title: Structural Aspects of Heparan Sulfate Binding to Robo1-Ig1-2.
abstract: Roundabout 1, or Robo1, is a cell surface signaling molecule important in axon guidance. Its interaction with heparan sulfate (HS) and members of the Slit protein family is essential to its activity, making characterization of these interactions by structural methods, such as NMR, highly desirable. However, the fact that Robo1 is a glycosylated protein prevents employment of commonly used bacterial hosts for expression of properly glycosylated forms with the uniform15N,13C, and2H labeling needed for NMR studies. Here, we apply an alternative methodology, based on labeling with a single amino acid type and high structural content NMR data, to characterize a two-domain construct of glycosylated Robo1 (Robo1-Ig1-2) interacting with a synthetic HS tetramer (IdoA-GlcNS6S-IdoA2S-GlcNS6S-(CH2)5NH2). Significant chemical shift perturbations of the crosspeak from K81 on titration with the tetramer provide initial evidence for the location of a binding site and allow determination of a 255 μM disassociation constant. The binding epitopes, bound conformation, and binding site placement of the HS tetramer have been further characterized by saturation transfer difference (STD), transferred nuclear Overhauser effect (trNOE), and paramagnetic perturbation experiments. A model of the complex has been generated using constraints derived from the various NMR experiments. Postprocessing energetic analysis of this model provides a rationale for the role each glycan residue plays in the binding event, and examination of the binding site in the context of a previous Robo-Slit structure provides a rationale for modulation of Robo-Slit interactions by HS.
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia , Athens, Georgia 30602, United States.
authors.forename: Qi
authors.initials: Q
authors.lastname: Gao
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia , Athens, Georgia 30602, United States.
authors.forename: Cheng-Yu
authors.initials: CY
authors.lastname: Chen
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia , Athens, Georgia 30602, United States.
authors.forename: Chengli
authors.initials: C
authors.lastname: Zong
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia , Athens, Georgia 30602, United States.
authors.forename: Shuo
authors.initials: S
authors.lastname: Wang
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia , Athens, Georgia 30602, United States.
authors.forename: Annapoorani
authors.initials: A
authors.lastname: Ramiah
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia , Athens, Georgia 30602, United States.
authors.forename: Pradeep
authors.initials: P
authors.lastname: Prabhakar
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia , Athens, Georgia 30602, United States.
authors.forename: Laura C
authors.initials: LC
authors.lastname: Morris
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia , Athens, Georgia 30602, United States.
authors.forename: Geert-Jan
authors.initials: GJ
authors.lastname: Boons
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia , Athens, Georgia 30602, United States.
authors.forename: Kelley W
authors.initials: KW
authors.lastname: Moremen
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia , Athens, Georgia 30602, United States.
authors.forename: James H
authors.initials: JH
authors.lastname: Prestegard
mesh_terms: D006031:Glycosylation; D006497:Heparan Sulfate / Q000737:chemistry / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D062105:Molecular Docking Simulation; D015394:Molecular Structure; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000737:chemistry / Q000378:metabolism*; D055672:Static Electricity; D000097905:Roundabout Proteins
chemical_list: D006497:Heparan Sulfate; D009419:Nerve Tissue Proteins; D011971:Receptors, Immunologic; D000097905:Roundabout Proteins