Protein NMR Studies of Substrate Binding to Human Blood Group A and B Glycosyltransferases.
Lena Lisbeth GrimmSophie WeissbachFriedemann FlüggeNora BegemannMonica M PalcicThomas PetersPublished in: Chembiochem : a European journal of chemical biology (2017)
Donor and acceptor substrate binding to human blood group A and B glycosyltransferases (GTA, GTB) has been studied by a variety of protein NMR experiments. Prior crystallographic studies had shown these enzymes to adopt an open conformation in the absence of substrates. Binding either of the donor substrate UDP-Gal or of UDP induces a semiclosed conformation. In the presence of both donor and acceptor substrates, the enzymes shift towards a closed conformation with ordering of an internal loop and the C-terminal residues, which then completely cover the donor-binding pocket. Chemical-shift titrations of uniformly 2 H,15 N-labeled GTA or GTB with UDP affected about 20 % of all crosspeaks in 1 H,15 N TROSY-HSQC spectra, reflecting substantial plasticity of the enzymes. On the other hand, it is this conformational flexibility that impedes NH backbone assignments. Chemical-shift-perturbation experiments with δ1-[13 C]methyl-Ile-labeled samples revealed two Ile residues-Ile123 at the bottom of the UDP binding pocket, and Ile192 as part of the internal loop-that were significantly disturbed upon stepwise addition of UDP and H-disaccharide, also revealing long-range perturbations. Finally, methyl TROSY-based relaxation dispersion experiments do not reveal micro- to millisecond timescale motions. Although this study reveals substantial conformational plasticity of GTA and GTB, the matter of how binding of substrates shifts the enzymes into catalytically competent states remains enigmatic.
Keyphrases
- molecular dynamics simulations
- endothelial cells
- binding protein
- amino acid
- magnetic resonance
- dna binding
- single molecule
- high resolution
- induced pluripotent stem cells
- molecular dynamics
- transcription factor
- single cell
- pet imaging
- crystal structure
- pluripotent stem cells
- case control
- gene expression
- computed tomography
- structural basis
- mass spectrometry
- density functional theory
- energy transfer