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Structural analysis of SHARPIN, a subunit of a large multi-protein E3 ubiquitin ligase, reveals a novel dimerization function for the pleckstrin homology superfold.

Stieglitz B, Haire LF, Dikic I, Rittinger K - J. Biol. Chem. (2012)

Bottom Line: In contrast, the N-terminal region does not show any homology with known protein interaction domains but has been suggested to be responsible for self-association of SHARPIN, presumably via a coiled-coil region.We show that in SHARPIN, this domain does not appear to be used as a ligand recognition domain because it lacks many of the surface properties that are present in other pleckstrin homology fold-based interaction modules.Instead, it acts as a dimerization module extending the functional applications of this superfold.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Structure, Medical Research Council National Institute for Medical Research, The Ridgeway, London NW7 1AA, United Kingdom.

ABSTRACT
SHARPIN (SHANK-associated RH domain interacting protein) is part of a large multi-protein E3 ubiquitin ligase complex called LUBAC (linear ubiquitin chain assembly complex), which catalyzes the formation of linear ubiquitin chains and regulates immune and apoptopic signaling pathways. The C-terminal half of SHARPIN contains ubiquitin-like domain and Npl4-zinc finger domains that mediate the interaction with the LUBAC subunit HOIP and ubiquitin, respectively. In contrast, the N-terminal region does not show any homology with known protein interaction domains but has been suggested to be responsible for self-association of SHARPIN, presumably via a coiled-coil region. We have determined the crystal structure of the N-terminal portion of SHARPIN, which adopts the highly conserved pleckstrin homology superfold that is often used as a scaffold to create protein interaction modules. We show that in SHARPIN, this domain does not appear to be used as a ligand recognition domain because it lacks many of the surface properties that are present in other pleckstrin homology fold-based interaction modules. Instead, it acts as a dimerization module extending the functional applications of this superfold.

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A, schematic presentation of all four PH domains observed in the asymmetric unit of the SHARPIN crystals. The loop region connecting strand β1 with β2 of subunits I, III, and IV are not observed in the density and indicated with dashed lines. B, overall structure shown as a ribbon representation of the non-crystallographic dimer of the SHARPIN PH domain. The electrostatic potential is projected on the surface of one subunit of the PH dimer. Valine 114 of the other subunit is depicted as ball-and-stick representation and points against the hydrophobic patch of the dimer interface. The structure is rotated by 180° with respect to the structure on the left.
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Figure 2: A, schematic presentation of all four PH domains observed in the asymmetric unit of the SHARPIN crystals. The loop region connecting strand β1 with β2 of subunits I, III, and IV are not observed in the density and indicated with dashed lines. B, overall structure shown as a ribbon representation of the non-crystallographic dimer of the SHARPIN PH domain. The electrostatic potential is projected on the surface of one subunit of the PH dimer. Valine 114 of the other subunit is depicted as ball-and-stick representation and points against the hydrophobic patch of the dimer interface. The structure is rotated by 180° with respect to the structure on the left.

