Vip3Aa binds to MAM and CCP domains.

Abstract

(A) Schematic representation of Sf-SR-C, indicating the different domains. Complement control protein (CCP), MAM, somatomedin B (somato), and Ser/Thr rich domains. Sig-seq: signal peptide sequence, TM: transmembrane. The numbers represent the number of amino acids at the position. (B) GST, GST-SR-C-F-1, GST-SR-C-F-2, and GST-SR-C-F-3 proteins were dotted on a PVDF membrane directly and were then probed with Vip3Aa-flag or with Vip3Aa-flag plus unlabeled Vip3Aa without Flag-tag (500-fold), followed by immunoblotting with an anti-Flag antibody. (C) GST-SR-C-F-1, GST-SR-C-F-2, and GST-SR-C-F-3 conjugated to GST-Sepharose affinity beads respectively, and then incubated with Vip3Aa-flag. GST was used as the control and immunoblotting used the anti-Flag antibody. (D) Dot blotting to detect the interaction of Sf-CCP with Vip3Aa-flag; Dm-CCP was used as the control and immunoblotting used the anti-Flag antibody. (E) Pulldown experiments: Vip3Aa-flag conjugated to protein G agarose beads using an anti-flag antibody, and incubated with Sf-CCP-His or Dm-CCP-His, followed by immunoblotting with an anti-His antibody. (F) and (G) The binding affinity of Vip3Aa with CCP (F) and MAM (G) domains of Sf-SR-C were analysed with MST. The labelled Vip3Aa was kept constant at 10 nM, and the CCP and MAM domains are titrated from 0.3 nM to 10 μM, respectively. Fitted binding curves and Kd values (mean ± SD) were derived from three independent experiments.</p