A New Method for Mapping Discontinuous Antibody Epitopes to Reveal Structural Features of Proteins

Abstract

Antibodies that bind to protein surfaces of interest can be used to report the three-dimensional structure of the protein as follows: Proteins are composed of linear polypeptide chains that fold together in complex spatial patterns to create the native protein structure. These folded structures form binding sites for antibodies. Antibody binding sites are typically "assembled" on the protein surface from segments that are far apart in the primary amino acid sequence of the target proteins. Short amino acid probe sequences that bind to the active region of each antibody can be used as witnesses to the antibody epitope surface and these probes can be efficiently selected from random sequence peptide libraries. This paper presents a new method to align these antibody epitopes to discontinuous regions of the one-dimensional amino acid sequence of a target protein. Such alignments of the epitopes indicate how segments of the protein sequence must be folded together in space and thus provide long-range constraints for solving the 3-D protein structure. This new antibody-based approach is applicable to the large fraction of proteins that are refractory to current approaches for structure determination and has the additional advantage of requiring very small amounts of the target protein. The binding site of an antibody is a surface, not just a continuous linear sequence, so the epitope mapping alignment problem is outside the scope of classical string alignment algorithms, such as Smith-Waterman. We formalize the alignment problem that is at the heart of this new approach, prove that the epitope mapping alignment problem is NP-complete, and give some initial results using a branch-and-bound algorithm to map two real-life cases. Initial results for two validation cases are presented for a graph-based protein surface neighbor mapping procedure that promises to provide additional spatial proximity information for the amino acid residues on the protein surface.

Site-Specific Inhibitors of NADPH Oxidase Activity and Structural Probes of Flavocytochrome b: Characterization of Six Monoclonal Antibodies to the p22phox Subunit

“…Current efforts are directed at sequencing large numbers of selected phage clones for each of the p22 phox -specific mAbs for attempts to identify residues involved in complex epitopes by the recently described computational method FINDMAP (39,40).…”

“…2. Concerning mAb NS5, preliminary analysis of Ͼ100 selected phage clones with the program FINDMAP (39,40) suggests that in addition to p22 phox 78 KLFGPF 83 , residues 51 LLEYPRG 57 might also contribute to a complex epitope (A. Jesaitis, unpublished data).…”

C-Terminal Tail Phosphorylation of N-Formyl Peptide Receptor: Differential Recognition of Two Neutrophil Chemoattractant Receptors by Monoclonal Antibodies NFPR1 and NFPR2

“…4 show the NFPR1-and NFPR2-selected nonapeptide phage sequences, respectively, aligned according to a visual correspondence of the consensus to specific regions of the predicted cytoplasmic domain of FPR. To confirm this alignment, phage sequences were also examined by the program FINDMAP, which can identify linear and complex epitopes of anti-protein Abs (37,42). Fig.…”

Equilibrium between Metarhodopsin-I and Metarhodopsin-II Is Dependent on the Conformation of the Third Cytoplasmic Loop

“…This technique rests upon the primary immunological principle that the antigen recognition site of an antibody is complementary to the structure of the antigen and thus reflects the local topological features of its epitope (24,26,28). A combination of techniques is used to access this structural information.…”