Mentions: To understand the structural basis of SHARPIN dimerization, we crystallized the fragment containing residues 1–127. Crystals of space group P43212 were grown in 4 m sodium formate from selenomethionine-derivatized protein, which diffracted to a maximal resolution of 2.0 Å with four molecules in the asymmetric unit. The structure was solved by single anomalous dispersion (Table 1 and supplemental Fig. S1). Remarkably, the structure of the four subunits in the asymmetric unit revealed that SHARPIN utilizes a PH fold for self-association (Fig. 2, A and B). Residues 20–120 of SHARPIN cover the canonical PH fold, which is a seven-stranded antiparallel β-sheet, strongly bent to form a β-barrel-like conformation and capped by a C-terminal α-helix. A helical turn that connects strand β4 and β5 of the collapsed β-barrel is the only variation of the conserved PH domain organization. Residues 1–20 adopt different conformations in the four copies in the asymmetric unit. The N terminus of one of the subunits is buried into the center of the symmetry axis of the tetramer and participates in intermolecular interactions with the other PH-like domains (Fig. 2A). The amino-terminal stretch of the diagonally opposite subunit makes contacts to symmetry related molecules. No density can be observed for the remaining other two N termini, indicating that they are disordered. Each of the protomers in the crystallographic tetramer forms two distinctive interfaces. Because our solution studies did not give any indication that SHARPIN assembles into higher order oligomers, it is likely that tetramerization is due to crystal packing. The subunits I and II are aligned head-to-head against the subunits IV and III, respectively, with a rotation of 180° degrees (Fig. 2A). This orientation causes the formation of a histidine-stacking interaction, which is sandwiched between two salt bridges and probably based on crystal contacts (supplemental Fig. S2). In comparison, the second interface (I–II and III–IV) (Fig. 2B) is formed predominantly by a perpendicular arrangement of the helical elements of two neighboring PH-like domains and involves the interplay of many more side chains (Fig. 3, A and B). Val-114 is in the center of a hydrophobic interface formed by Leu-115, Leu-21, and Phe-56 of the other dimer half (Fig. 3, A and B). The hydrophobic patch is limited by electrostatic interactions, which involve the side chains of two arginines positioned at the C-terminal α-helix. Arg-111 binds the backbone oxygen of Leu-21 in β1 and Arg-117 coordinates the carbonyl group of Gly-118 and the side chain of Glu-122, which are both located at the tip of the helix. To confirm the relevance of the observed interface for self-association, we introduced a negative charge into the hydrophobic patch of the dimerization side. The mutation V114D should impair the hydrophobic network of the dimer interface and abolish dimerization in solution if this was the physiologically relevant dimer interface. In a complementary approach to our AUC analysis, we performed size exclusion chromatography coupled to multi-angle light scattering measurements of the wild-type and mutant PH-like domain. In these experiments, the retention time of wild-type SHARPIN shifts from a monomeric to a dimeric species when the concentration is increased in line with a monomer-dimer equilibrium (Fig. 4A). In contrast, the V114D mutant elutes as a monomer even at the very high concentration of 2.5 mm. Similarly, no monomer-dimer equilibrium can be observed for V114D when probed by ITC (Fig. 4B). These results clearly indicate that complex formation of the PH-like dimer is indeed formed via the helical interface observed in the crystal structure.


Structural analysis of SHARPIN, a subunit of a large multi-protein E3 ubiquitin ligase, reveals a novel dimerization function for the pleckstrin homology superfold.

Stieglitz B, Haire LF, Dikic I, Rittinger K - J. Biol. Chem. (2012)

A, schematic presentation of all four PH domains observed in the asymmetric unit of the SHARPIN crystals. The loop region connecting strand β1 with β2 of subunits I, III, and IV are not observed in the density and indicated with dashed lines. B, overall structure shown as a ribbon representation of the non-crystallographic dimer of the SHARPIN PH domain. The electrostatic potential is projected on the surface of one subunit of the PH dimer. Valine 114 of the other subunit is depicted as ball-and-stick representation and points against the hydrophobic patch of the dimer interface. The structure is rotated by 180° with respect to the structure on the left.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3375506&req=5

