Stabilization of antibody structure upon binding epitope peptide: x-ray, microcalorimetry and molecular dynamics study


P. Mader1, J. Brynda1, V. Král1, P. Řezáčová1,2, R. Štouračová1,3, M. Fábry1, J. Závada1, Z. Závadová1, L. Rulíšek4, M. Kožíšek4, J. Sedláček1


1Institute of Molecular Genetics, Flemingovo nám. 2, CZ-166 37 Praha 6

2UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, Texas 75390

3University of Colorado at Denver, Health Sciences Center, 4200 E. Ninth Ave. Denver, CO 80262

4Institute of Organic Chemistry and Biochemistry, Flemingovo nám. 2, CZ-166 37 Praha 6


Among fourteen human carbonic anhydrases, only the CA IX isoform is strongly associated with certain types of cancer. A unique structural feature of CA IX is its extra component - the proteoglycan-like (PG) segment, located at the amino terminus of the molecule. Conceivably, this segment could be of relevance for oncogenesis, therefore it deserves a more detailed investigation. 

The PG amino acid sequence [Q16790; gi:5915865] comprises 59 residues:




A remarkable feature of the PG segment of the CA IX molecule is a high content of dicarboxylic amino acids (27 D + E out of total 59 residues) and a low content of basic ones (4 R + K). Most of the dicarboxylic amino acids are grouped in four identical repeats of the motif GEEDLP (bold) and in its three modified versions. The epitope peptide used in this study (underlined) seems to be an adequate structural representation of the protein antigen since predictions show lack of any secondary structure in the PG domain. Abnormal expression of CA IX in various commonly occurring carcinomas suggests its involvement in oncogenic pathways. CA IX is also a cell adhesion molecule (CAM) that can mediate attachment of cells to non‑adhesive solid support [1]. For a secondary structure-lacking, flexible peptide it might be intuitively expected that such ligand would adopt a complementary shape and undergo stabilization in the complex with its cognate antibody: this would be accompanied with a loss of the conformational freedom and with an unfavorable entropy contribution, measurable with microcalorimetry methods. We show here that upon the epitope peptide binding a substantial structural re-arrangement occurs also in one of the antibody hypervariable loops and that the accompanying local stabilization can be traced in comparisons of the crystal structures of the free and complexed antibody. Somewhat unexpectedly, all substantial 3D structural transitions occur in the hypervariable loops other than those that form counterparts of the epitope dicarboxylic amino acid residues and that provide for major enthalpy contributions.

1.     J. Závada, Z. Závadová, J. Pastorek, Z. Biesová, J. Ježek, J. Velek, Br J. cancer., 82, (2000), 1808-13.