Discovery of Novel 1,3-Diphenylpyrazine Derivatives as Potent S-Phase Kinase-Associated Protein 2 (Skp2) Inhibitors for the Treatment of Cancer.

F-box protein S-phase kinase-associated protein 2 (Skp2) is a component of cullin-RING ligases, which is responsible for recruiting and ubiquitinating substrates and subsequently plays its proteolytic and non-proteolytic role. High expression of Skp2 is frequently observed in multiple aggressive tumor tissues and associated with poor prognosis. Several of the Skp2 inhibitors have been reported in the last decades; however, few of them have shown detailed structure-activity relationship (SAR) and potent bioactivity. Herein, based on the hit compound 11a from our in-house library, we optimize and synthesize a series of new 2,3-diphenylpyrazine-based inhibitors targeting the Skp2-Cks1 interaction and further systematically study the SAR. Among them, compound 14i shows potent activity against the Skp2-Cks1 interaction with an IC50 value of 2.8 µM and against PC-3 and MGC-803 cells with IC50 values of 4.8 and 7.0 µM, respectively. Most importantly, compound 14i exhibited effectively anticancer effects on PC-3 and MGC-803 xenograft mice models without obvious toxicity.

Disorder in the Human Skp1 Structure is the Key to its Adaptability to Bind Many Different Proteins in the SCF Complex Assembly.

Skp1(S-phase kinase-associated protein 1 - Homo sapiens) is an adapter protein of the SCF(Skp1-Cullin1-Fbox) complex, which links the constant components (Cul1-RBX) and the variable receptor (F-box proteins) in Ubiquitin E3 ligase. It is intriguing how Skp1 can recognise and bind to a variety of structurally different F-box proteins. For practical reasons, previous efforts have used truncated Skp1, and thus it has not been possible to track the crucial aspects of the substrate recognition process. In this background, we report the solution structure of the full-length Skp1 protein determined by NMR spectroscopy for the first time and investigate the sequence-dependent dynamics in the protein. The solution structure reveals that Skp1 has an architecture: ß1-ß2-H1-H2-L1-H3-L2-H4-H5-H6-H7(partially formed) and a long tail-like disordered C-terminus. Structural analysis using DALI (Distance Matrix Alignment) reveals conserved domain structure across species for Skp1. Backbone dynamics investigated using NMR relaxation suggest substantial variation in the motional timescales along the length of the protein. The loops and the C-terminal residues are highly flexible, and the (R2/R1) data suggests µs-ms timescale motions in the helices as well. Further, the dependence of amide proton chemical shift on temperature and curved profiles of their residuals indicate that the residues undergo transitions between native state and excited state. The curved profiles for several residues across the length of the protein suggest that there are native-like low-lying excited states, particularly for several C-terminal residues. Our results provide a rationale for how the protein can adapt itself, bind, and get functionally associated with other proteins in the SCF complex by utilising its flexibility and conformational sub-states.

Establishment of high-throughput screening HTRF assay for identification small molecule inhibitors of Skp2-Cks1.

S-phase kinase associated protein 2 (Skp2), a member of the F-box family that constitute the largest known class of ubiquitin E3 specificity components, is responsible for recognizing and recruiting cyclin-dependent kinase inhibitor p27 for its ubiquitination in the presence of the small accessory protein cyclin-dependent kinase regulatory subunit 1(Cks1). Skp2 is overexpressed in esophageal carcinoma tissues and closely related with tumor poor prognosis, and perturbation of the Skp2-Cks1 interaction by inhibitors or RNAi could inhibit the proliferation and metastasis of tumor cells. Therefore, inhibition of Skp2 function by small-molecule compounds targeting Skp2-Cks1 interaction is emerging as a promising and novel anti-cancer strategy. In this study, we establish an improved high-throughput screening platform to screen Skp2 inhibitors targeting Skp2-Cks1interaction, which may provide a new therapeutic approach for the clinic.

HSP70 promotes tumor progression by stabilizing Skp2 expression in gastric cancer cells.

