Format

Send to

Choose Destination
Methods. 2015 Nov 1;89:45-53. doi: 10.1016/j.ymeth.2015.02.011. Epub 2015 Feb 26.

Structural analysis of the interleukin-8/glycosaminoglycan interactions by amide hydrogen/deuterium exchange mass spectrometry.

Author information

1
Department of Proteomics, Helmholtz-Centre for Environmental Research-UFZ, Permoserstraße 15, 04318 Leipzig, Germany.
2
Structural Bioinformatics, BIOTEC TU Dresden, Tatzberg 47-51, Dresden 01307, Germany.
3
Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstraße 16-18, D-04107 Leipzig, Germany.
4
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.
5
Department of Proteomics, Helmholtz-Centre for Environmental Research-UFZ, Permoserstraße 15, 04318 Leipzig, Germany. Electronic address: stefan.kalkhof@ufz.de.

Abstract

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.

KEYWORDS:

Amide hydrogen/deuterium exchange; Chemokine; Chondroitin sulfate; Extracellular matrix; Interleukin-8; Protein/glycosaminoglycan interaction

PMID:
25726910
DOI:
10.1016/j.ymeth.2015.02.011
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center