Limits...
Structure of Rpn10 and its interactions with polyubiquitin chains and the proteasome subunit Rpn12.

Riedinger C, Boehringer J, Trempe JF, Lowe ED, Brown NR, Gehring K, Noble ME, Gordon C, Endicott JA - J. Biol. Chem. (2010)

Bottom Line: We have solved the structure of full-length SpRpn10 by determining the crystal structure of the von Willebrand factor type A domain and characterizing the full-length protein by NMR.We demonstrate that the single Ub-interacting motif (UIM) of SpRpn10 forms a 1:1 complex with Lys(48)-linked diUb, which it binds selectively over monoUb and Lys(63)-linked diUb.We further show that the SpRpn10 UIM binds to SpRpn12, a subunit of the lid subparticle, with an affinity comparable with Lys(48)-linked diUb.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.

ABSTRACT
Schizosaccharomyces pombe Rpn10 (SpRpn10) is a proteasomal ubiquitin (Ub) receptor located within the 19 S regulatory particle where it binds to subunits of both the base and lid subparticles. We have solved the structure of full-length SpRpn10 by determining the crystal structure of the von Willebrand factor type A domain and characterizing the full-length protein by NMR. We demonstrate that the single Ub-interacting motif (UIM) of SpRpn10 forms a 1:1 complex with Lys(48)-linked diUb, which it binds selectively over monoUb and Lys(63)-linked diUb. We further show that the SpRpn10 UIM binds to SpRpn12, a subunit of the lid subparticle, with an affinity comparable with Lys(48)-linked diUb. This is the first observation of a UIM binding other than a Ub fold and suggests that SpRpn12 could modulate the activity of SpRpn10 as a proteasomal Ub receptor.

Show MeSH
Models for Lys48- and Lys63-linked diUb binding to SpRpn10. A, the two hydrophobic patches of Lys48-linked diUb are facing each other and form a groove that can only accommodate one UIM. With the proximal moiety bound, the distal Ub would be positioned on the opposite side of the UIM helix. B, binding of the distal Ub moiety to the UIM places the proximal Ub in close proximity to the SpRpn10 linker region. The groove formed between the two Ub moieties “traps” the UIM between two UIM binding sites, allowing for efficient rebinding. C, Lys63-linked diUb is quite extended, and the two hydrophobic UIM binding sites are located on opposite sides of the molecule. This relative disposition permits independent binding of two SpRpn10 molecules. Residues within the SpRpn10 linker sequence that undergo significant chemical shifts upon Lys48-linked diUb binding are colored dark cyan. The LALAL motif is colored cyan. Ub residues Leu8, Ile44, His68, and Val70 which contribute to the UIM binding site are colored light salmon. Additional Lys63-linked Ub residues that experience peak broadening upon SpRpn10 addition are colored dark salmon.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2962499&req=5

Figure 6: Models for Lys48- and Lys63-linked diUb binding to SpRpn10. A, the two hydrophobic patches of Lys48-linked diUb are facing each other and form a groove that can only accommodate one UIM. With the proximal moiety bound, the distal Ub would be positioned on the opposite side of the UIM helix. B, binding of the distal Ub moiety to the UIM places the proximal Ub in close proximity to the SpRpn10 linker region. The groove formed between the two Ub moieties “traps” the UIM between two UIM binding sites, allowing for efficient rebinding. C, Lys63-linked diUb is quite extended, and the two hydrophobic UIM binding sites are located on opposite sides of the molecule. This relative disposition permits independent binding of two SpRpn10 molecules. Residues within the SpRpn10 linker sequence that undergo significant chemical shifts upon Lys48-linked diUb binding are colored dark cyan. The LALAL motif is colored cyan. Ub residues Leu8, Ile44, His68, and Val70 which contribute to the UIM binding site are colored light salmon. Additional Lys63-linked Ub residues that experience peak broadening upon SpRpn10 addition are colored dark salmon.

Mentions: We generated a model for the Lys48-linked diUb·SpRpn10 complex using our SpRpn10 structure and that of a modeled open conformation of Lys48-linked diUb derived from a closed crystal structure (50) (Fig. 6, A and B). Binding of the proximal Ub moiety to the LALAL motif of SpRpn10 positions the distal moiety C-terminal to, and on the opposite side of the UIM helix (Fig. 6A). Binding of the distal moiety would bring the isopeptide bond and the proximal Ub into close proximity with the SpRpn10 linker and so could account for the Lys48 linkage-specific observed chemical shifts in this region. Even though this orientation might be favored, the NMR data support a model in which both alternative binding modes are represented in the bound population. Notably, unlike Lys63-linked diUb, the two UIM binding sites of Lys48-linked diUb face each other so that rebinding is favored. Taken together, the observed shifts in the SpRpn10 linker sequence and the relative positioning of the two Ub UIM binding sites could account for the increased affinity of SpRpn10 for Lys48-linked diUb (Fig. 6).