Gastric cancer (GC) has one of the highest tumor incidences worldwide. Heat shock protein 70 (HSP70) is highly expressed and plays a critical role in the occurrence, progression, metastasis, poor prognosis, and drug resistance of GC. However, the underlying mechanisms of HSP70 are not clear. To explore the regulatory role of HSP70 in GC, we performed cell counting kit-8 (CCK-8) and EdU staining assays to assess cell proliferation; immunohistochemistry and western blot analyses to assess protein expression; coimmunoprecipitation (Co-IP) assays to assess interactions between two proteins; and immunofluorescence to assess protein expression and localization. HSP70 was highly expressed in clinical samples from patients with GC and indicated a poor prognosis. HSP70 inhibition enhanced the sensitivity of GC cells to thermochemotherapy. Furthermore, we found that S phase kinase-associated protein 2 (Skp2) was highly expressed in GC and correlated with HSP70 in array data from The Cancer Genome Atlas (TCGA). Importantly, HSP70 inhibition promoted Skp2 degradation. Skp2 overexpression abrogated HSP70 inhibition-induced cell cycle arrest, suggesting that the role of HSP70 in GC depends on Skp2 expression. Our results illustrate a possible regulatory mechanism of HSP70 and may provide a therapeutic strategy for overcoming resistance to thermochemotherapy.

Co-operative binding of SKP1, Cullin1 and Cullin7 to FBXW8 results in Cullin1-SKP1-FBXW8-Cullin7 functional complex formation that monitors cellular function of ß-TrCP1.

F-box protein FBXW8 is known to interact with scaffolding protein Cullin1 and Cullin7 to form SCF (SKP1, Cullin and F-box protein) complex. However, detail understanding about the importance of both Cullins for SCF-FBXW8 complex formation as well as its ubiquitin ligase activity remains elusive. Here, we show that, through in vitro and in vivo studies, Cullin1 and Cullin7 increase each other's binding to FBXW8 synergistically. Interestingly, absence of either Cullin results in abrogation of binding of other Cullin to FBXW8. Binding of SKP1 to FBXW8 also increases in the presence of both the Cullins. Thus, SKP1, Cullin1 and Cullin7 are essential to form Cullin1-SKP1-FBXW8-Cullin7 functional ubiquitin ligase complex. Further, using computational, mutational and biochemical analysis, we found that Cullin1 binds to N-terminus of FBXW8 through SKP1 while Cullin7 associates with C-terminus of FBXW8 to form Cullin1-SKP1-FBXW8-Cullin7 functional complex in a cooperative manner. Results showed that Cullin1-SKP1-FBXW8-Cullin7 complex plays a key role in maintaining the basal level expression of ß-TrCP1. Moreover, Cullin1-SKP1-FBXW8-Cullin7 complex promotes cell migration by activating ß-catenin via directing proteasomal degradation of ß-TrCP1. Overall, our study reveals the intriguing molecular mechanism of assembly of SKP1, Cullin1, Cullin7 and FBXW8 to form Cullin1-SKP1-FBXW8-Cullin7 functional complex that control the function of ß-TrCP1.

Phytochemical library screening reveals betulinic acid as a novel Skp2-SCF E3 ligase inhibitor in non-small cell lung cancer.

Skp2 is overexpressed in multiple cancers and plays a critical role in tumor development through ubiquitin/proteasome-dependent degradation of its substrate proteins. Drugs targeting Skp2 have exhibited promising anticancer activity. Here, we identified a plant-derived Skp2 inhibitor, betulinic acid (BA), via high-throughput structure-based virtual screening of a phytochemical library. BA significantly inhibited the proliferation and migration of non-small cell lung cancer (NSCLC) through targeting Skp2-SCF E3 ligase both in vitro and in vivo. Mechanistically, BA binding to Skp2, especially forming H-bonds with residue Lys145, decreases its stability by disrupting Skp1-Skp2 interactions, thereby inhibiting the Skp2-SCF E3 ligase and promoting the accumulation of its substrates; that is, E-cadherin and p27. In both subcutaneous and orthotopic xenografts, BA significantly inhibited the proliferation and metastasis of NSCLC through targeting Skp2-SCF E3 ligase and upregulating p27 and E-cadherin protein levels. Taken together, BA can be considered a valuable therapeutic candidate to inhibit metastasis of NSCLC.

Bipartite binding of the N terminus of Skp2 to cyclin A.

