Assembly of model postsynaptic densities involves interactions auxiliary to stoichiometric binding

Abstract

The assembly of functional biomolecular condensates often involves liquid-liquid phase separation (LLPS) of proteins with multiple modular domains, which can be folded or conformationally disordered to various degrees. To understand the LLPS-driving domain-domain interactions, a fundamental question is how readily the interactions in the condensed phase can be inferred from interdomain interactions in dilute solutions. In particular, are the interactions leading to LLPS exclusively those underlying the formation of discrete interdomain complexes in homogeneous solutions? We address this question by developing a mean-field LLPS theory of two stoichiometrically constrained solute species. The theory is applied to the neuronal proteins SynGAP and PSD-95, whose complex coacervate serves as a rudimentary model for neuronal postsynaptic densities (PSDs). The predicted phase behaviors are compared with experiments. Previously, a three SynGAP/two PSD-95 ratio was determined for SynGAP/PSD-95 complexes in dilute solutions. However, when this 3:2 stoichiometry is uniformly imposed in our theory encompassing both dilute and condensed phases, the tie-line pattern of the predicted SynGAP/PSD-95 phase diagram differs drastically from that obtained experimentally. In contrast, theories embodying alternate scenarios postulating auxiliary SynGAP-PSD-95 as well as SynGAP-SynGAP and PSD-95-PSD-95 interactions, in addition to those responsible for stoichiometric SynGAP/PSD-95 complexes, produce tie-line patterns consistent with experiment. Hence, our combined theoretical-experimental analysis indicates that weaker interactions or higher-order complexes beyond the 3:2 stoichiometry, but not yet documented, are involved in the formation of SynGAP/PSD-95 condensates, imploring future efforts to ascertain the nature of these auxiliary interactions in PSD-like LLPS and underscoring a likely general synergy between stoichiometric, structurally specific binding and stochastic, multivalent "fuzzy" interactions in the assembly of functional biomolecular condensates.

Figure 1

Schematics of hypothetical multiple SynGAP/PSD-95 configurations that are based upon interactions among stoichiometric S₃P₂ complexes. (a) Caricatures of the SynGAP trimer (S₃, left) and PSD-95 (P, right). For S₃, the pink circle represents the SynGAP CC trimer, the three red circles represent the PDZ binding domains (PBMs) at the C termini of the three S chains, and the three yellow chains represent the rest of each of the three S sequences N-terminal to the CC domain. For P, the blue circle represents the three PDZ domains, and the green rectangle represents the C-terminal guanylate kinase (GK) domain. The SH3 domain sequentially situated between these two domains is not depicted explicitly. The blue curve represents the part of P N-terminal to the PDZ domains. Domain organizations of SynGAP and PSD-95 are described in more detail in Figs. 1 and 2 of (42). (b) S₃P₂ multimerization by connecting each S₃P₂ to two other S₃P₂s results in a linear chain of S₃P₂ complexes. This scenario corresponds to that in Fig. 7 of (50). As described in this reference, the binding between an S₃ with two Ps is mediated by PDZ-PBM contacts (shown by concentric red and blue circles); favorable interaction between two S₃P₂s is then effectuated by binding of a PDZ-PBM of one S₃P₂ to the GK (and SH3) of another S₃P₂ (indicated by a contact between a green rectangle with a set of concentric red and blue circles). (c) S₃P₂ multimerization by connecting each S₃P₂ to three other S₃P₂s results in a network of S₃P₂ complexes. The notation for binding interactions is the same as that in (b).

Figure 2

Prediction of a mean-field theory that assumes SynGAP/PSD-95 complex coacervation is driven solely by favorable interactions among stoichiometric S₃P₂ complexes (Eq. 13). (a) Simplified schematic representation of S₃, P, and S₃P₂. The three S₃ PBMs and their connecting chain segments to the CC trimer (large pink circle, see Fig. 1a) are now shown as small red circles, the PDZ3-SH3-GK domains of P are now shown as a blue circle encased in a green rectangle, and the yellow and blue N-terminal chain segments in Fig. 1a are not depicted explicitly. Each S₃P₂ is enclosed by a dashed circle to underscore its role as a unit of phase-separation-driving interaction in the mean-field theory. (b) Schematic picture of a hypothetical SynGAP/PSD-95 condensed phase. The formulation in Eq. 13 stipulates that although unbound S₃ and P may be present in the condensed phase, phase separation is only driven by interactions between units of S₃P₂ (magenta dashed lines). These interunit favorable interactions may include binding of a PDZ/PBM of one S₃P₂ to the SH3-GK of another S₃P₂ as envisioned in Fig. 1, b and c. (c) Predicted phase diagram for this hypothetical scenario based upon Eq. 13, using ${\mathcal{K}}^0$ = 10⁻¹⁰ and χ = 1.5 as illustration. The boundary of binodal phase separation is shown by the light blue lines. Each of the dashed dark blue lines is a tie line indicating a pair of coexisting phases on the boundary of the phase-separated region. The overall volume constraint of ${\phi }_{{\mathsf{S}}_3}$ + φ_P ≤ 1 is marked by the solid black line with slope = −1 for ${\phi }_{{\mathsf{S}}_3}$ + φ_P = 1, whereas the ${\phi }_{{\mathsf{S}}_3}$/φ_P = 1/2 stoichiometric ratio of the S₃P₂ complex is indicated by the black solid line with slope = 1/2.

