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, 401 (3), 389-402

Structural Basis for Par-4 Recognition by the SPRY Domain- And SOCS Box-Containing Proteins SPSB1, SPSB2, and SPSB4

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Structural Basis for Par-4 Recognition by the SPRY Domain- And SOCS Box-Containing Proteins SPSB1, SPSB2, and SPSB4

Panagis Filippakopoulos et al. J Mol Biol.

Abstract

The mammalian SPRY domain- and SOCS box-containing proteins, SPSB1 to SPSB4, belong to the SOCS box family of E3 ubiquitin ligases. Substrate recognition sites for the SPRY domain are identified only for human Par-4 (ELNNNL) and for the Drosophila orthologue GUSTAVUS binding to the DEAD-box RNA helicase VASA (DINNNN). To further investigate this consensus motif, we determined the crystal structures of SPSB1, SPSB2, and SPSB4, as well as their binding modes and affinities for both Par-4 and VASA. Mutation of each of the three Asn residues in Par-4 abrogated binding to all three SPSB proteins, while changing EL to DI enhanced binding. By comparison to SPSB1 and SPSB4, the more divergent protein SPSB2 showed only weak binding to Par-4 and was hypersensitive to DI substitution. Par-4((59-77)) binding perturbed NMR resonances from a number of SPSB2 residues flanking the ELNNN binding site, including loop D, which binds the EL/DI sequence. Although interactions with the consensus peptide motif were conserved in all structures, flanking sites in SPSB2 were identified as sites of structural change. These structural changes limit high-affinity interactions for SPSB2 to aspartate-containing sequences, whereas SPSB1 and SPSB4 bind strongly to both Par-4 and VASA peptides.

Figures

Fig. 1
Fig. 1
Identification of key interacting residues of hPar-4. (a) Comparison of HSQC spectra of uniformly 15N-labeled hPar-4(59–77), free (red) and in a 1:1 complex with mSPSB2(12–224) (blue). Spectra were recorded on 0.1-mM solutions in 95% H2O/5% 2H2O, pH 6.7, and 295 K, using a Bruker Avance500 spectrometer with a cryoprobe. (b) Weighted average chemical shift variations of 15N and 1H between free and bound forms of 15N-labeled hPar-4(59–77). The weighted averages of the three Asn residues, Asn70–72 (blue asterisks and blue bars), are a representation only, as the peaks in the complex may have shifted more than indicated. Green asterisks correspond to residues (Gly59, Pro61, and Pro74) not observed in these spectra, while the purple asterisk denotes a very weak peak from Thr60.
Fig. 2
Fig. 2
Identification of key interacting residues of mSPSB2. (a) Comparison of HSQC spectra of 0.1 mM uniformly N-labeled mSPSB2(12–224), free (red) and in a 1:1 complex with hPar-4(59–77) (blue). Spectra were recorded in 95% H2O/5% 2H2O, pH 6.7, 295 K, at 500 MHz. The red peaks are labeled with sequence-specific assignments for free mSPSB2(12–224) using the one-letter code and sequence positions (black). Conserved residues of GUSTAVUS and three SPSB proteins that are important for GUSTAVUS/VASA and mSPSB2/hPar-4 interactions are represented in cyan. Aliased resonances arising from Arg side chains of mSPSB2 are shown in red (free) and blue (complex) square boxes as these were due to different spectra widths used in the 15N dimension. (b) Weighted average chemical shift variations of 15N and 1H between free and bound forms of 15N-labeled mSPSB2(12–224). Cyan asterisks represent those residues that are conserved in GUSTAVUS, mSPSB1, mSPSB2, and mSPSB4 and have been shown to be involved in GUSTAVUS binding to VASA, except for mSPSB2 E55, where L66 is found in GUSTAVUS, mSPSB1, and mSPSB4. Orange asterisks indicate those residues that are known to be involved in mSPSB2/hPar-4 interaction according to a previous study. Horizontal lines show cutoffs for weighted average chemical shift differences of 0.02  and 0.04 ppm. The Trp207 peak is from the indole NH; the backbone amide resonance for this residue is close to the water resonance and difficult to follow.
Fig. 3
Fig. 3
Crystal structures of hSPSB1, SPSB2, and SPSB4. (a) Overview of the structures of hSPSB1 in complex with hPar-4 and VASA, as well as the hSPSB2 complex with VASA and apo-hSPSB4. Peptide ligands are shown as sticks. (b) Molecular surface representation of hSPSB structures. The electrostatic surface potential is conserved across the hPar-4 binding site, but more varied beyond this epitope. (c) Sequence alignment of SPRY domain structures in the SPSB family. Secondary-structure elements are shown for hSPSB1, including binding loops A–E. Key hPar-4 contact residues are indicated by asterisks. Red and yellow shading indicate positions with sequence identity and sequence similarity, respectively. An enhanced 3D visualization file displaying all structures is available for download (file S6).
Fig. 4
Fig. 4
Comparison of the VASA and hPar-4 binding modes to hSPSB1. (a) Overlay of the hSPSB1 structures in complex with VASA (dark green) and hPar-4 (light green). The surface of hSPSB1 is colored by electrostatic surface potential. The peptide NNN motifs bind a shallow pocket centered on hSPSB1 Arg77. The peptide backbone deviates at hPar-4 Glu68 where the shorter Asp184 side chain of VASA enables an intramolecular main-chain hydrogen bond with VASA Asn188. (b) Stick representation showing the side-chain hydrogen bonds formed by the central hPar-4 NNN motif. Similar bonds are formed in the VASA complex. (c) Intramolecular hydrogen bonding in hPar-4 and VASA. The VASA conformation is stabilized by an additional main-chain hydrogen bond between Asp184 and Asn188.
Fig. 5
Fig. 5
Comparison of the hSPSB1 and hSPSB2 binding modes. Overlay of the hSPSB1 (orange) and hSPSB2 (green) structures in complex with VASA. (a) The N-terminal region of VASA in both structures superimposes well, but the C-terminal region was disordered in the hSPSB2 structure. The hSPSB2 SPRY domain is shown in green ribbon. (b) Overlay of the two structures reveals a likely steric clash between the VASA C-terminal region (orange) and the side chains of hSPSB2 Glu55 and Gln116 (green). (c) hSPSB1 and hSPSB2 show structural changes in β7/loop D where hSPSB1 residues 125-HSVG-128 are replaced by hSPSB2 116-QTDH-119. At a neighboring position, only a small change is observed in the position of the hSPSB2 Tyr120 hydrogen bond with VASA Glu196, with little change in the overall bond length or geometry.
Fig. 6
Fig. 6
Structural changes upon ligand binding. (a) Overlay of the hSPSB2/VASA complex and apo-mSPSB2 structure. Side-chain rearrangements between the ligand-free and ligand-bound state are largely limited to hSPSB2 Trp207. The hSPSB2-bound VASA peptide is shown in ribbon representation for reference and colored orange. (b) Overlay of the ligand-free structures of hSPSB4 and mSPSB2 reveals more substantial structural changes in loops B, C, and E. The side chain of hSPSB4 Trp217 also shows a substantial rotamer change from mSPSB2 Trp207. Note that this residue has an unusual backbone amide chemical shift in apo-mSPSB2.

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