Skp2 and cyclin A are cell-cycle regulators that control the activity of CDK2. Cyclin A acts as an activator and substrate recruitment factor of CDK2, while Skp2 mediates the ubiquitination and subsequent destruction of the CDK inhibitor protein p27. The N terminus of Skp2 can interact directly with cyclin A but is not required for p27 ubiquitination. To gain insight into this poorly understood interaction, we have solved the 3.2 Å X-ray crystal structure of the N terminus of Skp2 bound to cyclin A. The structure reveals a bipartite mode of interaction with two motifs in Skp2 recognizing two discrete surfaces on cyclin A. The uncovered binding mechanism allows for a rationalization of the inhibitory effect of Skp2 on CDK2-cyclin A kinase activity toward the RxL motif containing substrates and raises the possibility that other intermolecular regulators and substrates may use similar non-canonical modes of interaction for cyclin targeting.

Discriminative SKP2 Interactions with CDK-Cyclin Complexes Support a Cyclin A-Specific Role in p27KIP1 Degradation.

The SCFSKP2 ubiquitin ligase relieves G1 checkpoint control of CDK-cyclin complexes by promoting p27KIP1 degradation. We describe reconstitution of stable complexes containing SKP1-SKP2 and CDK1-cyclin B or CDK2-cyclin A/E, mediated by the CDK regulatory subunit CKS1. We further show that a direct interaction between a SKP2 N-terminal motif and cyclin A can stabilize SKP1-SKP2-CDK2-cyclin A complexes in the absence of CKS1. We identify the SKP2 binding site on cyclin A and demonstrate the site is not present in cyclin B or cyclin E. This site is distinct from but overlapping with features that mediate binding of p27KIP1 and other G1 cyclin regulators to cyclin A. We propose that the capacity of SKP2 to engage with CDK2-cyclin A by more than one structural mechanism provides a way to fine tune the degradation of p27KIP1 and distinguishes cyclin A from other G1 cyclins to ensure orderly cell cycle progression.

The FBXL family of F-box proteins: variations on a theme.

The ubiquitin-proteasome system (UPS) is responsible for the rapid targeting of proteins for degradation at 26S proteasomes and requires the orchestrated action of E1, E2 and E3 enzymes in a well-defined cascade. F-box proteins (FBPs) are substrate-recruiting subunits of Skp1-cullin1-FBP (SCF)-type E3 ubiquitin ligases that determine which proteins are ubiquitinated. To date, around 70 FBPs have been identified in humans and can be subdivided into distinct families, based on the protein-recruiting domains they possess. The FBXL subfamily is defined by the presence of multiple leucine-rich repeat (LRR) protein-binding domains. But how the 22 FBPs of the FBXL family achieve their individual specificities, despite having highly similar structural domains to recruit their substrates, is not clear. Here, we review and explore the FBXL family members in detail highlighting their structural and functional similarities and differences and how they engage their substrates through their LRRs to adopt unique interactomes.

Biochemical and biophysical analyses of hypoxia sensing prolyl hydroxylases from Dictyostelium discoideum and Toxoplasma gondii.

In animals, the response to chronic hypoxia is mediated by prolyl hydroxylases (PHDs) that regulate the levels of hypoxia-inducible transcription factor α (HIFα). PHD homologues exist in other types of eukaryotes and prokaryotes where they act on non HIF substrates. To gain insight into the factors underlying different PHD substrates and properties, we carried out biochemical and biophysical studies on PHD homologues from the cellular slime mold, Dictyostelium discoideum, and the protozoan parasite, Toxoplasma gondii, both lacking HIF. The respective prolyl-hydroxylases (DdPhyA and TgPhyA) catalyze prolyl-hydroxylation of S-phase kinase-associated protein 1 (Skp1), a reaction enabling adaptation to different dioxygen availability. Assays with full-length Skp1 substrates reveal substantial differences in the kinetic properties of DdPhyA and TgPhyA, both with respect to each other and compared with human PHD2; consistent with cellular studies, TgPhyA is more active at low dioxygen concentrations than DdPhyA. TgSkp1 is a DdPhyA substrate and DdSkp1 is a TgPhyA substrate. No cross-reactivity was detected between DdPhyA/TgPhyA substrates and human PHD2. The human Skp1 E147P variant is a DdPhyA and TgPhyA substrate, suggesting some retention of ancestral interactions. Crystallographic analysis of DdPhyA enables comparisons with homologues from humans, Trichoplax adhaerens, and prokaryotes, informing on differences in mobile elements involved in substrate binding and catalysis. In DdPhyA, two mobile loops that enclose substrates in the PHDs are conserved, but the C-terminal helix of the PHDs is strikingly absent. The combined results support the proposal that PHD homologues have evolved kinetic and structural features suited to their specific sensing roles.