Figure 2: A, schematic presentation of all four PH domains observed in the asymmetric unit of the SHARPIN crystals. The loop region connecting strand β1 with β2 of subunits I, III, and IV are not observed in the density and indicated with dashed lines. B, overall structure shown as a ribbon representation of the non-crystallographic dimer of the SHARPIN PH domain. The electrostatic potential is projected on the surface of one subunit of the PH dimer. Valine 114 of the other subunit is depicted as ball-and-stick representation and points against the hydrophobic patch of the dimer interface. The structure is rotated by 180° with respect to the structure on the left.
Mentions: To understand the structural basis of SHARPIN dimerization, we crystallized the fragment containing residues 1–127. Crystals of space group P43212 were grown in 4 m sodium formate from selenomethionine-derivatized protein, which diffracted to a maximal resolution of 2.0 Å with four molecules in the asymmetric unit. The structure was solved by single anomalous dispersion (Table 1 and supplemental Fig. S1). Remarkably, the structure of the four subunits in the asymmetric unit revealed that SHARPIN utilizes a PH fold for self-association (Fig. 2, A and B). Residues 20–120 of SHARPIN cover the canonical PH fold, which is a seven-stranded antiparallel β-sheet, strongly bent to form a β-barrel-like conformation and capped by a C-terminal α-helix. A helical turn that connects strand β4 and β5 of the collapsed β-barrel is the only variation of the conserved PH domain organization. Residues 1–20 adopt different conformations in the four copies in the asymmetric unit. The N terminus of one of the subunits is buried into the center of the symmetry axis of the tetramer and participates in intermolecular interactions with the other PH-like domains (Fig. 2A). The amino-terminal stretch of the diagonally opposite subunit makes contacts to symmetry related molecules. No density can be observed for the remaining other two N termini, indicating that they are disordered. Each of the protomers in the crystallographic tetramer forms two distinctive interfaces. Because our solution studies did not give any indication that SHARPIN assembles into higher order oligomers, it is likely that tetramerization is due to crystal packing. The subunits I and II are aligned head-to-head against the subunits IV and III, respectively, with a rotation of 180° degrees (Fig. 2A). This orientation causes the formation of a histidine-stacking interaction, which is sandwiched between two salt bridges and probably based on crystal contacts (supplemental Fig. S2). In comparison, the second interface (I–II and III–IV) (Fig. 2B) is formed predominantly by a perpendicular arrangement of the helical elements of two neighboring PH-like domains and involves the interplay of many more side chains (Fig. 3, A and B). Val-114 is in the center of a hydrophobic interface formed by Leu-115, Leu-21, and Phe-56 of the other dimer half (Fig. 3, A and B). The hydrophobic patch is limited by electrostatic interactions, which involve the side chains of two arginines positioned at the C-terminal α-helix. Arg-111 binds the backbone oxygen of Leu-21 in β1 and Arg-117 coordinates the carbonyl group of Gly-118 and the side chain of Glu-122, which are both located at the tip of the helix. To confirm the relevance of the observed interface for self-association, we introduced a negative charge into the hydrophobic patch of the dimerization side. The mutation V114D should impair the hydrophobic network of the dimer interface and abolish dimerization in solution if this was the physiologically relevant dimer interface. In a complementary approach to our AUC analysis, we performed size exclusion chromatography coupled to multi-angle light scattering measurements of the wild-type and mutant PH-like domain. In these experiments, the retention time of wild-type SHARPIN shifts from a monomeric to a dimeric species when the concentration is increased in line with a monomer-dimer equilibrium (Fig. 4A). In contrast, the V114D mutant elutes as a monomer even at the very high concentration of 2.5 mm. Similarly, no monomer-dimer equilibrium can be observed for V114D when probed by ITC (Fig. 4B). These results clearly indicate that complex formation of the PH-like dimer is indeed formed via the helical interface observed in the crystal structure.

Bottom Line: In contrast, the N-terminal region does not show any homology with known protein interaction domains but has been suggested to be responsible for self-association of SHARPIN, presumably via a coiled-coil region.We show that in SHARPIN, this domain does not appear to be used as a ligand recognition domain because it lacks many of the surface properties that are present in other pleckstrin homology fold-based interaction modules.Instead, it acts as a dimerization module extending the functional applications of this superfold.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Structure, Medical Research Council National Institute for Medical Research, The Ridgeway, London NW7 1AA, United Kingdom.

ABSTRACT
SHARPIN (SHANK-associated RH domain interacting protein) is part of a large multi-protein E3 ubiquitin ligase complex called LUBAC (linear ubiquitin chain assembly complex), which catalyzes the formation of linear ubiquitin chains and regulates immune and apoptopic signaling pathways. The C-terminal half of SHARPIN contains ubiquitin-like domain and Npl4-zinc finger domains that mediate the interaction with the LUBAC subunit HOIP and ubiquitin, respectively. In contrast, the N-terminal region does not show any homology with known protein interaction domains but has been suggested to be responsible for self-association of SHARPIN, presumably via a coiled-coil region. We have determined the crystal structure of the N-terminal portion of SHARPIN, which adopts the highly conserved pleckstrin homology superfold that is often used as a scaffold to create protein interaction modules. We show that in SHARPIN, this domain does not appear to be used as a ligand recognition domain because it lacks many of the surface properties that are present in other pleckstrin homology fold-based interaction modules. Instead, it acts as a dimerization module extending the functional applications of this superfold.

Show MeSH