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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.

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SpRpn10 structure. A, structure of the SpRpn10 VWA domain, drawn with secondary structure elements colored yellow (β-sheets β1–β6) and blue (α-helices α1–α6). Amino acids that constitute the pseudo-MIDAS motif at the end of β1 are drawn as cylinders, labeled, and colored in coral. B, VWA structure in the vicinity of the pseudo-MIDAS motif. The MIDAS motif is characterized by the sequence DXSXS together with threonine and aspartate residues in the second (between α2 and α3) and third (between β4 and α4) loops, respectively. In the SpRpn10 VWA domain these residues are replaced by the sequence DNSEW (of which the side chains of Asp11, Ser13, and Trp15 are drawn) and amino acids Ala85 and Ser116. The hydrogen bonds formed by Asp11 show the importance of interactions in this region for the structural integrity of the “top loop” region of the fold. The 2Fo − Fc electron density map is contoured at 0.81 e−/Å3 and drawn in blue, the anomalous difference map at 10.01 e−/Å3 and colored orange. C, full-length model of SpRpn10 derived from the VWA crystal structure and the homology-modeled C terminus with the Ub binding LALAL motif highlighted in cyan.
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Figure 4: SpRpn10 structure. A, structure of the SpRpn10 VWA domain, drawn with secondary structure elements colored yellow (β-sheets β1–β6) and blue (α-helices α1–α6). Amino acids that constitute the pseudo-MIDAS motif at the end of β1 are drawn as cylinders, labeled, and colored in coral. B, VWA structure in the vicinity of the pseudo-MIDAS motif. The MIDAS motif is characterized by the sequence DXSXS together with threonine and aspartate residues in the second (between α2 and α3) and third (between β4 and α4) loops, respectively. In the SpRpn10 VWA domain these residues are replaced by the sequence DNSEW (of which the side chains of Asp11, Ser13, and Trp15 are drawn) and amino acids Ala85 and Ser116. The hydrogen bonds formed by Asp11 show the importance of interactions in this region for the structural integrity of the “top loop” region of the fold. The 2Fo − Fc electron density map is contoured at 0.81 e−/Å3 and drawn in blue, the anomalous difference map at 10.01 e−/Å3 and colored orange. C, full-length model of SpRpn10 derived from the VWA crystal structure and the homology-modeled C terminus with the Ub binding LALAL motif highlighted in cyan.

Mentions: SpRpn10 contains an N-terminal VWA domain connected via a short linker to a C-terminal sequence that encodes a single UIM (supplemental Fig. S4). Because attempts to crystallize the full-length protein were not successful, we independently determined the structure of the VWA domain by x-ray crystallography and characterized the UIM by NMR. We identified an untagged construct that encodes SpRpn10 residues 1–193 that crystallized readily (SpRpn10VWA) and solved its structure by sulfur single-wavelength anomalous dispersion at 1.3 Å (supplemental Table S1). The structure consists of a central six-stranded β-sheet of five parallel strands and one antiparallel strand (β3) sandwiched between two triplets of α-helices (Fig. 4A and supplemental Fig. S4C). The fold and topology are very similar to those of other VWA and I-domains despite their low sequence identities (supplemental Fig. S5).


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)

SpRpn10 structure. A, structure of the SpRpn10 VWA domain, drawn with secondary structure elements colored yellow (β-sheets β1–β6) and blue (α-helices α1–α6). Amino acids that constitute the pseudo-MIDAS motif at the end of β1 are drawn as cylinders, labeled, and colored in coral. B, VWA structure in the vicinity of the pseudo-MIDAS motif. The MIDAS motif is characterized by the sequence DXSXS together with threonine and aspartate residues in the second (between α2 and α3) and third (between β4 and α4) loops, respectively. In the SpRpn10 VWA domain these residues are replaced by the sequence DNSEW (of which the side chains of Asp11, Ser13, and Trp15 are drawn) and amino acids Ala85 and Ser116. The hydrogen bonds formed by Asp11 show the importance of interactions in this region for the structural integrity of the “top loop” region of the fold. The 2Fo − Fc electron density map is contoured at 0.81 e−/Å3 and drawn in blue, the anomalous difference map at 10.01 e−/Å3 and colored orange. C, full-length model of SpRpn10 derived from the VWA crystal structure and the homology-modeled C terminus with the Ub binding LALAL motif highlighted in cyan.
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Figure 4: SpRpn10 structure. A, structure of the SpRpn10 VWA domain, drawn with secondary structure elements colored yellow (β-sheets β1–β6) and blue (α-helices α1–α6). Amino acids that constitute the pseudo-MIDAS motif at the end of β1 are drawn as cylinders, labeled, and colored in coral. B, VWA structure in the vicinity of the pseudo-MIDAS motif. The MIDAS motif is characterized by the sequence DXSXS together with threonine and aspartate residues in the second (between α2 and α3) and third (between β4 and α4) loops, respectively. In the SpRpn10 VWA domain these residues are replaced by the sequence DNSEW (of which the side chains of Asp11, Ser13, and Trp15 are drawn) and amino acids Ala85 and Ser116. The hydrogen bonds formed by Asp11 show the importance of interactions in this region for the structural integrity of the “top loop” region of the fold. The 2Fo − Fc electron density map is contoured at 0.81 e−/Å3 and drawn in blue, the anomalous difference map at 10.01 e−/Å3 and colored orange. C, full-length model of SpRpn10 derived from the VWA crystal structure and the homology-modeled C terminus with the Ub binding LALAL motif highlighted in cyan.
Mentions: SpRpn10 contains an N-terminal VWA domain connected via a short linker to a C-terminal sequence that encodes a single UIM (supplemental Fig. S4). Because attempts to crystallize the full-length protein were not successful, we independently determined the structure of the VWA domain by x-ray crystallography and characterized the UIM by NMR. We identified an untagged construct that encodes SpRpn10 residues 1–193 that crystallized readily (SpRpn10VWA) and solved its structure by sulfur single-wavelength anomalous dispersion at 1.3 Å (supplemental Table S1). The structure consists of a central six-stranded β-sheet of five parallel strands and one antiparallel strand (β3) sandwiched between two triplets of α-helices (Fig. 4A and supplemental Fig. S4C). The fold and topology are very similar to those of other VWA and I-domains despite their low sequence identities (supplemental Fig. S5).

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.

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