dictyNews
Electronic Edition
Volume 43, number 23
September 29, 2017
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Abstracts
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O2 sensing–associated glycosylation exposes the F-box–combining site of the
Dictyostelium Skp1 subunit in E3 ubiquitin ligases
M. Osman Sheikh 1,2,3,6, David Thieker 2,6, Gordon Chalmers 2,4, Christopher
M. Schafer 3, Mayumi Ishihara 2, Parastoo Azadi 2, Robert J. Woods 1,2,
John N. Glushka 2, Brad Bendiak 5, James H. Prestegard 1,2, and
Christopher M. West 1,3
1 Department of Biochemistry & Molecular Biology, University of Georgia, Athens,
GA 30602 USA;
2 Complex Carbohydrate Research Center, University of Georgia, Athens,
GA 30602 USA;
3 Dept. of Biochemistry & Molecular Biology, Oklahoma Center for Medical
Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City,
OK 73104 USA;
4 Department of Computer Science, University of Georgia, Athens, GA 30602 USA;
5 Cell and Developmental Biology, University of Colorado Anschutz Medical
Campus, School of Medicine, Aurora, Colorado 80045 USA
6 contributed equally
J. Biol. Chem., in press
Skp1 is a conserved protein linking cullin-1 to F-box proteins in SCF (Skp1-
Cullin1-F-box) E3 ubiquitin ligases, which modify protein substrates with
polyubiquitin chains that typically target them for 26S proteasome-mediated
degradation. In Dictyostelium (a social amoeba), Toxoplasma gondii (the agent
for human toxoplasmosis), and other protists, Skp1 is regulated by a unique
pentasaccharide attached to hydroxylated Pro-143 within its C-terminal F-box–
binding domain. Prolyl hydroxylation of Skp1 contributes to O2-dependent
Dictyostelium development, but full glycosylation at that position is required for
optimal O2 sensing. Previous studies have shown that the glycan promotes
organization of the F-box–binding region in Skp1, and aids in Skp1’s association
with F-box proteins. Here, nuclear magnetic resonance and mass spectrometry
approaches were used to determine the glycan structure, and then a combination
of NMR and molecular dynamics simulations were employed to characterize the
impact of the glycan on the conformation and motions of the intrinsically flexible
F-box–binding domain of Skp1. MD trajectories of glycosylated Skp1 whose
calculated monosaccharide relaxation kinetics and rotational correlation times
agreed with the NMR data indicated that the glycan interacts with the loop
connecting two alpha-helices of the F-box–combining site. In these trajectories,
the helices separated from one another to create a more accessible and dynamic
F-box interface. These results offer an unprecedented view of how a glycan
modification influences a disordered region of a full-length protein. The increased
sampling of an open Skp1 conformation can explain how glycosylation enhances
interactions with F-box proteins in cells.
submitted by: Chris West [[log in to unmask]]
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[End dictyNews, volume 43, number 23]
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