Structure of Rpn10 and its interactions with polyubiquitin chains and the proteasome subunit Rpn12.

Riedinger C, Boehringer J, Trempe JF, Lowe ED, Brown NR, Gehring K, Noble ME, Gordon C, Endicott JA - J. Biol. Chem. (2010)

Models for Lys48- and Lys63-linked diUb binding to SpRpn10. A, the two hydrophobic patches of Lys48-linked diUb are facing each other and form a groove that can only accommodate one UIM. With the proximal moiety bound, the distal Ub would be positioned on the opposite side of the UIM helix. B, binding of the distal Ub moiety to the UIM places the proximal Ub in close proximity to the SpRpn10 linker region. The groove formed between the two Ub moieties “traps” the UIM between two UIM binding sites, allowing for efficient rebinding. C, Lys63-linked diUb is quite extended, and the two hydrophobic UIM binding sites are located on opposite sides of the molecule. This relative disposition permits independent binding of two SpRpn10 molecules. Residues within the SpRpn10 linker sequence that undergo significant chemical shifts upon Lys48-linked diUb binding are colored dark cyan. The LALAL motif is colored cyan. Ub residues Leu8, Ile44, His68, and Val70 which contribute to the UIM binding site are colored light salmon. Additional Lys63-linked Ub residues that experience peak broadening upon SpRpn10 addition are colored dark salmon.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2962499&req=5

Figure 6: Models for Lys48- and Lys63-linked diUb binding to SpRpn10. A, the two hydrophobic patches of Lys48-linked diUb are facing each other and form a groove that can only accommodate one UIM. With the proximal moiety bound, the distal Ub would be positioned on the opposite side of the UIM helix. B, binding of the distal Ub moiety to the UIM places the proximal Ub in close proximity to the SpRpn10 linker region. The groove formed between the two Ub moieties “traps” the UIM between two UIM binding sites, allowing for efficient rebinding. C, Lys63-linked diUb is quite extended, and the two hydrophobic UIM binding sites are located on opposite sides of the molecule. This relative disposition permits independent binding of two SpRpn10 molecules. Residues within the SpRpn10 linker sequence that undergo significant chemical shifts upon Lys48-linked diUb binding are colored dark cyan. The LALAL motif is colored cyan. Ub residues Leu8, Ile44, His68, and Val70 which contribute to the UIM binding site are colored light salmon. Additional Lys63-linked Ub residues that experience peak broadening upon SpRpn10 addition are colored dark salmon.
Mentions: We generated a model for the Lys48-linked diUb·SpRpn10 complex using our SpRpn10 structure and that of a modeled open conformation of Lys48-linked diUb derived from a closed crystal structure (50) (Fig. 6, A and B). Binding of the proximal Ub moiety to the LALAL motif of SpRpn10 positions the distal moiety C-terminal to, and on the opposite side of the UIM helix (Fig. 6A). Binding of the distal moiety would bring the isopeptide bond and the proximal Ub into close proximity with the SpRpn10 linker and so could account for the Lys48 linkage-specific observed chemical shifts in this region. Even though this orientation might be favored, the NMR data support a model in which both alternative binding modes are represented in the bound population. Notably, unlike Lys63-linked diUb, the two UIM binding sites of Lys48-linked diUb face each other so that rebinding is favored. Taken together, the observed shifts in the SpRpn10 linker sequence and the relative positioning of the two Ub UIM binding sites could account for the increased affinity of SpRpn10 for Lys48-linked diUb (Fig. 6).

Bottom Line: We have solved the structure of full-length SpRpn10 by determining the crystal structure of the von Willebrand factor type A domain and characterizing the full-length protein by NMR.We demonstrate that the single Ub-interacting motif (UIM) of SpRpn10 forms a 1:1 complex with Lys(48)-linked diUb, which it binds selectively over monoUb and Lys(63)-linked diUb.We further show that the SpRpn10 UIM binds to SpRpn12, a subunit of the lid subparticle, with an affinity comparable with Lys(48)-linked diUb.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.

ABSTRACT
Schizosaccharomyces pombe Rpn10 (SpRpn10) is a proteasomal ubiquitin (Ub) receptor located within the 19 S regulatory particle where it binds to subunits of both the base and lid subparticles. We have solved the structure of full-length SpRpn10 by determining the crystal structure of the von Willebrand factor type A domain and characterizing the full-length protein by NMR. We demonstrate that the single Ub-interacting motif (UIM) of SpRpn10 forms a 1:1 complex with Lys(48)-linked diUb, which it binds selectively over monoUb and Lys(63)-linked diUb. We further show that the SpRpn10 UIM binds to SpRpn12, a subunit of the lid subparticle, with an affinity comparable with Lys(48)-linked diUb. This is the first observation of a UIM binding other than a Ub fold and suggests that SpRpn12 could modulate the activity of SpRpn10 as a proteasomal Ub receptor.

Show MeSH