Abstract
Skp1 is a conserved protein linking cullin-1 to F-box proteins in SCF (Skp1/Cullin-1/F-box protein) 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, NMR and MS 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. Molecular dynamics 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 α-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.
Highlights
ResultsThe Skp glycoprotein was purified from ϳ2.4 ϫ 1012 Dictyostelium cells as described under “Experimental procedures,” yielding ϳ2 mg (100 nmol) of the native molecule
The described hydrogen bonds between the protein and glycan were not observed during this simulation; additional replicates would be required to form conclusions regarding interactions within the glycoprotein
Knowledge of the internal linkages of the glycan enabled a series of molecular dynamics simulations of the overall conformation of the glycan alone and when linked to the Skp1 protein
Summary
The Skp glycoprotein was purified from ϳ2.4 ϫ 1012 Dictyostelium cells as described under “Experimental procedures,” yielding ϳ2 mg (100 nmol) of the native molecule. Less than 10 nmol was recovered using the preparation isolated directly from Dictyostelium As outlined, this quantity was sufficient for partial analysis. Mass spectrometry (MALDI-TOF-MS) of a portion of the intact glycopeptide sample indicated an oligosaccharide having three hexoses, one deoxyhexose, and one N-acetylhexosamine residue (Fig. 1C), attached at a hydroxyproline that was present in the NDFTPEEEEQIRK sequence of Skp. Each glycopeptide contained a ϩ2 m/z mass increment due to the presence of two [1-13C]Gal residues, as com-
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