High Structural Resolution Hydroxyl Radical Protein Footprinting Reveals an Extended Robo1-Heparin Binding Interface

摘要

Hydroxyl radical protein footprinting (HRPF) coupled with MS has become increasingly popular as a labeling technique to probe intact protein structure, protein-protein interactions, protein folding, and protein-small molecule ligand interactions in solution. 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 (HS), a sulfated linear polysaccharide modified in a complex variety of ways, serves as an essential coreceptor in Slit-Robo signaling. For the first time, we utilized ETD-based high spatial resolution hydroxyl radical protein footprinting to identify two separate binding sites for heparin interaction with Robo1. We also identified residues in the hydrophobic core that show altered solvent accessibility upon heparin binding, suggesting a heparin binding-induced conformational change that may be involved in GAG-induced ternary complex formation. A model for heparin/heparan sulfate binding and activation of the Slit-Robo complex is proposed.