Mechanistic dissection of increased enzymatic rate in a phase-separated compartment

Abstract

Biomolecular condensates concentrate macromolecules into discrete cellular foci without an encapsulating membrane. Condensates are often presumed to increase enzymatic reaction rates through increased concentrations of enzymes and substrates (mass action), although this idea has not been widely tested and other mechanisms of modulation are possible. Here we describe a synthetic system where the SUMOylation enzyme cascade is recruited into engineered condensates generated by liquid-liquid phase separation of multidomain scaffolding proteins. SUMOylation rates can be increased up to 36-fold in these droplets compared to the surrounding bulk, depending on substrate K_M. This dependency produces substantial specificity among different substrates. Analyses of reactions above and below the phase-separation threshold lead to a quantitative model in which reactions in condensates are accelerated by mass action and changes in substrate K_M, probaby due to scaffold-induced molecular organization. Thus, condensates can modulate reaction rates both by concentrating molecules and physically organizing them.

Extended Data Figure 1.. Sensitivity of enhanced condensate activity to K_M, substrate concentration, and partition coefficient.

(A) Modeled ratio of total reaction rate in a phase separated solution, with and without recruitment of enzyme and substrate to the scaffold (Total_S and Total_US, respectively, as a function of substrate concentration (plotted as [S]/K_M,US) and partition coefficient (PC). Modeled for K_M,US = 70 and K_M,S = 17, as measured for FRB-polySH3₃+polyPRM₅, with identical PC values for enzyme and substrate. Modeling assumes simple, hyperbolic Michaelis-Menten kinetics (see Methods). Color scale is a relative representation of the z-axis values and goes from low (blue) to high (red). Inset is a plot of Total_S:Total_US rate as a function of substrate concentration at a fixed partition coefficient of 50. (B) Modeled ratio of Total_S to Total_US as a function of PC and the change in K_M upon recruitment of enzyme and substrate to the scaffold, K_M,US/K_M,S. Total substrate concentration, [S]_T, set to 0.1 * K_M,US. (C) Same as (B), except [S]_T set to 10 * K_M,US.

Extended Data Figure 2.. Total scaffold rate can be less than total unscaffolded activity in certain regimes if enzyme partitioning is much less than substrate partitioning.

(A)-(C) Modeled ratio of total reaction rate in a phase separated solution, with and without recruitment of enzyme and substrate to the scaffold as a function of substrate concentration and substrate partition coefficient (PC_S). Both reactions have K_M = 70. Enzyme partitioning (PC_E) is 1 (A), 10 (B), and 100 (C); enzyme concentration, [E] = 0.1[S]. Model based on 0.01 droplet volume fraction.

Extended Data Figure 3.. Droplet rate increases rapidly relative to bulk as a function of partition coefficient.

(A) Modeled ratio of droplet and bulk reaction rates as a function of substrate concentration and partition coefficient (PC). Both reactions are scaffolded and have K_M = 17. Enzyme partitioning is identical to substrate partitioning, and [E] = 0.1[S]. (B) Modeled fractional activity contributed by the droplet phase as a function of substrate concentration and partition coefficient (PC). Conditions same as in (A), with a 0.01 droplet volume fraction.

Figure 1.. Design of an inducible, condensate-targeted enzyme cascade.

(A) Schematic of the SUMOylation cascade. SUMO is initially conjugated to E1 through a thioester bond, then transferred to E2 through a second thioester, and finally to a lysine on the target protein. (B) Inducible recruitment of the SUMOylation cascade to polySH3-polyPRM condensates using the FRB-FKBP-rapamycin system. FRB is fused to polySH3, which is concentrated in the condensates. Upon addition of rapamycin, FKBP-E2 and FKBP-substrate enrich in the condensates, while untagged SUMO remains evenly distributed. Although not illustrated in the diagram, untagged E1 weakly enriches due to binding E2 (see text).

Figure 2.. Condensates increase the total SUMOylation rate.

(A) Confocal fluorescence microscopy images of mCherry-FKBP-E2 (top row) and FKBP-EGFP-substrate (bottom row) in the presence of FRB-polySH3₃:polyPRM₅ condensates upon addition of DMSO control (left column) or rapamycin (right column). These images are representative of 3 independent experiments. Scale bar is 50um. (B) SDS-PAGE gel stained with Coomassie blue illustrating production of SUMOylated substrate as a function of time with either DMSO (−) or rapamycin (+). Black square denotes E2, black star denotes FRB-polySH3₃, and black circle denotes E1. This gel is representative of 3 independent experiments. (C) Quantification of data in panel B, showing intensity of the SUMOylated substrate band as a function of time. DMSO = black circles, rapamycin = red squares. Data are plotted as the mean and SD from n=3 independent experiments. (D) Fluorescence-detected SDS-PAGE gel depicting production of SUMOylated substrate as a function of time when E2, substrate, both or neither are fused to FKBP (and thus are recruited into FRB-polySH3₃-polyPRM₅ droplets). + indicates FKBP-fusion, - indicates non-fused. This gel is representative of 4 independent experiments. (E) Quantification of data in panel D, showing intensity of the SUMOylated substrate band as a function of time. E2+substrate recruited, inverted red triangles; E2 recruited, cyan triangles; substrate recruited, magenta squares; neither recruited black circles. Absolute amounts quantified by an internal standard. Data are plotted as the mean and SD from n=4 independent experiments. (F) Fluorescent SDS page gel of SUMOylation of PML peptide or FKBP-PML peptide when co-incubated with FRB-polySH3₃:polyPRM₅ condensates and either DMSO or rapamycin. SUMOylated FKBP-peptide is the upper band, and SUMOylated peptide is the lower band. This gel is representative of 2 independent experiments. All figure panels have associated raw data.

