TY - JOUR
T1 - Protein NMR Studies of Substrate Binding to Human Blood Group A and B Glycosyltransferases
AU - Grimm, Lena Lisbeth
AU - Weissbach, Sophie
AU - Flügge, Friedemann
AU - Begemann, Nora
AU - Palcic, Monica M.
AU - Peters, Thomas
N1 - Publisher Copyright:
© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.
PY - 2017/7/4
Y1 - 2017/7/4
N2 - 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 2H,15N-labeled GTA or GTB with UDP affected about 20 % of all crosspeaks in 1H,15N 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-[13C]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.
AB - 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 2H,15N-labeled GTA or GTB with UDP affected about 20 % of all crosspeaks in 1H,15N 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-[13C]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.
UR - http://www.scopus.com/inward/record.url?scp=85017417708&partnerID=8YFLogxK
U2 - 10.1002/cbic.201700025
DO - 10.1002/cbic.201700025
M3 - Journal articles
C2 - 28256109
AN - SCOPUS:85017417708
VL - 18
SP - 1260
EP - 1269
JO - Chembiochem : a European journal of chemical biology
JF - Chembiochem : a European journal of chemical biology
SN - 1439-4227
IS - 13
ER -