Structural Aspects of Glycosylated Protein–Glycan Interactions by NMR

Abstract

Characterization of glycosylated proteins and their response to interaction with glycans is challenging for any method, because of the heterogeneity of glycosylation and the extensive conformational sampling by most glycans. For NMR there are extra challenges because expression in mammalian cell cultures, which can preserve near‐native glycosylation, requires expensive substrates to achieve uniform isotopic labeling with 15N or 13C, and mammalian cells do not tolerate high levels of deuteration (something required to improve spectral resolution). We have developed a sparse‐labeling approach that preserves high resolution without deuteration, proves to be economical in most applications, and returns enough structural data to define geometries of multiple domain proteins and ligand protein complexes. The approach is based on labeling proteins with a single type of amino acid isotopically labeled in methyl groups with 13C (13C‐valine or 13C alanine). NMR signals from methyl groups are special in terms of the sensitivity and resolution that they provide. The addition of paramagnetic tags to specific sites in a protein provides long range distance and angular information on the location of labeled methyl groups. In combination with a newly developed approach to assign methyl resonances to specific sites in a protein, this information proves adequate to define relative domain orientations and facilitate the identification of ligand binding sites. Application to terminal domains from a cell surface signaling molecule, ROBO1, is used to illustrate the approach. It also provides an example of how binding of a heparan sulfate oligomer alters the relative orientation of domains.Support or Funding InformationThis work was supported by grants from the National Institutes of Health, P41GM103390 and R01GM033225.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.