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.
Highlights
The molecular basis of full-length heparin activation of Slit-Robo is poorly understood, despite its importance in nervous system development
Our results show a modest protection of hydrophobic residues Met[180] and Met[189] in the Ig2 domain upon binding to unfractionated heparin (Fig. 7)
In the static x-ray crystal structure, Met[189] is highly protected from solvent, yet we detect heavy oxidation of this residue as well. These results suggest that Met[189] may be somewhat more solvent-exposed in the absence of heparin than is indicated by the crystal structure
Summary
The molecular basis of full-length heparin activation of Slit-Robo is poorly understood, despite its importance in nervous system development. Previous studies have shown that heparin octasaccharides bind to Drosophila Robo directly, and the heparin dp[8] binding sites have been identified by mutagenesis and partial direct crystallographic observation of a bound heparin oligosaccharide (21) These observations indicate that HS/heparin plays a direct and essential role in Slit-Robo signaling. We have successfully demonstrated the accuracy of electron transfer dissociation (ETD)-based methods for quantifying multiple adjacent sites of isomeric oxidation products as well as the general applicability of ETD-based dissociation to quantify oxidized peptide isomers with multiple sites of oxidation on both a series of synthetic peptide oxidation isomers and peptides from Robo[1] Ig1-2 protein generated by HRPF (31) This technology is applied to characterizing the interaction of Robo[1] Ig1-2 with unfractionated heparin. 10730 JOURNAL OF BIOLOGICAL CHEMISTRY action by high resolution HRPF, providing binding interface information down to the amino acid residue level in some cases
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