Crystal structure and thermodynamic analysis of diagnostic mAb 106.3 complexed with BNP 5-13 (C10A).
B-type natriuretic peptide (BNP) is a naturally secreted regulatory hormone that influences blood pressure and vascular water retention in human physiology. The plasma BNP concentration is a clinically recognized biomarker for various cardiovascular diseases. Quantitative detection of BNP can be achieved in immunoassays using the high-affinity monoclonal IgG1 antibody 106.3, which binds an epitope spanning residues 5-13 of the mature bioactive peptide. To understand the structural basis of this molecular recognition, we crystallized the Fab fragment complexed with the peptide epitope and determined the three-dimensional structure by X-ray diffraction to 2.1 A resolution. The structure reveals the detailed interactions that five of the complementarity-determining regions make with the partially folded peptide. Thermodynamic measurements using fluorescence spectroscopy suggest that the interaction is enthalpy driven, with an overall change in free energy of binding, DeltaG = -54 kJ/mol, at room temperature. The parameters are interpreted on the basis of the structural information. The kinetics of binding suggest a diffusion-limited mechanism, whereby the peptide easily adopts a bound conformation upon interaction with the antibody. Moreover, comparative analysis with alanine-scanning results of the epitope explains the basis of selectivity for BNP over other related natriuretic peptides.
Longenecker
Kenton L
KL
Advanced Technology, Global Pharmaceutical Research and Development, Abbott Park, Illinois 60064, USA.
Ruan
Qiaoqiao
Q
Fry
Elizabeth H
EH
Saldana
Sylvia C
SC
Brophy
Susan E
SE
Richardson
Paul L
PL
Tetin
Sergey Y
SY
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000936:Antigen-Antibody Complex / Q000737:chemistry*; D002460:Cell Line; D018360:Crystallography, X-Ray; D000939:Epitopes / Q000737:chemistry; D051379:Mice; D020097:Natriuretic Peptide, Brain / Q000737:chemistry*; D011487:Protein Conformation; D013816:Thermodynamics
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D020097:Natriuretic Peptide, Brain