Human protein CD69 studied by combination of vibrational spectroscopy and molecular modeling

K. Hofbauerová^1,2, V. Kopecký Jr.², R. Ettrich³, O. Vaněk⁴, J. Pavlíček⁴ and K. Bezouška^1,4

¹Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, Prague 4, CZ-14220, Czech Republic; hofbauer@biomed.cas.cz

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

³Laboratory of High Performance Computing, Institute of Physical Biology of USB and Institute of Systems Biology and Ecology of AS CR, Zámek 136, Nové Hrady, CZ-37333, Czech Republic

⁴Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030, Prague 2, CZ-12840, Czech Republic

CD69 (Cluster of Differentiation) is the earliest leukocyte activation antigen playing a pivotal role in the cellular signalling [1, 2]. It belongs to the type II of transmembrane proteins possessing an extracellular C-terminal protein motif related to C-type animal lectins. CD69 is a disulfide-linked homodimer with two constitutively phosphorylated and variously glycosylated chains. It is expressed on the cell surface only 30–60 minutes after stimulation.

To initiate structural and ligand binding studies, we have prepared soluble monomer form of human CD69 protein (Gly⁷⁰–Lys¹⁹⁹) by recombinant expression of its extracellular domain, using pRSET B vector, in Escherichia coli followed by in vitro refolding. To investigate potential of CD69 for ligand binding a large scale of ligands with different stereo-chemical properties was selected – GlcNAc a basic carbohydrate, tri and penta-antennary carbohydrates, and a short peptide with the sequence LELTEGY. The influence of binding of those ligands on the secondary structure of CD69 as well as on local environments of amino acids with aromatic side chains was followed by Raman spectroscopy and Fourier transform infrared spectroscopy (FTIR). Raman and FTIR spectroscopy are very sensitive methods for investigation of differences between bound and native stage of the protein with respect to its secondary structure content. Moreover, they can provide detail information of certain amino acid residues. Such a big ligands as antennary sugars or peptides can have an influence on protein dynamics, wherefore thermal dynamics of CD69 with and without ligands was investigated by Raman spectroscopy.

The protein homology model, based on template crystal structure of CD69 1E87 [2], was used for identification of ligand binding sites. Refinement was achieved through algorithmic analysis and a minimization was done with the TRIPOS force field in the SYBYL/MAXIMIN2 (Tripos) module. Docking of a calcium ion was explored with the DOCK module of SYBYL. Interactions with ligands were studied using the AUTODOCK program, which is suite for automated docking of flexible ligands to receptors. The binding was investigated by usage of molecular dynamics calculations, which reveals small changes in protein CD69 structure upon ligand binding. Results from theoretical calculations were critically confronted with experimental measurements, whereas good agreement was reached.

1. J. Pavlíček, B. Sopko, R. Ettrich, V. Kopecký Jr., V. Baumruk, P. Man, V. Havlíček, M. Vrbacký, L. Martínková, V. Křen, M. Pospíšil, K. Bezouška, Biochemistry, 42, (2003), 9295.

2. A. S. Llera, F. Viedma, F. Sanchez-Madrid, J. Tormo, J. Biol. Chem., 276, (2001), 7312.

The work was supported by Ministry of Education of the Czech Republic (MSM 0021620835, MSM 6007665808, MSM 0021620808), by Institutional Research Concept (AVOZ 60870520, AVOZ 50200510), by Grant Agency of the Czech Republic (301/05/P567), and by Grant Agency of the Academy of Sciences of the Czech Republic (A5020403).