1,3Petr Kolenko, 1Jan Dohnálek, 2Renata Štouračová, 1Tereza Skálová,

1Galina Tiščenko, 1Jarmila Dušková, 1Jindřich Hašek. Mail to:


1 Institute of Macromolecular Chemistry, Academy of Sciences of CR

2 Institute of Molecular Genetics, Academy of Sciences of CR

3 Faculty of Nuclear Sciences and Physical Eng., Czech Technical University of Prague


Immunoglobulines play an irreplaceable role in the immunity system in all higher organisms. Because of its role in activation of immunological reaction against infected cells, immunoglobulines are routinely used in medical applications. There is a very reliable way of IgG purification based on its affinity to the B fragment of the protein A from Staphylococcus Aureus. However, the costs of production of protein A are significant and thus a new method of rapid and cheap production of purified immunoglobulines in non-denaturating conditions is worth of our special interest. Any highly selective separation process should be based on molecular recognition between a specially designed ligand and the immunoglobulin surface.

In spite of an immense diversity of Fab fragments containing the hypervariable regions (responsible for molecular recognition of antibody) at the antigen binding sites, the immunoglobulines of the same  type (e.g. IgG2b) share very similar aminoacid sequences of Fc fragment. The object of our interest is Fc-fragment, because of its invariant structure. Explication of structure of this fragment and its interaction with other molecules can help in design of polymer sorbents for affinity chromatography.


Structure determination

Monoclonal antibody, class IGg2b was cleaved by papain and purified in 4C on BioLOGIC LP System(BIORAD) using protein A Sepharose column (Biorad). The measured crystal was grown under the following conditions:

Reservoir: HEPES pH 7.5, PEG 2000 20 % (w/v). Drop: 1 ml of reseivoir solution plus 1 ml of  protein 8 mg/ml, PBS (phosphate buffer saline) pH 7.5. Cryoprotectant: 20 % glycerol.

The diffracted intensities (total 34029 independent reflections) was collected at the ID-29 beamline at the synchrotron ESRF in Grenoble with the diffraction limit 2.2 Å. The measured crystal (a triangle platelet 0.3 x 0.1 x 0.02 mm) was flash cooled to 100 K. Space group is C2, the unit cell a=135,73 Å, b=62,75 Å, c=69,81 Å, β=103,35°. Data reduction was performed by program package HKL (Denzo, Scalepack, Xdisp) /1/. The phase problem was solved by molecular replacement (program AMORE /2/) using the structure model 1IGT taken from PDB /7/ (sequence similarity 79 %). The other data processing was done mostly using the program package CCP4 /3/. The structure refinement was done by program REFMAC /4/. Manual corrections were done by program XtalView /5/, the solvent water molecules determined with help of ARP/wARP /6/. The methods used were similar as described in /9/. The final R factors are R=0.19,  Rfree=0.25. The refined structure satisfies all criteria set by program Procheck /7/ .

Both protein chains of the Fc fragment IgG2b (residues from Gly125A to Arg331A and Gly125B to Arg331B) were uniquely identified in the maps of electron density (except of conformational alternatives evident at side chains of several residues. The oligosaccharide chains joining the two protein chains were taken from the protein structure database PDB /8/ - the structure of immunoglobulin IgG2a (PDB code 1I1C). Both chains -NAG1(FUC)-NAG2-MAN3(MAN-NAG-GAL)-MAN4-NAG5  are chemically bound to Fc fragment through asparagines Asn185A and Asn185B. The MAN-NAG-GAL branch of both oligosaccharide chains has contacts to its own protein chain only. The chains -MAN3-MAN4-NAG5 of both Fc fragment halfs form several hydrogen bonds joining thus both CH2 domains together.



Fig. 1. Schematic view of the Fc fragment of  mouse immunoglobulin IgG2b. Each of the protein chains A,B (upper and lower) divides into two compact domains Cg2 (loosely joined together by non-covalent interactions of oligosaccharide chains [black lines] - left side) and more compact dimer of two Cg3 domains (right side). Small spheres are water molecules in positions stabilized by hydrogen bridges to the molecular complex.



A detailed structure of the intact Fc fragment of IgG2b in buffer with pH 7.5 determined in this paper is important for elucidation of the structure changes and the interactions with proteins possessing high selectivity for Fc fragment surface. These include complement components responsible for immunological response, viral proteins (protein A, protein G,...), cell surface receptors and specially designed molecules suitable for highly efficient separation of immunoglobulines by affinity chromatography.

The project is supported by MSMT - 1K05008.


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