Figure 3.. Rate enhancement is substrate-dependent.

(A) Fluorescent SDS-PAGE gel depicting the production of SUMOylated substrate as a function of time with FKBP-RanGAP, FKBP-E2, and FRB-polySH3₃:polyPRM₅ condensates with DMSO (−) or rapamycin (+). This gel is representative of 3 independent experiments. (B) Quantification of data in panel A, showing intensity of the SUMOylated substrate band as a function of time. DMSO, black circles; rapamycin, red squares. Data are plotted as the mean and SD from n=3 independent experiments. (C) Fluorescent SDS-PAGE gel depicting the production of SUMOylated substrate as a function of time with FKBP-RanGAP or FKBP-RanGAP*, FKBP-E2, and FRB-polySH3₃:polyPRM₅ condensates with DMSO (−) or rapamycin (+). This gel is representative of 2 independent experiments. All figure panels have associated raw data.

Figure 4.. SUMOylation is greatly accelerated in the droplet phase.

(A) Schematic of workflow to measure SUMOylation rate in the droplet phase. Total SUMOylation rate is measured by simply mixing all components and adding ATP. Bulk rate is measured by centrifuging the total mixture prior to addition of ATP to sediment droplets, transferring the supernatant to a new tube, and then initiating the reaction with ATP. The difference between Total and Bulk rate yields the Droplet rate. (B) Representative plot showing the production of SUMOylated RanGAP* over time in the total (+rap), bulk (+rap), and total (+DMSO) solutions. Error bars on total (+rap) and bulk (+rap) represent the SEM of 3 experiments. (C) Schematic depicting the two approaches used to calculate droplet volume fraction. Top panel shows the equation used based on conservation of mass. [ ] represent concentrations in the indicated phase measured by fluorescence imaging. Bottom panel illustrates the direct measurement approach based on confocal imaging of a three-dimensional volume. (D) Volume-normalized rate toward RanGAP* for the droplet, droplet equivalent concentration (DEC), and bulk phases. Error bars represent the SEM from 6 independent experiments. The statistical significance was assessed by a two-tailed, unpaired Student’s t-test. ** represents a p-value < 0.01 (0.0022), and **** represents a p-value < 0.0001 (1.4 × 10⁻⁶). Droplet rate was determined from the difference between the average total reaction rate and average bulk reaction rate, with errors propagated accordingly. Panels B and D have associated raw data.

Figure 5.. Excess activity is due to a scaffold-induced decrease in K_M.

(A) Time course showing the production of SUMOylated substrate with E1, E2, RanGAP*, and SUMO1 at droplet equivalent concentrations (black circles), +3% Ficoll 70 (magenta squares), or + 3% PEG 3350 (green triangles). Each datapoint shown in duplicate. (B) Bar chart showing the volume-normalized reaction rate of bulk (black) or bulk equivalent concentration (BEC, blue). Error bars represent the SEM from 6 experiments. Statistical significance was assessed by a two-tailed, unpaired Student’s t-test. **** represents a p-value < 0.0001 (1.3 × 10⁻⁹). (C) Rate of production of SUMOylated RanGAP* as a function of substrate concentration with (red squares) or without (black circles; same data as shown in Supplementary Figure 3D) sub-threshold concentrations of FRB-polySH3₃-polyPRM₅. Each symbol represents the mean and standard deviation from n=3 (<150uM) and n=2 (≥150uM) independent experiments. Points without errors bars have standard deviations too small to show. (D) Ratio of scaffolded:unscaffolded reaction rate (black curve) calculated by dividing the fit of the red curve from the fit of the black curve in Panel C. Overlaid on this curve are the scaffolded:unscaffolded rate ratios from bulk vs BEC (blue square) and droplet vs DEC (red circles). Error bars represent SEM from a total of 6 independent measurements for the bulk:BEC point and the droplet:DEC datapoints at ~7 and 32uM. The 36uM datapoint is from 3 independent experiments. All figure panels have associated raw data.

Figure 6.. Activity enhancement is scaffold-specific.

(A) Rate of production of SUMOylated RanGAP* as a function of RanGAP* concentration with (red) or without (black; same data as shown in Supplementary Figure 3D) subcritical concentrations of FRB-polySH3₅-polyPRM₅. Each symbol represents the mean and standard deviation from n=3 (<150uM) and n=2 (≥150uM) independent experiments. Points without errors bars have standard deviations too small to show. (B) Volume-normalized reaction rates of FRB-polySH3₅-polyPRM₅ droplets (black) and droplet equivalent concentration (DEC, red). Error bars represent SEM from 6 independent experiments. Statistical significance was assessed by a two-tailed, unpaired Student’s t-test with a p-value cutoff of 0.05. Droplet rate was determined from the difference between the average total reaction rate and average bulk reaction rate, with errors propagated accordingly. (C) Droplet:DEC rate ratio of FRB-polySH3₅-polyPRM₅ droplets at total RanGAP* concentrations of 0.5 µM (black), 1 µM (blue), and 2 µM (green), corresponding to droplet concentrations of ~9 µM, ~32 µM, and ~40 µM, respectively. Error bars represent SEM from n=4, n=6, and n=3 independent experiments, respectively. Statistical significance was assessed by unpaired one way ANOVA with a p-value cutoff of 0.05. (D) Fluorescence emission spectrum of FKBP-YPet-RanGAP* upon 445nm excitation of CyPet-FKBP-E2. Spectra recorded in the presence of FRB-polySH3₅ (black circles), FRB-polySH3₅ + polyPRM₅ (cyan squares), FRB-polySH3₃ (magenta triangles) or FRB-polySH3₃ + polyPRM₅ (inverted red triangles). Each point represents the mean and SD (error bars) of 2 independent experiments. All figure panels have associated raw data.