Skp1 Dimerization Conceals Its F-Box Protein Binding Site.

Skp1 is an adapter that links F-box proteins to cullin-1 in the Skp1/cullin-1/F-box (SCF) protein family of E3 ubiquitin ligases that targets specific proteins for polyubiquitination and subsequent protein degradation. Skp1 from the amoebozoan Dictyostelium forms a stable homodimer in vitro with a Kd of 2.5 µM as determined by sedimentation velocity studies yet is monomeric in crystal complexes with F-box proteins. To investigate the molecular basis for the difference, we determined the solution NMR structure of a doubly truncated Skp1 homodimer (Skp1ΔΔ). The solution structure of the Skp1ΔΔ dimer reveals a 2-fold symmetry with an interface that buries ∼750 Å2 of predominantly hydrophobic surface. The dimer interface overlaps with subsite 1 of the F-box interaction area, explaining why only the Skp1 monomer binds F-box proteins (FBPs). To confirm the model, Rosetta was used to predict amino acid substitutions that might disrupt the dimer interface, and the F97E substitution was chosen to potentially minimize interference with F-box interactions. A nearly full-length version of Skp1 with this substitution (Skp1ΔF97E) behaved as a stable monomer at concentrations of ≤500 µM and actively bound a model FBP, mammalian Fbs1, which suggests that the dimeric state is not required for Skp1 to carry out a basic biochemical function. Finally, Skp1ΔF97E is expected to serve as a monomer model for high-resolution NMR studies previously hindered by dimerization.

FBXL5 Regulates IRP2 Stability in Iron Homeostasis via an Oxygen-Responsive [2Fe2S] Cluster.

Cellular iron homeostasis is dominated by FBXL5-mediated degradation of iron regulatory protein 2 (IRP2), which is dependent on both iron and oxygen. However, how the physical interaction between FBXL5 and IRP2 is regulated remains elusive. Here, we show that the C-terminal substrate-binding domain of FBXL5 harbors a [2Fe2S] cluster in the oxidized state. A cryoelectron microscopy (cryo-EM) structure of the IRP2-FBXL5-SKP1 complex reveals that the cluster organizes the FBXL5 C-terminal loop responsible for recruiting IRP2. Interestingly, IRP2 binding to FBXL5 hinges on the oxidized state of the [2Fe2S] cluster maintained by ambient oxygen, which could explain hypoxia-induced IRP2 stabilization. Steric incompatibility also allows FBXL5 to physically dislodge IRP2 from iron-responsive element RNA to facilitate its turnover. Taken together, our studies have identified an iron-sulfur cluster within FBXL5, which promotes IRP2 polyubiquitination and degradation in response to both iron and oxygen concentrations.

Chaperone and Immunoglobulin-Binding Activities of Skp Protein from Yersinia pseudotuberculosis.

Here, we determined qualitative and quantitative characteristics of the chaperone and immunoglobulin-binding activities of recombinant Skp protein (rSkp) from Yersinia pseudotuberculosis using the methods of dynamic light scattering and surface plasmon resonance. Commercial human polyclonal IgG and Fc and Fab fragments of human IgG were used as substrate proteins. The activity of rSkp strongly depended on the medium pH. The most stable low-molecular-weight complexes with a hydrodynamic radius up to 10 nm were formed by rSkp and protein substrates at acidic pH values. Under these conditions, rSkp exhibited the lowest propensity to self-association and the highest affinity for human IgG and its Fc and Fab fragments, as well as prevented their aggregation most efficiently (i.e., demonstrated the maximal chaperone activity). As the medium pH increased, the affinity of rSkp for IgG and its fragments decreased; rSkp was not able to completely prevent the aggregation of protein substrates, but significantly slowed it down. The obtained information may be of practical interest, since the stability of therapeutic IgG preparations affects their safety and efficacy in medical applications.

