabilized by its long C-terminal tail. Structure-based mutational studies revealed two positive-charge clusters, near the center and apex of the hexamer, that are involved in SAA association with heparin. The binding of high-density lipoprotein involves only the apex region of SAA and can be inhibited by heparin. Peptide amyloid formation assays identified the N-terminal helices 1 and 3 as amyloidogenic peptides of SAA1.1. Both peptides are secluded in the hexameric structure of SAA1.1, suggesting that the native SAA is nonpathogenic. Furthermore, dissociation of the SAA hexamer appears insufficient to initiate amyloidogenic transition, and proteolytic cleavage or removal of the C-terminal tail of SAA resulted in formation of various-sized structural aggregates containing ∼5-nm regular repeating protofibril-like units. The combined structural and functional studies provide mechanistic insights into the pathogenic contribution of glycosaminoglycan in SAA1.1-mediated AA amyloid formation.

Keywords: HDL binding site; heparan sulfate binding site.

Fig. 1.

Monomeric structure of SAA1.1. (A) Ribbon representation of the crystal structure of human SAA1.1 with α-helices 1 (residues 1–27), 2 (residues 32–47), 3 (residues 50–69), and 4 (residues 73–88) and the C-terminal tail (residues 89–104) colored in yellow, blue, cyan, green, and sand, respectively. (B) Schematic illustration of observed SAA, ApoE, and cytokine four–α-helix bundles. Open and filled circles represent helices pointing up and down, respectively, from the plane of the paper. (C) The packing between the four-helix bundle and the C-terminal tail of SAA1.1, with hydrogen bonds and salt bridges shown as dotted lines. (D) Sequence alignment of SAA proteins. The secondary-structure elements (lines indicate loops; cylinders indicate helices) are indicated above the sequence. The lengths of representative SAA1 peptides identified in AA amyloid fibrils are indicated by red arrows. The four-helix bundle and the C-terminal tail (CTL) interface residues of SAA1 are shaded in yellow and green, respectively. The predicted amyloidogenic residues in human SAA1 are highlighted in black boxes. The dimerization and trimerization residues in the SAA1 hexamer are shaded in red and cyan, respectively. The cluster mutation sites at the center pore and apex of the SAA1 hexamer are indicated by purple and blue dots, respectively. The semiinvariant and invariant residues are indicated by dots and asterisks, respectively, under their sequences.

Fig. 2.

Hexameric structure of SAA1. (A) Two orthogonal views of the hexameric SAA1 structure in space group R32. The SAA1 hexamer was centered on the crystallographic threefold axis with a twofold axis-related dimer in the asymmetric unit. (B and C) Trimer interface (B) and dimer interface (C) residues in the SAA1 hexamer. The key contact residues are indicated by sticks in the enlarged views. The buried N-terminal helix 1 is shown as a colored surface.

Fig. 3.

Oligomeric state of SAA1.1. (A) Analytical size-exclusion profiles of _his6SAA1.1 (blue), refolded SAA1.1 (red), and ammonium sulfate-treated SAA1.1 (green) on a Superdex 200 10/30 column. (B) Sedimentation coefficient distribution for _his6SAA1.1 (blue) and the refolded SAA1 at 0.36 mg/mL (black), 0.18 mg/mL (red), and 0.06 mg/mL (cyan). (C) Size-exclusion chromatography comparison of native (blue) and Gn⋅HCl-treated _his6SAA1.1 (green) with refolded SAA1.1 (red) on a Superdex 200 16/60 column. (D) Size-exclusion profile of MBP-SAA1₈₉ protein. (A and D) (Insets) SDS gels of the corresponding samples. mAU, milliabsorption unit.

Fig. 4.

Interaction of SAA1 with HDL and heparin. (A and B) Electrostatic surface representation showing the positively charged patches near the center (A) and apex (B) of the SAA1.1 hexamer. (C) Cartoon of HDL and heparin binding sites on SAA. (D) Concentration-dependent binding of wild-type SAA (black) and the center (red) and apex (green) charge cluster mutants to immobilized heparin as measured by Biacore. RU, response units. (E) Binding of recombinant hexameric (black), trimeric (red), and monomeric (green) SAA1.1, acute-phase serum SAA (cyan), and MBP-SAA1₈₉ (blue) to HDL. (F) The binding of wild-type (black, red, pink), center mutant (green, blue), and apex mutant (cyan) to immobilized HDL in the absence (black, green, cyan) or presence (red, blue) of soluble heparin or in the presence of soluble HDL (pink). The error bars are calculated as SDs of three experiments.

Fig. 5.

Amyloidogenic core of SAA1 and amyloid formation. (A) Amyloid formation of SAA1 peptides using ThT binding assays. (B) EM images of fibrils formed by helix 1 (Upper) and 3 (Lower, and the fibrils are indicated by arrows) peptides. (C) Binding of ThT to hexameric (green, red) and trimeric (blue) SAA1.1 in the presence (green) and absence of cathepsin B. ThT alone is shown in black. (D) Binding of ThT to 70 µg/mL aggregated (black) or monomeric (red) MBP-SAA1₈₉, hexameric SAA1.1 (green), MBP (blue), and aggregated IgG₁ (pink). (E) EM image of negatively stained _his6SAA1 protein (Top) and the class averaging of _his6SAA1 particles (Middle). (Bottom) Surface rendering of the _his6SAA1 hexamer in two views, with each trimer colored in green and blue. (F) EM image of negatively stained MBP-SAA1₈₉ oligomers (Upper) and their class average (Lower). (G) Images of MBP-SAA1₈₉ aggregates showing repeating nods in either circular or linear assemblies. (Scale bars, 20 nm.)