title: Alternate arrangement of PpL B3 domain and SpA D domain creates synergistic double-site binding to VH3 and Vkappa regions of fab.
abstract: 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 (V(H)3-V(kappa)I) than to KM41 (V(H)1-V(kappa)III) or KM36 (V(H)3-V(kappa)III). 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 V(H)3 and V(kappa). Thus, our results indicated for the first time that alternate arrangement of B3 and D domains creates synergistic double-site binding to V(H)3 and V(kappa) regions of fragment of antigen binding. The different competitive inhibition pattern of binding of LD5 to scFv KM38 by 4L + SpA suggested strict use of antibody conformation for this simultaneous double-site binding. The demonstration of this novel binding property would promote to achieve the designed hybrid IBPs for useful immunological applications.
authors.affiliation: Department of Microbiology, Second Military Medical University, Shanghai, China.
authors.forename: Shaohua H
authors.initials: SH
authors.lastname: Jiang
authors.forename: Jufang F
authors.initials: JF
authors.lastname: Wang
authors.forename: Rong
authors.initials: R
authors.lastname: Xu
authors.forename: Yanjun J
authors.initials: YJ
authors.lastname: Liu
authors.forename: Xiaoning N
authors.initials: XN
authors.lastname: Wang
authors.forename: Jie
authors.initials: J
authors.lastname: Cao
authors.forename: Ping
authors.initials: P
authors.lastname: Zhao
authors.forename: Yijun J
authors.initials: YJ
authors.lastname: Shen
authors.forename: Tong
authors.initials: T
authors.lastname: Yang
authors.forename: Hua
authors.initials: H
authors.lastname: Yang
authors.forename: Jiaan A
authors.initials: JA
authors.lastname: Jia
authors.forename: Qiuli L
authors.initials: QL
authors.lastname: Chen
authors.forename: Wei
authors.initials: W
authors.lastname: Pan
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000276:immunology; D001666:Binding Sites, Antibody; D015153:Blotting, Western; D003198:Computer Simulation; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000378:metabolism; D007145:Immunoglobulin kappa-Chains / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000502:physiology; D013205:Staphylococcal Protein A / Q000737:chemistry* / Q000276:immunology
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; C116593:L-protein, Peptococcus magnus; D013205:Staphylococcal Protein A