High structural resolution hydroxyl radical protein footprinting reveals an extended Robo1-heparin binding interface
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Authors
Li, Zixuan
Moniz, Heather
Wang, Shuo
Ramiah, Annapoorani
Zhang, Fuming
Moremen, Kelley W.
Linhardt, Robert J.
Sharp, Joshua S.
Issue Date
2015-04-24
Type
Article
Language
ENG
Keywords
Biology , Chemistry and chemical biology , Chemical and biological engineering , Biomedical engineering
Alternative Title
Abstract
Interaction of transmembrane receptors of the Robo family and the secreted protein Slit provides important signals in the development of the central nervous system and regulation of axonal midline crossing. Heparan sulfate, a sulfated linear polysaccharide modified in a complex variety of ways, serves as an essential co-receptor in Slit-Robo signaling. Previous studies have shown that closely related heparin octasaccharides bind to Drosophila Robo directly, and surface plasmon resonance analysis revealed that Robo1 binds more tightly to full-length unfractionated heparin. For the first time, we utilized electron transfer dissociation-based high spatial resolution hydroxyl radical protein footprinting to identify two separate binding sites for heparin interaction with Robo1: one binding site at the previously identified site for heparin dp8 and a second binding site at the N terminus of Robo1 that is disordered in the x-ray crystal structure. Mutagenesis of the identified N-terminal binding site exhibited a decrease in binding affinity as measured by surface plasmon resonance and heparin affinity chromatography. Footprinting also indicated that heparin binding induces a minor change in the conformation and/or dynamics of the Ig2 domain, but no major conformational changes were detected. These results indicate a second low affinity binding site in the Robo-Slit complex as well as suggesting the role of the Ig2 domain of Robo1 in heparin-mediated signal transduction. This study also marks the first use of electron transfer dissociation-based high spatial resolution hydroxyl radical protein footprinting, which shows great utility for the characterization of protein-carbohydrate complexes.
Description
Journal of Biological Chemistry, 290, 10729-10740
Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.
Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.
Full Citation
High structural resolution hydroxyl radical protein footprinting reveals an extended Robo1-heparin binding interface, Z. Li, H. Moniz, S. Wang, A. Ramiah, F. Zhang, K. W. Moremen, R. J. Linhardt, J. S. Sharp, Journal of Biological Chemistry, 290, 10729-10740, 2015.
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Terms of Use
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Volume
Issue
PubMed ID
DOI
ISSN
1083351X
219258
219258