O. Vanek1
, P. Novak2, V. Kopecky Jr.3, V. Kren2, J. Brynda4, K. Bezouska1,2


1 Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 8,

CZ-12840 Prague 2

2 Institute of Microbiology, Academy of Sciences of the Czech Republic, Videnska 1083, CZ-14220 Prague 2

3 Institute of Physics, Faculty of Mathematics and Physics, Charles University in Prague, Ke Karlovu 5, CZ-12116 Prague 2

4 Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo namesti 2, CZ-16636 Prague 6


CD69 protein [1], an early activation antigen of human lymphocytes, is one of the most studied surface receptor molecules involved in tumour recognition by natural killer cells. Although there is certain knowledge about its function from immunological point of view, detailed structural description of target recognition by natural killer cells is missing, including identification of CD69 natural ligand. CD69 receptor belongs to a group of C-type lectin-like lymphocyte receptors and till now several types of possible ligands have been identified or proposed: calcium cation, various carbohydrate structures and even peptides. Crystal structures of carbohydrate recognition domain (CRD) of CD69 molecule have been already published [2,3], but crystallization conditions used does not favour ligand binding. In this work we present our structural approach to describe CD69 binding properties. After recombinant production and optimalization of in-vitro refolding of soluble CRD domain of CD69 protein, we analyzed its homogeneity by FT-ICR mass spectrometry and its secondary structure was determined by drop coating deposition Raman spectroscopy method [4]. Further, ligand binding was examined both by Raman spectroscopy and by protein crystallization. We tested several types of proposed CD69 ligands, from single monosaccharide ligand N-acetyl-D-glucosamine, to more complex structures, such as antenary oligosaccharides isolated from hen egg white protein ovomucoid, synthetic peptidomimetic ligands based on calixarene core, or heptapeptide ligand from mycobacterial heat shock protein hsp60, which all binds with high affinity to CRD domain of CD69 receptor. Comparison between computational docking model of calcium binding and observed results by crystallography is discussed.


This work was supported by Ministry of Education of the Czech Republic No. MSM 0021620808, by Institutional Research Concept No. AVOZ 50200510, by Grant Agency of the Czech Republic No. 301/05/P567, and by Grant Agency of the Academy of Sciences of the Czech Republic No. A5020403.


[1] J. Pavlicek, B. Sopko, R. Ettrich, V. Kopecky Jr., V. Baumruk, P. Man, V. Havlicek, M. Vrbacky, L. Martinkova, V. Kren, M. Pospisil, K. Bezouska, Biochemistry 42 (2003) 9295-9306.

[2] K. Natarajan, M. W. Sawicki, D. H. Margulies, R. A. Mariuzza, Biochemistry 39 (2000) 14779-14786

[3] A. S. Llera, F. Viedma, F. Sanchez-Madrid, J. Tormo, J. Biol. Chem. 276 (2000) 7312-7319

[4] D. Zhang, Y. Xie, M. F. Mrozek, C. Ortiz,V. J. Davisson, D. Ben-Amotz, Anal. Chem. 75 (2003) 57035709.