Figure 3

Experimental measurements of SynGAP/PSD-95 phase separation. (a) Sedimentation assay showing the dilute phase (marked by “S” at the top of the columns) and the condensed phase (marked by “P” at the top of the columns) distribution of PSD-95 and SynGAP mixed at different concentrations of PSD-95 and a fixed concentration of SynGAP at 90 μM. (b) Phase diagram of PSD-95 and SynGAP condensates. The open circles in phase diagram indicate no phase separation, and solid circles represent condensed-phase formation. The results enclosed in the dashed red box correspond to those from (a). The inclined dashed line indicate the dilute-solution 3:2 ratio for [SynGAP]/[PSD-95]. (c) Standard curve of Cy3-labeled PSD-95 obtained from dilute solutions. (d) Confocal images showing an indicative confocal slice of condensed droplets of each concentration of Cy3-labeled PSD-95 mixed with unlabeled SynGAP at a fixed concentration at 180 μM. (e) Heat map plot showing the PSD-95 concentrations in condensed phase at different combinations of PSD-95 and SynGAP concentrations. Each number represents the PSD-95 concentration (μM) in the condensed phase. The data enclosed in the dashed red box correspond to those from (d). a.u., arbitrary unit.

Figure 4

Experimental trend of SynGAP/PSD-95 coexisting phases. Shown data are inferred from confocal microscopy and centrifugation measurements as described in the text. Initial (overall) concentrations ([P⁰], [S⁰]) are plotted as green data points. The phase-separated dilute phase ([P^dil], [S^dil]) are plotted as orange data points, and the condensed phase ([P^cond], [S^cond]) are plotted as blue data points. Dashed lines are tie lines connecting coexisting phases. Results are shown in linear (a) as well as log-log (b) scales for clarity, as the green data points are not visible in the linear plot in (a) because they are very close to the orange data points. Note that because the dilute-phase ([P^dil], [S^dil]) concentrations (orange data points) are all very close to the origin, the slopes of tie lines in the log-log plot in (b) are all approximately equal to 1. The diversity of the actual tie-line slopes as seen in (a) is manifested by the offsets of the log-log tie lines (different intercepts) in (b).

Figure 5

Alternate scenarios of SynGAP/PSD-95 phase separation with auxiliary interactions beyond those underpinning the assembly of stoichiometric S₃P₂ in dilute solution. The schematic representations of S₃ and P here are the same as those in Fig. 2a. (a) Favorable LLPS-driving interactions are envisioned to be restricted to those between S₃ and P (red dashed lines) as modeled by the χ_SP term in Eq. 27 for a simple FH model. (b) LLPS is envisioned to be driven also by favorable interactions among S₃ (blue dashed lines) and among P (green dashed lines) as modeled by the χ_SS and χ_PP terms in Eq. 28a. The bias afforded by this model toward formation of S₃P₂ complexes in the dilute phase is not exhibited in this schematic depiction of the condensed phase. (c–e) Mean-field FH theory predictions for SynGAP/PSD-95 phase behaviors in the alternate scenario in (a) (c and d) and the alternate scenario in (b) (e). The black solid lines with slopes = 1/2 and −1, the dashed tie lines, and the light blue phase boundaries in the three phase diagrams carry the same meanings as those in Fig. 2c. The FH parameters are (c) χ_SP = 10.0 in Eq. 27; (d) ${\mathcal{K}}^0$ = 10⁻¹⁰, χ_SP = 10.0, and χ_SS = χ_PP = 0 in (28a), (28b); and (e) ${\mathcal{K}}^0$ = 10⁻¹⁰, χ_SP = 4.0, and χ_SS = χ_PP = 3.5 in (28a), (28b).