Shape transformations in peptide-DNA coacervates driven by enzyme-catalyzed deacetylation

Abstract

Biomolecular condensates formed by liquid-liquid phase separation (LLPS) are important organizers of biochemistry in living cells. Condensate formation can be dynamically regulated, for example, by protein binding or enzymatic processes. However, how enzymatic reactions can influence condensate shape and control shape transformations is less well understood. Here, we design a model condensate that can be formed by the enzymatic deacetylation of a small peptide by sirtuin-3 in the presence of DNA. Interestingly, upon nucleation condensates initially form gel-like aggregates that gradually transform into spherical droplets, displaying fusion and wetting. This process is governed by sirtuin-3 concentration, as more enzyme results in a faster aggregate-to-liquid transformation of the condensates. The counterintuitive transformation of gel-like to liquid-like condensates with increasing interaction strength between the peptide and DNA is recapitulated by forming condensates with different peptides and nucleic acids at increasing salt concentrations. Close to the critical point where coacervates dissolve, gel-like aggregates are formed with short double stranded DNA, but not with single stranded DNA or weakly binding peptides, even though the coacervate salt resistance is similar. At lower salt concentrations the interaction strength increases, and spherical, liquid-like condensates are formed. We attribute this behavior to bending of the DNA by oppositely charged peptides, which becomes stronger as the system moves further into the two-phase region. Overall, this work shows that enzymes can induce shape transformations of condensates and that condensate material properties do not necessarily reveal their stability.

Fig. 1. (a) Schematic design of coacervation induced by deacetylation, SIRT3 (yellow) deacetylates peptide substrates (black), which results in a more positively charged peptide (red) that phase separates with dsDNA (blue, green) to form condensates. (b) Peptides GRK1 (top, m = 1, sequence: GRKRG), GRK2 (top, m = 2, sequence: GRKRGRKRG) and KKASL3 (bottom, sequence: KKASLKKASLKKASL) used to assess the activity of SIRT3. Incubation of the peptide with the enzyme and NAD⁺ leads to deacetylation of the acetylated lysine side chains and formation of O-AADPR. Positively charged residues are colored red. (c) SIRT3 purified by Ni-NTA affinity chromatography resolved by SDS/PAGE and detected by Coomassie, the calculated weight for SIRT3 is 33.4 kDa. (d) Mass spectrum of deacetylated KKASL3 (M + H⁺ = 1601.9) after 8 h of incubation with SIRT3 and NAD⁺. (e) Phase diagram of GRK2_Ac before (light blue) and after (dark blue) the deacetylation reaction with SIRT3. Shaded regions indicate standard deviation (n = 3). Mixture contains 2 mM GRK2, 10 mM NAD⁺, 1 mM DTT and 2.5 μM SIRT3 in 50 mM HEPES pH 7.4. For the pre-reaction sample, SIRT3 was incubated at 95 °C to ensure denaturation of the enzyme.

Fig. 2. (a) Formation of amorphous gel-like condensates of GRK2_Ac and dsDNA resulting from the deacetylation reaction. Over time, the gels relax and get a spherical shape, indicative of liquid droplets. (b) Circularity of condensates formed during the reaction at different SIRT3 concentrations. Points are mean circularities and shaded areas indicate the standard deviation. (c) The time it takes to reach spherical droplets decreases with a higher SIRT3 concentration. (d) GRK2, SIRT3 and dsDNA all localize to the coacervate phase. Molecules were labeled and their partition coefficient (K_P) was calculated by their relative fluorescence intensity inside the coacervate phase compared to the dilute phase. Mixtures contain 2 mM GRK2, 10 mM NAD⁺, 1 mM DTT, 60 mM NaCl and varying SIRT3 concentrations in 50 mM HEPES pH 7.4. Scale bars are 10 μm.

Fig. 3. (a) GRK2_Ac/dsDNA coacervates form gel-like structures near their coacervate salt resistance. (b) Circularities of GRK2/dsDNA coacervates at increasing NaCl concentrations. (c) FRAP measurements of GRK2_Ac/dsDNA coacervates at no salt and 60 mM NaCl. The recovery half-life at 0 mM NaCl was 3.07 seconds, while at 60 mM, it took 12.5 seconds to reach half-maximum recovery. Data were fitted with an exponential decay function f(t) = A(1 – e^−bt) + C. (d) dsDNA forms nonspherical coacervates with K₂₀, ELP-K₇₂ and SRRRR near their CSR. (e) Circularities of dsDNA coacervates near their CSR. R₁₀ and protamine form circular droplets, while other polycations form nonspherical droplets. When interactions between polycation and dsDNA are relatively strong, no gel-like aggregates are found. Scale bars are 10 μm.

Fig. 4. Schematic overview of complex dsDNA coacervates. (a) Strong polycations, such as R₁₀ and protamine can bend dsDNA at high and low salt concentrations to maximize charge neutralization, resulting in spherical liquid condensates. (b) Weak polycations, such as GRK2_Ac, K₂₀ and PDDA cannot bend dsDNA at high salt concentrations, resulting in spherical condensates with low salt and gel-like aggregates at high salt concentrations.