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Molecular basis and regulation of OTULIN-LUBAC interaction.

Elliott PR, Nielsen SV, Marco-Casanova P, Fiil BK, Keusekotten K, Mailand N, Freund SM, Gyrd-Hansen M, Komander D - Mol. Cell (2014)

Bottom Line: Moreover, LUBAC-OTULIN complex formation is regulated by OTULIN phosphorylation in the PIM.Phosphorylation of OTULIN prevents HOIP binding, whereas unphosphorylated OTULIN is part of the endogenous LUBAC complex.Our work exemplifies how coordination of ubiquitin assembly and disassembly activities in protein complexes regulates individual Ub linkage types.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK.

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A PUB-Domain-Interacting Motif in OTULIN(A) Structure of PNGase bound to the p97 PIM peptide (PDB ID 2HPL) (Zhao et al., 2007) reveals the position of the PIM pocket.(B) Surface conservation analysis of the HOIP PUB domain colored according to the sequence alignment in Figure S1A. The PIM pocket is highly conserved, whereas other regions, including the surface generated by the N-terminal PUB domain extension, are not conserved.(C) Primary sequence alignment of the HOIP binding region in OTULIN (Figure S3A) (Rivkin et al., 2013). Alignment shows that the patch with highest evolutionary conservation resembles the p97 PIM.(D) Affinity measurements using HOIP PUB domain against FITC-Ahx-labeled p97 PIM peptide (aa 797–806) and OTULIN PIM peptide (aa 49–67). Experiments were performed in triplicate, and errors represent SD from the mean.(E) Binding of PUB domains from HOIP (aa 1–184, red), PNGase (aa 11–109, green), and UBXD1 (aa 150–264, magenta) to a fluorescent p97 PIM peptide (aa 797–806) as in (D). KD values are indicated, and errors represent SD from the mean from triplicate experiments.(F) Binding of PUB domains as in (E) to the OTULIN PIM peptide.(G) 15N-transverse relaxation optimized spectroscopy (TROSY) spectra of HOIP alone (black), HOIP bound to OTULIN PIM peptide at a 1:1 molar ratio (red), and HOIP bound to p97 PIM peptide at a 1:1 (blue) and 1:4 ratio (green). Selected perturbed resonances are shown. For the full spectra, see Figure S4A.(H) Chemical shift map by HOIP residue number for perturbation by p97 and OTULIN PIM peptides.(I) 15N-TROSY spectra of HOIP alone (black), HOIP bound to OTULIN PIM peptide (red), HOIP bound to full-length OTULIN (blue), and HOIP with OTULIN catalytic domain (aa 80–352, yellow), all at a 1:1 molar ratio. The same resonances as in (G) are shown. For the full spectra, see Figures S4B, 4C, and 3.(J) Difference map of chemical shifts between HOIP bound to PIM peptide or full-length OTULIN derived from respective spectra in (I).
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fig2: A PUB-Domain-Interacting Motif in OTULIN(A) Structure of PNGase bound to the p97 PIM peptide (PDB ID 2HPL) (Zhao et al., 2007) reveals the position of the PIM pocket.(B) Surface conservation analysis of the HOIP PUB domain colored according to the sequence alignment in Figure S1A. The PIM pocket is highly conserved, whereas other regions, including the surface generated by the N-terminal PUB domain extension, are not conserved.(C) Primary sequence alignment of the HOIP binding region in OTULIN (Figure S3A) (Rivkin et al., 2013). Alignment shows that the patch with highest evolutionary conservation resembles the p97 PIM.(D) Affinity measurements using HOIP PUB domain against FITC-Ahx-labeled p97 PIM peptide (aa 797–806) and OTULIN PIM peptide (aa 49–67). Experiments were performed in triplicate, and errors represent SD from the mean.(E) Binding of PUB domains from HOIP (aa 1–184, red), PNGase (aa 11–109, green), and UBXD1 (aa 150–264, magenta) to a fluorescent p97 PIM peptide (aa 797–806) as in (D). KD values are indicated, and errors represent SD from the mean from triplicate experiments.(F) Binding of PUB domains as in (E) to the OTULIN PIM peptide.(G) 15N-transverse relaxation optimized spectroscopy (TROSY) spectra of HOIP alone (black), HOIP bound to OTULIN PIM peptide at a 1:1 molar ratio (red), and HOIP bound to p97 PIM peptide at a 1:1 (blue) and 1:4 ratio (green). Selected perturbed resonances are shown. For the full spectra, see Figure S4A.(H) Chemical shift map by HOIP residue number for perturbation by p97 and OTULIN PIM peptides.(I) 15N-TROSY spectra of HOIP alone (black), HOIP bound to OTULIN PIM peptide (red), HOIP bound to full-length OTULIN (blue), and HOIP with OTULIN catalytic domain (aa 80–352, yellow), all at a 1:1 molar ratio. The same resonances as in (G) are shown. For the full spectra, see Figures S4B, 4C, and 3.(J) Difference map of chemical shifts between HOIP bound to PIM peptide or full-length OTULIN derived from respective spectra in (I).

Mentions: The PNGase PUB domain was shown to have two functional surfaces. The first one is the PIM pocket derived from a PNGase crystal structure in complex with a five-residue DDLYG PIM peptide corresponding to the p97 C terminus (Zhao et al., 2007). In this interaction, two key residues in the PIM peptide (Leu804 and Tyr805) form mainly hydrophobic interactions with a hydrophobic pocket, the PIM pocket (Zhao et al., 2007) (Figures 1F and 2A, see below). A second functional surface of the PNGase PUB domain is a binding site for Ub or the Ub-like domain of human Rad23 located on the opposite face of the PIM pocket (Kamiya et al., 2012).