Antitumor Activity of Ohmyungsamycin A through the Regulation of the Skp2-p27 Axis and MCM4 in Human Colorectal Cancer Cells.

Ohmyungsamycin A (1), a novel cyclic peptide discovered from a marine Streptomyces sp., was previously reported with antibacterial and anticancer activities. However, the antitumor activities and the underlying molecular mechanisms of 1 remain to be elucidated. Compound 1 inhibited the proliferation and tumor growth of HCT116 human colorectal cancer cells based on both in vitro cell cultures and an in vivo animal model. A cDNA microarray analysis revealed that 1 downregulated genes involved in cell cycle checkpoint control. Compound 1 also induced G0/G1 cell cycle arrest that was mediated by the regulation of S-phase kinase-associated protein 2 (Skp2)-p27 axis and minichromosome maintenance protein 4 (MCM4). Furthermore, a longer exposure of 1 exhibited an accumulation of a sub-G1 phase cell population, which is characteristic of apoptotic cells. The induction of apoptosis by 1 was also associated with the modulation of caspase family proteins. Compound 1 effectively suppressed tumor growth in a xenograft mouse model subcutaneously implanted with HCT116 cells. In addition, analysis of tumors revealed that 1 upregulated the expression of the CDK inhibitor p27 but downregulated the expression of Skp2 and MCM4. These findings demonstrate the involvement of 1 in cell cycle regulation and the induction of apoptosis in human colorectal cancer cells.

The Putative Association of TOB1-AS1 Long Non-coding RNA with Immune Tolerance: A Study on Multiple Sclerosis Patients.

The hallmark of multiple sclerosis (MS) pathogenesis is the breakdown of peripheral tolerance in the immune system. However, its molecular mechanism is not completely understood. Since long non-coding RNAs (lncRNAs) has played important roles in regulation of immunological pathways, here, we evaluated the expression of a novel lncRNA, TOB1-AS1, and its putative associated coding genes in the mechanism of maintaining immune tolerance in peripheral blood of MS patients to assess their possible roles in MS pathogenesis. In this study, 39 MS patients and 32 healthy matched controls were recruited. Real-time PCR standard curve method was used to quantify transcript levels of TOB1-AS1, TOB1, SKP2, and TSG. In addition, the potential sex hormone receptor binding sites on target genes promoter were analyzed using JASPR software. This work demonstrates a negative correlation between TOB1-AS1 expression and EDSS of patients. Also, a robust dysregulation of co-expression of TOB1-AS1 lncRNA and the coding genes in MS patients compared to controls was observed. Such dysregulation in this pathway may be related to MS pathogenesis and response to interferon treatment.

Finding of bands of higher molecular weight than expected in three proteins in bovine preimplantation embryos.

SummaryWe report here the existence of bands of higher molecular weight after western blot analysis in three proteins - Skp1, p27 and IκBα in bovine preimplantation embryos. This finding is specific to preimplantation embryos (from the 2-cell stage to the blastocyst stage) and not differentiated fibroblast cells in which these bands were of expected molecular weight. We suggest that these bands of higher molecular weight represent a complex of proteins that are characteristic of preimplantation embryos.

Identification and structural characterization of deleterious non-synonymous single nucleotide polymorphisms in the human SKP2 gene.

In SCF (Skp, Cullin, F-box) ubiquitin-protein ligase complexes, S-phase kinase 2 (SKP2) is one of the major players of F-box family, that is responsible for the degradation of several important cell regulators and tumor suppressor proteins. Despite of having significant evidence for the role of SKP2 on tumorgenesis, there is a lack of available data regarding the effect of non-synonymous polymorphisms. In this communication, the structural and functional consequences of non-synonymous single nucleotide polymorphisms (nsSNPs) of SKP2 have been reported by employing various computational approaches and molecular dynamics simulation. Initially, several computational tools like SIFT, PolyPhen-2, PredictSNP, I-Mutant 2.0 and ConSurf have been implicated in this study to explore the damaging SNPs. In total of 172 nsSNPs, 5 nsSNPs were identified as deleterious and 3 of them were predicted to be decreased the stability of protein. Guided from ConSurf analysis, P101L (rs761253702) and Y346C (rs755010517) were categorized as the highly conserved and functional disrupting mutations. Therefore, these mutations were subjected to three dimensional model building and molecular dynamics simulation study for the detailed structural consequences upon the mutations. The study revealed that P101L and Y346C mutations increased the flexibility and changed the structural dynamics. As both these mutations are located in the most functional regions of SKP2 protein, these computational insights might be helpful to consider these nsSNPs for wet-lab confirmatory analysis as well as in rationalizing future population based studies and structure based drug design against SKP2.

