Structural analysis of the interleukin-8/glycosaminoglycan interactions by amide hydrogen/deuterium exchange mass spectrometry.
The recruitment of different chemokines and growth factors by glycosaminoglycans (GAGs) such as chondroitin sulfate or hyaluronan plays a critical role in wound healing processes. Thus, there is a special interest in the design of artificial extracellular matrices with improved properties concerning GAG interaction with common regulating proteins. In this study, amide hydrogen/deuterium (H/D) exchange mass spectrometry (HDX MS) combined with molecular modeling and docking experiments was used to obtain structural models of proinflammatory chemokine interleukin-8 (IL-8) in complex with hexameric chondroitin sulfate. Experiments on the intact protein showed a difference in deuterium labeling of IL-8 due to chondroitin sulfate binding. The extent of deuteration was reduced from 24% to 13% after 2 min exchange time, which corresponds to a reduced exchange of approximately 10 backbone amides. By local HDX MS experiments, H/D exchange information on the complete sequence of IL-8 could be obtained. A significantly reduced H/D exchange, especially of the C-terminal α-helical region comprising amino acids 70-77 and to the loop comprising amino acids 27-29 was observed in the presence of chondroitin sulfate. HDX MS data were used to model the IL-8/chondroitin sulfate complex. The binding interface of IL-8 and chondroitin sulfate determined this way correlated excellently with the corresponding NMR based atomistic model previously published. Our results demonstrate that HDX-MS in combination with molecular modeling is a valuable approach for the analysis of protein/GAG complexes at physiological pH, temperature, and salt concentration. The fact that HDX-MS requires only micrograms of protein and GAGs makes it a very promising technique to address protein-GAG interactions.
Department of Proteomics, Helmholtz-Centre for Environmental Research-UFZ, Permoserstraße 15, 04318 Leipzig, Germany.
Tommy
T
Hofmann
Structural Bioinformatics, BIOTEC TU Dresden, Tatzberg 47-51, Dresden 01307, Germany.
Sergey A
SA
Samsonov
Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstraße 16-18, D-04107 Leipzig, Germany.
Annelie
A
Pichert
Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstraße 16-18, D-04107 Leipzig, Germany.
Katharina
K
Lemmnitzer
Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstraße 16-18, D-04107 Leipzig, Germany.
Jürgen
J
Schiller
Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstraße 16-18, D-04107 Leipzig, Germany.
Daniel
D
Huster
Structural Bioinformatics, BIOTEC TU Dresden, Tatzberg 47-51, Dresden 01307, Germany.
M Teresa
MT
Pisabarro
Department of Proteomics, Helmholtz-Centre for Environmental Research-UFZ, Permoserstraße 15, 04318 Leipzig, Germany; Department of Metabolomics, Helmholtz-Centre for Environmental Research - UFZ, Permoserstraße 15, 04318 Leipzig, Germany; Department of Chemistry and Bioscience, Aalborg University, Fredrik Bajers Vej 7H, 9220 Aalborg East, Denmark.
Martin
M
von Bergen
Department of Proteomics, Helmholtz-Centre for Environmental Research-UFZ, Permoserstraße 15, 04318 Leipzig, Germany. Electronic address: stefan.kalkhof@ufz.de.
Stefan
S
Kalkhof
D000577:Amides / Q000737:chemistry*; D000595:Amino Acid Sequence; D041961:Deuterium Exchange Measurement / Q000379:methods*; D006025:Glycosaminoglycans / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006859:Hydrogen / Q000737:chemistry*; D016209:Interleukin-8 / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D000577:Amides; C508606:CXCL8 protein, human; D006025:Glycosaminoglycans; D016209:Interleukin-8; D006859:Hydrogen
Amide hydrogen/deuterium exchange; Chemokine; Chondroitin sulfate; Extracellular matrix; Interleukin-8; Protein/glycosaminoglycan interaction