Fig. 2: Restoration of HTRA1 assembly and activity via protein-based complementation.

a Structures of wt HTRA1 (gray; PDB ID 3TJO), R274Q (orange; PDB ID 6Z0E), and D174R-R274Q (green; PDB ID 6Z0X; 6Z0Y) in S328A background. Mutants are shown in an overlay with wt. Panel 1: similar to wt HTRA1, R274Q and D174R-R274Q were crystallized in their trimeric state, superimposing well as seen for the aligned aromatic residues of the central hydrophobic core. Panels 2’–2”’: salt bridges in the HTRA1 protomer interface. Asterisks denote residues on adjacent protomers. The side chains at positions 174, 177, and 274 were well defined by electron density allowing their direct comparison. b Proteolytic activity (β-casein degradation) of D174R-R274Q (1 µM), and of D174R-S328A (0.33–3 µM) and R274Q (1 µM) mixed at increasing molar ratios. The activity of R274Q (1 µM) and D174R-S328A (1 µM) serve as controls. Data are representative of 2 independent experiments. c Cartoons of cis- and trans-complementation. D174R-R274Q assembles as an active homotrimer (complementation in cis); R274Q (monomeric, inactive) and D174R-S328A (trimeric, inactive) assemble as proteolytically active heterotrimers (complementation in trans). d, e Oligomeric states of R274Q and D174R-S328A alone or in combination. d Representative SEC-MALS UV chromatograms of R274Q, D174R-S328A and D174R-S328A + R274Q (mixed at a 1:1 ratio) depicted in overlay. The calculated molecular weight of mono-, di- and trimeric HTRA1 species is indicated. e Left panel: representative native MS spectrum of D174R-S328A (1.5 µM) mixed with R274Q (2 µM). Mono- and trimeric species and charge states are labeled. Right panel: titration of D174R-S328A (1.5 µM) with R274Q. R274Q dimers (<9%) are not depicted. The mean abundance of oligomeric species is depicted and error bars indicate SD (n = 3 independent datasets). Source data are provided as a Source Data file.