O2 sensing–associated glycosylation exposes the F-box–combining site of the Dictyostelium Skp1 subunit in E3 ubiquitin ligases

Abstract

Skp1 is a conserved protein linking cullin-1 to F-box proteins in SCF (kp1/ullin-1/-box protein) E3 ubiquitin ligases, which modify protein substrates with polyubiquitin chains that typically target them for 26S proteasome-mediated degradation. In (a social amoeba), (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 O-dependent development, but full glycosylation at that position is required for optimal O 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.

ToxoplasmaF-Box Protein 1 is Required for Daughter Cell Scaffold Function During Parasite Replication

“…Previously, we reported that 154 Pro of TgSKP1 is hydroxylated and glycosylated and that these post-translational modifications are predicted to change the conformation of the F-box protein-binding region of TgSKP1 as they do in the social amoeba Dictyostelium discoideum when a conserved proline residue in DdSKP1 is similarly modified [22]. An O 2 -regulated prolyl hydroxylase modifies 154 Pro and under low O 2 conditions can be observed by Western blotting as a decrease in the apparent molecular weight of TgSKP1 [19, 20, 28] (Figure 1B; TgSKP1 Input Fractions).…”

CRISPR/Cas9 gene editing for the creation of an MGAT1 deficient CHO cell line to control HIV-1 vaccine glycosylation

“…Data were obtained from AB SCIEX MALDI TOF/TOF 5800 (Applied Biosystem MDS Analytical Technologies, Foster City, CA) mass spectrometer in reflector positive-ion mode. Data analysis was performed by using Data Explorer V4.5, and the assignment of glycan structure was based on the primary mass (m/z) coupled with MS/MS fragmentation profile using the Expasy database online and the glycowork bench software analysis (84, 85).…”

Characterization of a cytoplasmic glucosyltransferase that extends the core trisaccharide of the Toxoplasma Skp1 E3 ubiquitin ligase subunit

“…FBPs possess a 40-amino acid F-box domain that binds to the C-terminal region of Skp1 (7,8). Recent studies show that glycosylation influences the organization and range of motions of this region of Skp1, in part by hydrogen bonding along the polypeptide in cis (9). Glycosylation is regulated by PhyA, whose action on Skp1 is rate-limited by O 2 availability in the cell (6,10).…”

“…Structural studies of Dictyostelium Skp1 suggest that the fulllength glycan encourages an ensemble of conformations that promote interactions with at least certain FBPs (9). The trisaccharide form of Dictyostelium Skp1 exhibits intermediate interaction with two of the FBPs (6), and, if an analogous mechanism operates in Toxoplasma, this might explain why the glt1 deletion results in a growth phenotype intermediate between complete absence of the glycan and its full assembly.…”