Identification of the dimerisation interface of human interleukin-8 by IL-8-variants containing the photoactivatable amino acid benzoyl-phenylalanine.
The three-dimensional structure of human interleukin-8 (hIL-8) was determined by the use of NMR and X-ray methodology. At high concentrations interleukin-8 and many other chemokines form a non-covalent homodimer. Several studies have been performed to investigate the relevance of the dimer on receptor activation and led to contradictory results. In order to obtain a better understanding of the dimerisation process, covalently linked homo- and heterodimers were produced by photo-induced dimerisation of hIL-8 analogues that contain the photo-activatable amino acid p-benzoyl-phenylalanine (Bpa) at different positions. Whereas the N-terminal fragment (1-54) was expressed as recombinant thioester, the C-terminal fragments (55-77) that contain Bpa either at position 65 or 74 were obtained by solid-phase peptide synthesis. The segments were combined by expressed protein ligation and led to full length IL-8 variants containing the non-proteinogenic amino acid Bpa at single positions. IP(3) activity tests showed high biological activity for the CXCR1-GFP receptor for both variants comparable to that of the native ligand. The refolded and purified ligation-products were used for dimer formation by UV-irradiation. The analysis of the reaction mixture was performed by gel-electrophoresis and mass spectrometry and showed that dimer formation of IL-8 occurred in a position dependent manner. [Bpa(74)]hIL-8 has a high tendency to form covalent dimers whereas no dimer formation was observed for the variant with Bpa at position 65. Accordingly one residue of the dimerisation interface could be identified.
David
Ralf
R
Institute of Biochemistry, Faculty of Bioscience, Pharmacy and Psychology, University of Leipzig, Leipzig, Germany.
Beck-Sickinger
Annette G
AG
D019943:Amino Acid Substitution; D001665:Binding Sites; D019281:Dimerization; D006801:Humans; D016209:Interleukin-8 / Q000737:chemistry* / Q000528:radiation effects*; D009154:Mutation; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000528:radiation effects; D010777:Photochemistry / Q000379:methods; D011485:Protein Binding / Q000528:radiation effects; D014466:Ultraviolet Rays
D016209:Interleukin-8; D010649:Phenylalanine; C053764:benzoylphenylalanine