Fig. 1. LC8 and RavP colocalize in cells and form a tight complex in vitro.

(A) Representative crystal structure of an LC8 dimer (protomers shown in shades of red). Partners bind along the noted groove and form a β-strand in each groove. (B) Domain organization for RavP showing the dimerization domain (DD, PDB 3L32) and the C-terminal domain (CTD, PDB 1VYI) in orange and green, respectively, as well as the predicted N-terminal helix (NTH, grey). Interacting partners are listed below the domain architecture diagram. The recognition motif for LC8 is underscored in red, and expanded above. Black lines denote predicted disorder (Linkers 1 and 2). (C) Confocal analysis of representative HeLa-LC8-GFP cells with RavP staining. (Top) Cells were infected with RAV-CVS11 at an MOI of 10 for 48 hours. Co-transfection of pTIT-RavN and pTIT-RavL with pTIT-RavP-WT (Middle) or pTIT-RavP-AAA (Bottom) proceeded for 48 hours. P was stained with a rabbit polyclonal antibody followed by incubation with Alexa-568 goat anti-rabbit IgG. White arrows denote the smaller Negri body-like structures seen during transfections, which are magnified in the inset. DAPI was used to stain the nuclei (Blue). (D) ITC thermogram for LC8 interactions with RavP, collected at 25 °C in 50 mM NaCl, 50 mM sodium phosphate, 1 mM NaN₃, pH 7.5, and fit to a single-site binding model. This is a tight interaction with an affinity of 82 nM (See also Table S1). (E) Molecular mass of RavP and LC8-RavP complex measured by SEC-MALLS. The lines show the elution profile monitored by refractive index (left axis) for RavP (black) and the RavP-LC8 complex (red). The crosses show the molecular mass (right axis) derived from MALLS and refractometry measurements. The molecular mass calculated for dimeric RavP is 70.7 kDa (black line and crosses) and the heterotetrameric LC8-RavP is 94.8 kDa (red line and crosses).