High structural resolution hydroxyl radical protein footprinting reveals an extended Robo1-heparin binding interface.
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.
From the Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602 and.
Zixuan
Z
Li
From the Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602 and.
Heather
H
Moniz
From the Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602 and.
Shuo
S
Wang
From the Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602 and.
Annapoorani
A
Ramiah
the Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180.
Fuming
F
Zhang
From the Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602 and.
Kelley W
KW
Moremen
the Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180.
Robert J
RJ
Linhardt
From the Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602 and jsharp@ccrc.uga.edu.
Joshua S
JS
Sharp
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D004579:Electron Transport; D006493:Heparin / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D017665:Hydroxyl Radical; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism; D046911:Macromolecular Substances / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D023302:Protein Footprinting; D054730:Protein Interaction Domains and Motifs; D011971:Receptors, Immunologic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D000097905:Roundabout Proteins; D000098552:Slit Homolog 2 Protein
D006493:Heparin; D017665:Hydroxyl Radical; D036341:Intercellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; D009419:Nerve Tissue Proteins; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; D000097905:Roundabout Proteins; D000098552:Slit Homolog 2 Protein
Glycosaminoglycan; Heparin; Heparin-binding Protein; Hydroxyl Radical Protein Footprinting; Mass Spectrometry (MS); Protein-Carbohydrate Interaction; Structural Biology