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. 2020 Apr 21;59(15):1527-1536.
doi: 10.1021/acs.biochem.0c00094. Epub 2020 Apr 13.

Skp1 Dimerization Conceals Its F-Box Protein Binding Site

Hyun W Kim, Alexander Eletsky, Karen J Gonzalez, Hanke van der Wel, Eva-Maria Strauch, James H Prestegard, Christopher M West

Skp1 Dimerization Conceals Its F-Box Protein Binding Site

Hyun W Kim et al. Biochemistry. .

Abstract

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.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1.
Figure 1.
Sedimentation velocity analysis of Dictyostelium Skp1. (A) c(s) distribution reveals concentration dependence of dimerization. The concentration range is depicted by a rainbow spectrum with the lowest concentration in red and the highest in purple. (B) An isotherm was constructed with weighted s-values (Sw); the fitted model indicates a Kd of 2.5 µM. The color of each data point corresponds to the respective c(s) distribution in panel A.
Figure 2.
Figure 2.
Structure of the Skp1 dimer. (A) Domain diagrams of the constructs examined. Note that versions derived from His6Skp1 have a SerMet-extension beyond the native Ser- resulting from Met removal. See Figure S1 for details. (B) Superimposition of Cα-traces of 20 calculated conformers of Skp1ΔΔ. (C) Ribbon representation of the lowest energy Skp1ΔΔ conformer (PDB ID 6V88). Dimer subunits are colored in green or magenta. A 2-fold axis of rotational symmetry lies vertically between the subunits. (D) Ribbon representation of a single Skp1ΔΔ, with the residues contributing to intermolecular contacts (<5 Å) shown in green with stick representations of their side chains. (E) Surface representation of the Skp1ΔΔ dimer is shown with the rear subunit colored in green and red, and the front in transparent gray. Red shading represents the homodimer contact region. (F) Surface representation of a hypothetical Skp1ΔΔ/F-box heterodimer model, generated by substitution of a single Skp1ΔΔ subunit for Skp1 in a human Skp1/FBXW7 complex (PDB ID 5V4B). Coloration is as in E, with FBXW7 residues 2263-2355 in gray.
Figure 3.
Figure 3.
Computational scanning mutagenesis of the Skp1ΔΔ homodimer interface. (A) Alanine-scanning mutagenesis using Rosetta. Changes in binding free energy upon replacement with alanine are shown according to the interface residue positions. (B) Skp1 dimer protein-protein interface. Residues with the highest binding free energy change (Phe97 and Ile123) are emphasized in stick representation and in red; other mutated residues are in blue. See panel C for color code explanation. (C) Heatmap of the changes in binding free energy upon all amino acid substitutions. Effects of amino acid replacements are shown for each interface position. The colors represent the changes in the binding free energy of the dimer (interface ΔΔG score). Values greater than one (warmer colors) indicate destabilizing mutations, and values less than one (colder colors) imply stabilizing mutation. Compare with effects on the monomer state (Fig. S8).
Figure 4.
Figure 4.
Skp1ΔF97E is a stable and functional monomer in solution. (A) c(s) distributions of 100 µM Skp1Δ or Skp1ΔF97E are shown in cyan and black, respectively. (B) 1H/15N-HSQC of 100 µM Skp1ΔF97E at 900 MHz and 35°C, with a 4 h collection time. The 500 μM spectrum (not shown) was indistinguishable. (C, D) Skp1ΔF97E binds the model F-box protein Fbs1. His6Fbs1 (1.5 μM) and an estimated 2.25 µM Skp1Δ (C) or Skp1Δ(F97E) (D) were analyzed on a Superdex 200 gel filtration column. Elution was monitored by A280, which favors detection of Fbs1 relative to Skp1 because of its higher extinction coefficient.
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References

    1. West CM,; Blader IJ, (2015) Oxygen Sensing by Protozoans: How They Catch Their Breath. Curr. Opin. Microbiol 26, 41–47. - PMC - PubMed
    1. van der Wel H,; Gas-Pascual E,; West CM, (2019) Skp1 Isoforms are Differentially Modified by a Dual Function Prolyl 4-Hydroxylase/N-Acetylglucosaminyltransferase in a Plant Pathogen. Glycobiology 29, 705–714. - PMC - PubMed
    1. Mandalasi M,; Kim HW,; Thieker D,; Sheikh MO,; Gas-Pascual E,; Rahman K,; Zhao P,; Daniel N,; van der Wel H,; Ichikawa TH,; Glushka JN,; Wells L,; Woods RJ,; Wood ZA,; West CM, (2020) A Glycogenin Homolog Controls Toxoplasma gondii Growth Via Glycosylation of an E3 Ubiquitin Ligase. bioRxiv. https://www.biorxiv.org/content/10.1101/764241v2 - DOI - PMC - PubMed
    1. Sheikh MO,; Thieker D,; Chalmers G,; Schafer CM,; Ishihara M,; Azadi P,; Woods RJ,; Glushka JN,; Bendiak B,; Prestegard JH,; West CM, (2017) O2 Sensing–Associated Glycosylation Exposes the F-Box–Combining Site of the Dictyostelium Skp1 Subunit in E3 Ubiquitin Ligases. J. Biol. Chem 292, 18897–18915. - PMC - PubMed
    1. Henzl MT,; Thalmann I,; Thalmann R, (1998) OCP2 Exists as a Dimer in the Organ of Corti. Hear. Res 126, 37–46. - PubMed
-