TY - JOUR
T1 - Structural analysis of the interleukin-8/glycosaminoglycan interactions by amide hydrogen/deuterium exchange mass spectrometry
AU - Hofmann, Tommy
AU - Samsonov, Sergey A.
AU - Pichert, Annelie
AU - Lemmnitzer, Katharina
AU - Schiller, Jürgen
AU - Huster, Daniel
AU - Pisabarro, M. Teresa
AU - von Bergen, Martin
AU - Kalkhof, Stefan
PY - 2015/11/1
Y1 - 2015/11/1
N2 - 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.
AB - 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.
KW - Amide hydrogen/deuterium exchange
KW - Chemokine
KW - Chondroitin sulfate
KW - Extracellular matrix
KW - Interleukin-8
KW - Protein/glycosaminoglycan interaction
UR - http://www.scopus.com/inward/record.url?scp=84946411083&partnerID=8YFLogxK
U2 - 10.1016/j.ymeth.2015.02.011
DO - 10.1016/j.ymeth.2015.02.011
M3 - Journal article
AN - SCOPUS:84946411083
SN - 1046-2023
VL - 89
SP - 45
EP - 53
JO - Methods
JF - Methods
ER -