Crystal structure of a SFPQ/PSPC1 heterodimer provides insights into preferential heterodimerization of human DBHS family proteins

Abstract

Members of the Drosophila behavior human splicing (DBHS) protein family are nuclear proteins implicated in many layers of nuclear functions, including RNA biogenesis as well as DNA repair. Definitive of the DBHS protein family, the conserved DBHS domain provides a dimerization platform that is critical for the structural integrity and function of these proteins. The three human DBHS proteins, splicing factor proline- and glutamine-rich (SFPQ), paraspeckle component 1 (PSPC1), and non-POU domain-containing octamer-binding protein (NONO), form either homo- or heterodimers; however, the relative affinity and mechanistic details of preferential heterodimerization are yet to be deciphered. Here we report the crystal structure of a SFPQ/PSPC1 heterodimer to 2.3-Å resolution and analyzed the subtle structural differences between the SFPQ/PSPC1 heterodimer and the previously characterized SFPQ homodimer. Analytical ultracentrifugation to estimate the dimerization equilibrium of the SFPQ-containing dimers revealed that the SFPQ-containing dimers dissociate at low micromolar concentrations and that the heterodimers have higher affinities than the homodimer. Moreover, we observed that the apparent dissociation constant for the SFPQ/PSPC1 heterodimer was over 6-fold lower than that of the SFPQ/NONO heterodimer. We propose that these differences in dimerization affinity may represent a potential mechanism by which PSPC1 at a lower relative cellular abundance can outcompete NONO to heterodimerize with SFPQ.

Keywords: DBHS protein family; NONO; PSPC1; RNA binding protein; SFPQ; analytical ultracentrifugation; crystal structure; dimerization; multifunctional protein.

Figure 1.

Domain architecture of human DBHS family proteins.

Figure 2.

Analytical ultracentrifugation analyses of SFPQ homo- and heterodimers. A–C, continuous size (c(s)) distributions are plotted as a function of sedimentation coefficient for (A) 5.0 μm SFPQ homodimer, (B) 5.0 μm SFPQ/NONO heterodimer, and (C) 5.0 μm SFPQ/PSPC1 heterodimer. Insets, residuals for the c(s) distribution best fits plotted as a function of radial position. All c(s) distribution best fits yielded r.m.s. deviation values <0.0055 and Runs test Z values <13. D–F, sedimentation equilibrium analyses of (D) SFPQ homodimer, (E) SFPQ/NONO heterodimer, and (F) SFPQ/PSPC1 heterodimer. Plotted are the data at 12,000 rpm (circles) and 18,000 rpm (triangles) for each protein at an initial concentration of 1.5 μm with solid lines representing the nonlinear best fits to homo- or heterodimerization equilibrium models. Insets, residuals plotted as a function of radial position for the best fits obtained at both rotor speeds. Note that data and fits shown are representative of global nonlinear least squares analyses employing multiple protein concentrations (0.5–15 μm) and rotor speeds that yielded the dimerization dissociation constants reported in Table 1. Global reduced χ² values were <1.0 for all nonlinear best fits.

Figure 3.

Crystal structure of a SFPQ/PSPC1 heterodimer. A, construct boundary used in this study is indicated with a square box, including residues 276–535 for SFPQ and 61–320 for PSPC1. Residues modeled in the final structure are colored in gray for SFPQ (part of NOPS and terminal residues disordered), in gold (RRM1), dark blue (RRM2), orange (NOPS) and red (coiled-coil) for PSPC1. B, stereo view of the heterodimer; SFPQ in surface presentation and PSPC1 in schematic presentation. C, side and top view of the heterodimer; SFPQ in blue, PSPC1 in yellow.

Figure 4.

Comparison of the SFPQ/PSPC1 heterodimer with the SFPQ homodimer. A, superposition of the SFPQ (blue)/PSPC1 (yellow) heterodimer with the SFPQ homodimer (brown, PDB code 4WII). Regions 1–4 harboring a stretch of amino acids with sequence variations and the associated local differences in structure are indicated. B, comparison of the two RRM arrangement in SFPQ/PSC1 (left, SFPQ in blue and PSPC1 in yellow) and SFPQ homodimer (right, brown). C, close-up view of the interface of the two RRMs in B; D and E, structural plasticity of the NOPS domain. NOPS domains are colored in yellow (PSPC1) and cyan (SFPQ), whereas interacting RRM2 from the partner subunit (RRM2′) shown in blue (SFPQ) and pink (PSPC1) in D. The corresponding regions in the SFPQ homodimer (PDB 4WII) are shown in E, with the NOPS domains in salmon pink (Chain A) and gray (Chain B) and the interacting RRM2′ in brown (Chain B) and green (Chain A). F, critical dimerization residues in the NOPS domain (boxed in A) show different side chain conformations in the SFPQ/PSPC1 heterodimer. The side chains of the conserved aromatic residues in NOPS are superposed; side chains for SFPQ shown in blue with its counterpart in dark gray with residue numbering for SFPQ. G–I, close-up views of the NOPS and RRM2 interactions in the SFPQ homodimer (G) and SFPQ/PSPC1 heterodimer (H and I) show variations in hydrophobic residues involved in the dimer interface. Italic residue numbers belong to PSPC1, whereas residue numbers with prime are for the partner subunit of the SFPQ homodimer.