Stratifin Inhibits SCFFBW7 Formation and Blocks Ubiquitination of Oncoproteins during the Course of Lung Adenocarcinogenesis.

PURPOSE: Aberrant overexpression of SFN (stratifin) plays an oncogenic role in lung adenocarcinoma. We have shown previously that SKP1, an adapter component of E3 ubiquitin ligase forming an SCF complex, is a unique SFN-binding protein in lung adenocarcinoma cells. EXPERIMENTAL DESIGN: In silico simulation and in vitro mutagenesis analysis were performed to identify the SFN-binding domain on SKP1. We examined expression, localization, and stability of SKP1 after knockdown of SFN using lung adenocarcinoma cells including A549. In silico library screening and experimental validation were used for drug screening. Daily oral administration of each candidate drugs to A549-injected tumor-bearing mice was performed to evaluate their in vivo antitumor efficacy. RESULTS: Suppression of SFN upregulated the stability of SKP1 and accelerated its cytoplasm-to-nucleus translocation. Consistently, IHC analysis revealed that cytoplasmic expression of SKP1 was significantly associated with SFN positivity, tumor malignancy, and poorer patient outcome. After SFN suppression, ubiquitination of oncoproteins, including p-cyclin E1, p-c-Myc, p-c-Jun, and cleaved Notch 1, which are target proteins of SCFFBW7, was strongly induced. These results indicate that SFN-SKP1 binding results in SCFFBW7 dysfunction and allows several oncoproteins to evade ubiquitination and subsequent degradation. Because inhibition of SFN-SKP1 binding was expected to have antitumor efficacy, we next searched for candidate SFN inhibitors. Aprepitant and ticagrelor were finally selected as potential SFN inhibitors that dose dependently reduced SFN-SKP1 binding and tumor progression in vivo. CONCLUSIONS: As overexpression of SFN is detectable in most adenocarcinoma, we believe that SFN inhibitors would be novel and promising antitumor drugs for lung adenocarcinoma.

Glycosylation Promotes the Random Coil to Helix Transition in a Region of a Protist Skp1 Associated with F-Box Binding.

Cullin-ring-ligases mediate protein polyubiquitination, a signal for degradation in the 26S proteasome. The CRL1 class consists of Skp1/cullin-1/F-box protein/Rbx1 (SCF) complexes that cyclically associate with ubiquitin-E2 to build the polyubiquitin chain. Within the SCF complex, the 162-amino acid DdSkp1 from Dictyostelium bridges cullin-1 with an F-box protein (FBP), the specificity factor for substrate selection. The hydroxylation-dependent glycosylation of Pro143 of DdSkp1 by a pentasaccharide forms the basis of a novel O2-sensing mechanism in the social amoeba Dictyostelium and other protists. Previous evidence indicated that glycosylation promotes increased α-helical content correlating with enhanced interaction with three F-box proteins. To localize these differences, we used nuclear magnetic resonance (NMR) methods to compare nonglycosylated DdSkp1 and a glycoform with a single GlcNAc sugar (Gn-DdSkp1). We report NMR assignments of backbone 1HN, 15N, 13Cα, and 13CO nuclei as well as side-chain 13Cß and methyl 13C/1H nuclei of Ile(δ1), Leu, and Val in both unmodified DdSkp1 and Gn-DdSkp1. The random coil index and 15N{1H} HNOE indicate that the C-terminal region, which forms a helix-loop-helix motif centered on Pro143 at the crystallographically defined binding interface with F-box domains, remains dynamic in both DdSkp1 and Gn-DdSkp1. Chemical shifts indicate that the variation of conformation in Gn-DdSkp1, relative to DdSkp1, is limited to this region and characterized by increased helical fold. Extension of the glycan chain results in further changes, also limited to this region. Thus, glycosylation may control F-box protein interactions via a local effect on DdSkp1 conformation, by a mechanism that may be general to many unicellular eukaryotes.

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

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.