Molecular characterization of disrupted in schizophrenia-1 risk variant S704C reveals the formation of altered oligomeric assembly

Abstract

DISC1 (Disrupted in schizophrenia-1) plays essential roles in neuronal proliferation, neuronal migration and axon guidance and has been implicated in schizophrenia and related psychiatric disorders. DISC1 forms a functional complex with nuclear distribution element-like protein-1 (NDEL1), a key component that regulates microtubule organization during cell division and neuronal migration. DISC1 polymorphisms at the binding interface of DISC1-NDEL1 complex have been implicated in schizophrenia. However, it is unknown how schizophrenia risk polymorphisms perturb its interaction with NDEL1 and how they change the inherent biochemical properties of DISC1. Here, we characterize the oligomerization and binding property of DISC1 and its natural schizophrenia risk variant, S704C. Our results show that DISC1 forms octamers via dimers as building blocks and directly interacts with tetramers of NDEL1. The schizophrenia risk variant S704C affects the formation of octamers of DISC1 and exhibits higher-order self-oligomerization. However, the observed formation of new oligomeric species did not influence its binding with NDEL1. These results suggest that the improper oligomeric assembly of DISC1-S704C may underlie the observed phenotypic variation due to the polymorphism.

FIGURE 1.

Expression and purification of DISC1 and NDEL1. DISC1 and NDEL1 were co-expressed with a trigger factor in a BL21(Star) strain. Homogenously purified DISC1 with MBP (molecular mass ∼135 kDa, without MBP (molecular mass ∼95 kDa) (a) and GB1-NDEL1 (molecular mass ∼52 kDa) (b) are shown in the SDS-PAGE gel. Folding of DISC1 and NDEL1 was assessed using circular dichroism spectroscopy. Both DISC1 (c) and NDEL1 (d) show significant helical content. Size-exclusion chromatography suggests DISC1 exists as a higher-order oligomers (> hexamer) (e), whereas the NDEL1 profile suggests the protein is a tetramer (f). a.u., arbitrary unit.

FIGURE 2.

Pulldown assays further confirm that the specificity of the DISC1/NDEL1 interaction is mapped to a key helical region on the C terminus of DISC1. a, to map the NDEL1 binding region on recombinant DISC1, two DISC1 constructs (MBP-DISC1-(680–802) and MBP-DISC1-(680–832) were made based on the previous yeast two-hybrid studies (24). These expressed DISC1 proteins were subjected to pulldown assay using NDEL1-CTF (shown in an arrow). MBP-DISC1-CTF-(680–802), which lacks the crucial helical region (802–832), could not pull down NDEL1. b, proper folding of full-length DISC1 was confirmed using MBP-DISC1 as a bait to pull down full-length NDEL1. The interaction was being competed using C-terminal region of DISC1.

FIGURE 3.

Distribution of oligomers in wild-type DISC1 and NDEL1. a, analytical ultracentrifugation data were fitted using the Monte Carlo simulation method using Ultrascan software. Wild-type DISC1 has been fitted to two oligomeric populations, a dimer (∼270 kDa) and an octamer (∼1.1 MDa). b, Monte Carlo data fitting of analytical ultracentrifugation data on NDEL1 predicts the existence of a unique tetramer population (∼200 kDa).

FIGURE 4.

Size-exclusion chromatography confirms the DISC1/NDEL1 oligomers did not dissociate during the interaction. Size-exclusion chromatography of full-length DISC1 and full-length NDEL1 complex characterization (left). A chromatogram of the full-length DISC1-NDEL1 complex (blue), DISC1 alone (red), and NDEL1 alone (green) reassure that the full-length DISC1 or NDEL1 and DISC1-NDEL1 form higher-order oligomers as consistent with analytical ultracentrifugation data (Fig. 3). right, DISC1-CTF and full-length NDEL1 complex characterization. Shown is a chromatogram of the DISC1-CTF-NDEL1 complex (red), DISC1-CTF alone (purple), and NDEL1 alone (green). A shift in the retention time indicates a shift toward higher molecular weight upon direct binding of DISC1/DISC1-CTF to NDEL1. No change in oligomerization of DISC1 and/or NDEL1 was observed. Purified components were used in a pre-calibrated Superdex-200 column. a.u., arbitrary unit.

FIGURE 5.

Isothermal titration calorimetry experiments confirm the direct binding of DISC1/NDEL1 with a binding constant of 2.3 μm.

FIGURE 6.

Size-exclusion chromatography experiments of wild-type DISC1 and DISC1-S704C suggest DISC1-S704C forms a higher molecular weight oligomeric species compared with wild-type. a, SDS-PAGE gel shows homogenous purification of MBP-DISC1 and S704C variant. b, size-exclusion chromatography of wild-type DISC1 (continuous line) and DISC1S704C variant (dashed line).

FIGURE 7.

Distribution of oligomers with the MBP-DISC1-S704C variant. Analytical ultracentrifugation data of MBP-DISC1-S704C were fitted using Monte Carlo simulation method (Ultrascan software). The data have been fitted to two oligomeric populations, a dimer (∼240 kDa) and an oligomer that is higher than a nonamer (∼1.3 MDa). The observed molecular weight of wild-type DISC1 is indicated by the dotted line.

FIGURE 8.

The DISC1-S704C variant shifts the molecular weight (Mol. Wt) of DISC1-S704C-NDEL1 into higher-order oligomers without affecting the binding affinity with NDEL1. a, size-exclusion chromatography profile of full-length DISC1-S704C alone (solid line) and the full-length DISC1-S704C-NDEL1 complex suggest a stable complex formation between DISC1-S704C and NDEL1. b, surface plasmon resonance experiments of the interactions between NDEL1 and wild-type DISC1 or DISC-S704C. Experiments were performed using the immobilized GB1-NDEL1 and titrated against wild-type DISC1 (red) and DISC1-S704C (green) at two different concentrations (2 and 10 μm). No major difference in NDEL1 binding between of two DISC1 variants, as both yield similar response units (RU).

FIGURE 9.

Proposed mechanisms of DISC1-S704C-associated schizophrenia phenotype. a, hypothesis 1: schematic diagram of DISC1 primary sequence and its potential NDEL1 binding site (left). Schizophrenia risk DISC1 SNPs are located at the NDEL1 binding site, suggesting a probable perturbation at the DISC1-S704C-NDEL1 interactions (right). b, hypothesis 2: oligomerization condition of WT DISC1 extracted from the postmortem brain control sample (left) and DISC1-S704C (right) in the disease sample. Insoluble oligomerization condition of DISC1-S704C and a compromised NDEL1 binding were proposed due to the disease risk polymorphism.

FIGURE 10.

Our working model for DISC1 self-association and NDEL1 binding. Model for full-length DISC-1 self-association and NDEL1 interactions. In our model, we propose that the dimers (a) of DISC1 act as building blocks to assemble DISC1 octamers (b). The DISC1-S704C SNP near the NDEL1 binding site does not prevent the formation of DISC1-S704C dimer (d) and leads to improper oligomerization (e). Furthermore, irrespective of the nature of oligomerization, NDEL1 interacts with octamer (wild-type DISC1) (c) and higher-order oligomers (DISC1 S704C) of DISC1 (f), suggesting that the nature of oligomeric assembly is dispensable for NDEL1 binding.