Alternate arrangement of PpL B3 domain and SpA D domain creates synergistic double-site binding to VH3 and Vkappa 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 (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.
Jiang
Shaohua H
SH
Department of Microbiology, Second Military Medical University, Shanghai, China.
Wang
Jufang F
JF
Xu
Rong
R
Liu
Yanjun J
YJ
Wang
Xiaoning N
XN
Cao
Jie
J
Zhao
Ping
P
Shen
Yijun J
YJ
Yang
Tong
T
Yang
Hua
H
Jia
Jiaan A
JA
Chen
Qiuli L
QL
Pan
Wei
W
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
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