Structural studies of M75 Fab fragment in complex with its epitope peptide
P. Mader, R. ouračová, J. Brynda, J. Závada
Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, CZ - 166 37 Prague 6, Czech Republic
The poster describes structural elements and thermodynamics of association of an antibody, M75, to an epitope peptide derived from its protein antigen, proteoglycan-like (PG) segment of human carbonic anhydrase IX (CA IX). 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:
SSGEDDPLGEEDLPSEEDSPREEDPPGEEDLPGEEDLPGEEDLPEVKPKSEEEGSLKL
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 support1. 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.
Acknowledgment. The research was supported
by the Grant Agency of the Czech Republic (project 203/02/0405).