Molecular basis and regulation of OTULIN-LUBAC interaction.

Elliott PR, Nielsen SV, Marco-Casanova P, Fiil BK, Keusekotten K, Mailand N, Freund SM, Gyrd-Hansen M, Komander D - Mol. Cell (2014)

A PUB-Domain-Interacting Motif in OTULIN(A) Structure of PNGase bound to the p97 PIM peptide (PDB ID 2HPL) (Zhao et al., 2007) reveals the position of the PIM pocket.(B) Surface conservation analysis of the HOIP PUB domain colored according to the sequence alignment in Figure S1A. The PIM pocket is highly conserved, whereas other regions, including the surface generated by the N-terminal PUB domain extension, are not conserved.(C) Primary sequence alignment of the HOIP binding region in OTULIN (Figure S3A) (Rivkin et al., 2013). Alignment shows that the patch with highest evolutionary conservation resembles the p97 PIM.(D) Affinity measurements using HOIP PUB domain against FITC-Ahx-labeled p97 PIM peptide (aa 797–806) and OTULIN PIM peptide (aa 49–67). Experiments were performed in triplicate, and errors represent SD from the mean.(E) Binding of PUB domains from HOIP (aa 1–184, red), PNGase (aa 11–109, green), and UBXD1 (aa 150–264, magenta) to a fluorescent p97 PIM peptide (aa 797–806) as in (D). KD values are indicated, and errors represent SD from the mean from triplicate experiments.(F) Binding of PUB domains as in (E) to the OTULIN PIM peptide.(G) 15N-transverse relaxation optimized spectroscopy (TROSY) spectra of HOIP alone (black), HOIP bound to OTULIN PIM peptide at a 1:1 molar ratio (red), and HOIP bound to p97 PIM peptide at a 1:1 (blue) and 1:4 ratio (green). Selected perturbed resonances are shown. For the full spectra, see Figure S4A.(H) Chemical shift map by HOIP residue number for perturbation by p97 and OTULIN PIM peptides.(I) 15N-TROSY spectra of HOIP alone (black), HOIP bound to OTULIN PIM peptide (red), HOIP bound to full-length OTULIN (blue), and HOIP with OTULIN catalytic domain (aa 80–352, yellow), all at a 1:1 molar ratio. The same resonances as in (G) are shown. For the full spectra, see Figures S4B, 4C, and 3.(J) Difference map of chemical shifts between HOIP bound to PIM peptide or full-length OTULIN derived from respective spectra in (I).
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fig2: A PUB-Domain-Interacting Motif in OTULIN(A) Structure of PNGase bound to the p97 PIM peptide (PDB ID 2HPL) (Zhao et al., 2007) reveals the position of the PIM pocket.(B) Surface conservation analysis of the HOIP PUB domain colored according to the sequence alignment in Figure S1A. The PIM pocket is highly conserved, whereas other regions, including the surface generated by the N-terminal PUB domain extension, are not conserved.(C) Primary sequence alignment of the HOIP binding region in OTULIN (Figure S3A) (Rivkin et al., 2013). Alignment shows that the patch with highest evolutionary conservation resembles the p97 PIM.(D) Affinity measurements using HOIP PUB domain against FITC-Ahx-labeled p97 PIM peptide (aa 797–806) and OTULIN PIM peptide (aa 49–67). Experiments were performed in triplicate, and errors represent SD from the mean.(E) Binding of PUB domains from HOIP (aa 1–184, red), PNGase (aa 11–109, green), and UBXD1 (aa 150–264, magenta) to a fluorescent p97 PIM peptide (aa 797–806) as in (D). KD values are indicated, and errors represent SD from the mean from triplicate experiments.(F) Binding of PUB domains as in (E) to the OTULIN PIM peptide.(G) 15N-transverse relaxation optimized spectroscopy (TROSY) spectra of HOIP alone (black), HOIP bound to OTULIN PIM peptide at a 1:1 molar ratio (red), and HOIP bound to p97 PIM peptide at a 1:1 (blue) and 1:4 ratio (green). Selected perturbed resonances are shown. For the full spectra, see Figure S4A.(H) Chemical shift map by HOIP residue number for perturbation by p97 and OTULIN PIM peptides.(I) 15N-TROSY spectra of HOIP alone (black), HOIP bound to OTULIN PIM peptide (red), HOIP bound to full-length OTULIN (blue), and HOIP with OTULIN catalytic domain (aa 80–352, yellow), all at a 1:1 molar ratio. The same resonances as in (G) are shown. For the full spectra, see Figures S4B, 4C, and 3.(J) Difference map of chemical shifts between HOIP bound to PIM peptide or full-length OTULIN derived from respective spectra in (I).
Mentions: The PNGase PUB domain was shown to have two functional surfaces. The first one is the PIM pocket derived from a PNGase crystal structure in complex with a five-residue DDLYG PIM peptide corresponding to the p97 C terminus (Zhao et al., 2007). In this interaction, two key residues in the PIM peptide (Leu804 and Tyr805) form mainly hydrophobic interactions with a hydrophobic pocket, the PIM pocket (Zhao et al., 2007) (Figures 1F and 2A, see below). A second functional surface of the PNGase PUB domain is a binding site for Ub or the Ub-like domain of human Rad23 located on the opposite face of the PIM pocket (Kamiya et al., 2012).

Bottom Line: Moreover, LUBAC-OTULIN complex formation is regulated by OTULIN phosphorylation in the PIM.Phosphorylation of OTULIN prevents HOIP binding, whereas unphosphorylated OTULIN is part of the endogenous LUBAC complex.Our work exemplifies how coordination of ubiquitin assembly and disassembly activities in protein complexes regulates individual Ub linkage types.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK.

Show MeSH