TLS ANALYSIS of ANTIBODY STRUCTURE SHOWS STABILIZATION by EPITOPE BINDING
Jiří Brynda1,
Vlastimil Král1, Pavel Mader1, Renata Šťouračová1, Milan
Fábry1, Magdaléna Hořejší1, Pavlína Řezáčová1,
Lubomír Rulíšek2, Milan Kožíšek3, Jan Závada4
and Juraj Sedláček1
1Department of Recombinant Expression and Structural Biology, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, 166 37 Prague, Czech Republic
2Gilead & IOCB Research Center, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, 166 10 Prague, Czech Republic
3Department of Protein Biochemistry, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, 166 10 Prague, Czech Republic
4Department of Cellular and Viral Genetics, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, 166 37 Prague, Czech Republic
Specific antibodies interfere with function
of human carbonic anhydrase IX (CA IX), and attract attention as tools for
anti-cancer interventions. This work presents a comparison between structural
elements and thermodynamic parameters of association of an antibody fragment,
Fab M75 [1], to a peptide representing its epitope in the proteoglycan-like
domain of CA IX. Comparisons
of crystal structures of free and liganded Fab fragment reveal major re‑adjustments
of H1 and H3 CDR loops. In contrast, shapes and positions of H2 and L2 CDR
loops remain unaltered and their positively charged residues may thus present a
fixed frame for epitope recognition and for the consecutive induced fit.
Attainment of the altered H3 CDR loop conformation in the complex structure is
accompanied with evident local stabilization, i.e. with decreased mobility, as
measured with residual atom displacement parameters (ADPs). Analysis of domain
mobility with translation-libration-screw (TLS) parameterization shows that librations of entire heavy chain variable domain (VH)
become decreased in magnitude and reoriented in the complex. This effect corresponds well with extensive
involvement of the heavy chain in the ligand binding. Isothermal titration
microcalorimetry (ITC) [2] yields high unfavorable entropy term, attributable,
in part, to local stabilization of the H3 CDR loop. Molecular dynamic
simulations indicate major roles for the charged residues of epitope.
1. Zavada, J., Zavadova, Z., Pastorek, J., Biesova, Z., Jezek, J.,
& Velek, J. (2000) Br. J. Cancer, 82, 1808-1813.
2. Jelesarov, I., & Bosshard, H.R. (1999). J. Mol. Recognit. 12, 3-18.