Fig. 4: Biochemical and structural characterization of the furin cleavage site in flaviviruses.

a Peptide substrates for furin activity assays were designed using the DABCYL-EDANS FRET pair. Each substrate includes five amino acids flanking both sides of the scissile bond to mimic the natural cleavage context. b, c The pH profile of each substrate was determined at a fixed concentration close to its Km at pH 7.0: HSRRSRRSLT (2.5 μM), HSKRSRRSIA (3 μM), EGSRSRRSVL (8 μM), and EGSRTRRSVL (5 μM). Reactions were performed in triplicate, and data are shown as mean ± SD. d, The prM/E heterodimer. The left panel shows the prM/E protomer from the experimental cryo-EM structure of an immature particle of TBEV at neutral pH (PDB 8PUV36). The two other panels show AlphaFold predictions, the middle panel is a prediction of the prM/E heterodimer, the right panel is the prediction of a (prM/E)2 dimer, but only one prM/E protomer is shown, for clarity (see Supplementary Fig. 4c). The limits of the linker region containing FCS are indicated by two stars (yellow and green) in the left panel. A dotted ribbon in the left panel (near the green star) indicates a disordered region of the linker. The framed region is zoomed beneath each panel, slightly rotated for clarity. A red arrow points to the scissile bond, showing that it is located at the site where the zipper enters the groove underneath the E protein. The side chains of residues discussed in the text are shown as sticks, including Asn255 in E and the hydrogen bonds it makes drawn in dotted lines. The side chain of Tyr255 is also drawn, to show that the polypeptide chain of the linker, in between residues 82 and 87, interferes with contacts with Pro210 (in the second protomer, not shown) across the E dimer interface. The experimental structure is displayed as ribbons colored coded according to temperature (B) factors in the order lowest-blue < cyan < green < yellow < red-highest. The AF3 predictions are shown color-coded by plDDT (orange < 50, yellow >  50, cyan > 70, blue > 90). Schematic elements in panel a were created in BioRender. Ruzek, D. (https://BioRender.com/4n4b27g).