Fig. 4: Interactions of enteroviral 3CD with CLs.

a Schematics of a CVB3 CL construct used for BLI measurements of 3C, 3D, and 3CD proteins. The 3' overhang sequence in the CL construct hybridizes with a 5' biotinylated complementary DNA oligo, allowing the construct to immobilize in streptavidin biosensors. The saturated BLI responses (wavelength shift) generated by the interactions of sensor-immobilized CL for various concentrations of (b) CVB3 3C and (c) 3CD (filled circles) were fitted with a binding isotherm (dotted curve). The reported error in K_ds represents the standard error of the fitting. See Supplementary Fig. 13 and the methods for details. d A model of CVB3 3CD predicted by the AlphFold3, showing 3C and 3D domains connected by a flexible linker. e Superposition of the AlphaFold3-predicted 3CD model with each 3C molecule of the CL-3C complex crystal structure, showing no apparent 3D domain interaction with the CL. f The alignment of the previously determined PV1 3CD crystal structure⁴⁴ with each 3C molecule of the CVB3 CL-3C complex crystal structure shows potential contacts of the linker region in both 3CD molecules with the CL. g The apparent K_d for a mutant CVB3 3CD with E190A and K193A mutations in the linker region decreased significantly compared to the wild-type 3CD. h A proposed model for binding interactions of two 3C domains with CVB3 cre involved in VPg uridylation. The structure of the cre was predicted by AlphaFold 3 and superimposed with the sD region of the CVB3 CL. Each panel and corresponding labels, where appropriate, have been colored analogously for facile comparisons. Source data are provided as a Source Data file.