Figure 4. Topology of the E_A and E_B Regions in the α345 Noncollagenous-1 Hexamer, Structural Determinants for the Binding of Alport Alloantibodies and Goodpasture Autoantibodies In Vitro, and Accessible Surface Area of the E_A-α3 and E_A-α5 Regions of the Noncollagenous-1 Hexamer.

The α345 noncollagenous-1 (NC1) hexamer is composed of two trimeric caps, each consisting of α3NC1 (red), α4NC1 (blue), and α5NC1 (green) subunits (Panel A). Two of the six sulfilimine bonds (S = N) that stabilize the trimer–trimer interface are shown (light yellow). The location and structure of the four homologous regions are also shown: E_A (yellow) and E_B (orange) in the α3NC1 subunit, and E_A (pink) and E_B (purple) in the α5NC1 subunit. Three regions, E_A and E_B in α3NC1 and E_A in α5NC1, become critical parts of the neoepitopes for Goodpasture autoantibodies. The topology of the E_A regions in α3NC1 and α5NC1 is similar, as indicated in the ribbon diagrams (Panel A, bottom), with the characteristic folding pattern of a β-sheet stabilized with a disulfide bond. Ala¹⁹, Gln²⁴, and Gln²⁸ (pink) within the E_A region of α5NC1, exposed in the α345NC1 hexamer, are candidates for the binding of Alport alloantibodies (Panel B, bottom right). In contrast, Leu²⁷ and Val²⁹ (gray) are sequestered by their lateral interaction with the α4NC1 domain, and when exposed as a result of hexamer dissociation, they become critical to the binding of Goodpasture autoantibodies. Dissociation of the sulfilimine-cross-linked hexamer into α35 dimer subunits is concomitant with a conformational change that results in the formation of the neoepitopes encompassing the E_A regions of the α5NC1 and α3NC1 monomers and the binding of their respective autoantibodies (Panel B, bottom left). The accessible surface area of the E_A-α3 region (Panel C, top) and the E_A-α5 region (Panel C, bottom) was calculated for a probe, which mimics the antibody molecule (radius, 9 Å); the area of individual residues in the α345NC1 hexamer (black bars) and the α3NC1/α5NC1 model monomers (gray bars) is shown. An increase in the surface area of the monomers indicates that residues are buried in the hexamer (Val²⁷ and Leu²⁹ in E_A-α3 and Leu²⁷ and Val²⁹ in E_A-α5). In contrast, residues with similar areas within the hexamer and monomers are exposed in the hexamer (Ala¹⁹, Gln²⁴, and Gln²⁸ in E_A-α5).