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 uniform 15N, 13C, and 2H 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.

NMR assignments of sparsely labeled proteins using a genetic algorithm

“…There are instances of missing data and different levels of internal motion. For the two domain construct from Robo1, our example of a sparsely labeled glycoprotein, 15 N- 1 H HSQC spectra for the lysine and phenylalanine labeled versions are shown in Supplementary Materials Figure S1 (Gao et al 2016). These spectra give examples of the resolution that can be expected for a sparsely labeled, non-deuterated 23 kDa protein.…”

“…The NMR data for Robo1-Ig1–2 are from our paper reporting its interaction with heparan sulfate (Gao et al 2016). There are several X-ray structures for the Ig1–2 construct, but these show significant differences in inter-domain orientation.…”

“…There are several X-ray structures for the Ig1–2 construct, but these show significant differences in inter-domain orientation. For the purpose of this application domain motions were simulated in a long MD trajectory (1μs) (Gao et al 2016) and an x-ray structure (PDB 2V9R) was selected that closely approximated the domain orientation in the most highly populated state of this trajectory. The PDB accession codes for the structures and a summary of experimental data used for all test proteins are summarized in Table 1.…”

“…Resonance assignment used a largely manual approach in which each type of prediction is sequentially used to exclude possible assignments (Gao et al 2016). However, it is difficult to set strict exclusion limits in a sequential strategy.…”

“…The existing assignments in this case are by traditional triple resonance methods on uniformly labeled proteins, but we mimic a sparsely labeled set by selecting resonances for a subset of amino acids. We also test the procedure on a sparsely labeled two domain fragment of the Robo1 protein whose assignment is achieved by a sequential approach (Gao et al 2016). Robo1 is a glycosylated protein whose activity in developmentally related cell-signaling is regulated by interaction with certain heparan sulfate (HS) epitopes.…”