Alternate Arrangement of PpL B3 Domain and SpA D Domain Creates Synergistic Double-Site Binding to VH3 and Vκ Regions of Fab

In our previous study, a kind of novel hybrid immunoglobulin (Ig)–binding proteins (IBPs) was obtained with the characteristic structure of alternately arranged Finegoldia magna (formerly Peptostreptococcus magnus) protein L (P. magnus protein L, PpL) B3 domain (B3) and Staphylococcal protein A (SpA) D domain (D). In this study, two representative molecules of these novel proteins, LD3 (B3-D-B3) and LD5 (B3-D-B3-D-B3) (LD3/5), showed substantially higher affinity for IgG-F(ab′)2, IgM, and IgA than 4L (B3-B3-B3-B3) or SpA, which were also demonstrated by surface plasmon resonance detection. Further, LD5 showed much stronger binding to single-chain Fv (scFv) KM38 (VH3-VκI) than to KM41 (VH1-VκIII) or KM36 (VH3-VλIII). Competitive inhibition studies showed that 4L alone or in combination with SpA (4L + SpA) was a weaker inhibitor than LD3/5 in inhibiting LD3/5's binding to IgG-F(ab′)2, IgM, or IgA. The computer modeling suggested that the B3-D arrangement in LD3/5 could simultaneously bind to VH3 and Vκ. Thu...