Single-molecule characterization of target search dynamics of DNA-binding proteins in DNA-condensed droplets.

Target search models of DNA-binding proteins in cells typically consider search mechanisms that include 3D diffusion and 1D sliding, which can be characterized by single-molecule tracking on DNA. However, the finding of liquid droplets of DNA and nuclear components in cells cast doubt on extrapolation from the behavior in ideal non-condensed DNA conditions to those in cells. In this study, we investigate the target search behavior of DNA-binding proteins in reconstituted DNA-condensed droplets using single-molecule fluorescence microscopy. To mimic nuclear condensates, we reconstituted DNA-condensed droplets using dextran and PEG polymers. In the DNA-condensed droplets, we measured the translational movement of four DNA-binding proteins (p53, Nhp6A, Fis and Cas9) and p53 mutants possessing different structures, sizes, and oligomeric states. Our results demonstrate the presence of fast and slow mobility modes in DNA-condensed droplets for the four DNA-binding proteins. The slow mobility mode capability is correlated strongly to the molecular size and the number of DNA-binding domains on DNA-binding proteins, but only moderately to the affinity to single DNA segments in non-condensed conditions. The slow mobility mode in DNA-condensed droplets is interpreted as a multivalent interaction mode of the DNA-binding protein to multiple DNA segments.

Suppression of TDP-43 aggregation by artificial peptide binder targeting to its low complexity domain.

TAR DNA-binding protein 43 (TDP-43), aggregation prone protein, is a potential target of drug discovery for amyotrophic lateral sclerosis. The molecular binders, targeting the disordered low complexity domain (LCD) relevant to the aggregation, may suppress the aggregation. Recently, Kamagata et al. developed a rational design of peptide binders targeting intrinsically disordered proteins based on contact energies between residue pairs. In this study, we designed 18 producible peptide binder candidates to TDP-43 LCD by using this method. Fluorescence anisotropy titration and surface plasmon resonance assays demonstrated that one of the designed peptides bound to TDP-43 LCD at 30 µM. Thioflavin-T fluorescence and sedimentation assays showed that the peptide binder suppressed the aggregation of TDP-43. In summary, this study highlights the potential applicability of peptide binder design for aggregation prone proteins.

One-Dimensional Search Dynamics of Tumor Suppressor p53 Regulated by a Disordered C-Terminal Domain.

Tumor suppressor p53 slides along DNA and finds its target sequence in drastically different and changing cellular conditions. To elucidate how p53 maintains efficient target search at different concentrations of divalent cations such as Ca2+ and Mg2+, we prepared two mutants of p53, each possessing one of its two DNA-binding domains, the CoreTet mutant having the structured core domain plus the tetramerization (Tet) domain, and the TetCT mutant having Tet plus the disordered C-terminal domain. We investigated their equilibrium and kinetic dissociation from DNA and search dynamics along DNA at various [Mg2+]. Although binding of CoreTet to DNA becomes markedly weaker at higher [Mg2+], binding of TetCT depends slightly on [Mg2+]. Single-molecule fluorescence measurements revealed that the one-dimensional diffusion of CoreTet along DNA consists of fast and slow search modes, the ratio of which depends strongly on [Mg2+]. In contrast, diffusion of TetCT consisted of only the fast mode. The disordered C-terminal domain can associate with DNA irrespective of [Mg2+], and can maintain an equilibrium balance of the two search modes and the p53 search distance. These results suggest that p53 modulates the quaternary structure of the complex between p53 and DNA under different [Mg2+] and that it maintains the target search along DNA.

The Disordered Linker in p53 Participates in Nonspecific Binding to and One-Dimensional Sliding along DNA Revealed by Single-Molecule Fluorescence Measurements.

The tumor suppressor p53 is a multidomain transcription factor that can quickly bind to its target DNA by sliding along the DNA strand. We hypothesized that the intrinsically disordered and positively charged linker of p53 regulates its search dynamics first by directly interacting with DNA and second by modulating hopping of the core domain. To test the two hypotheses, we prepared five variants of p53 in which the length and charge of the linker were modulated. The affinity for and sliding along nonspecific DNA of p53 were altered by the charge of the linker, but not by the linker length. In particular, charge neutralization significantly reduced the affinity, suggesting that the linker directly contacts the DNA. Charge neutralization eliminated the slow mode of sliding, in which the core domain was assumed to contact nonspecific DNA. In contrast, the affinity of p53 for the target DNA was not affected by linker mutations. These results demonstrate that the linker participates in a target search of p53 by contacting nonspecific DNA and recruiting the core domain to contact DNA.

Rational design of phase separating peptides based on phase separating protein sequence of p53.

Artificial phase-separating (PS) peptides can be used in various applications such as microreactors and drug delivery; however, the design of artificial PS peptides remains a challenge. This can be attributed to the limitation of PS-relevant residues that drive phase separation by interactions of their pairs in short peptides and the difficulty in the design involving interaction with target PS proteins. In this study, we propose a rational method to design artificial PS peptides that satisfy the requirements of liquid droplet formation and co-phase separation with target PS proteins based on the target PS protein sequence. As a proof of concept, we designed five artificial peptides from the model PS protein p53 using this method and confirmed their PS properties using differential interference contrast and fluorescence microscopy. Single-molecule fluorescent tracking demonstrated rapid diffusion of the designed peptides in their droplets compared to that of p53 in p53 droplets. In addition, size-dependent uptake of p53 oligomers was observed in the designed peptide droplets. Large oligomers were excluded from the droplet voids and localized on the droplet surface. The uptake of high-order p53 oligomers into the droplets was enhanced by the elongated linker of the designed peptides. Furthermore, we found that the designed peptide droplets recruited p53 to suppress gel-like aggregate formation. Finally, we discuss aspects that were crucial in the successful design of the artificial PS peptides.

Structure-dependent recruitment and diffusion of guest proteins in liquid droplets of FUS.

Liquid droplets of a host protein, formed by liquid-liquid phase separation, recruit guest proteins and provide functional fields. Recruitment into p53 droplets is similar between disordered and folded guest proteins, whereas the diffusion of guest proteins inside droplets depends on their structural types. In this study, to elucidate how the recruitment and diffusion properties of guest proteins are affected by a host protein, we characterized the properties of guest proteins in fused in sarcoma (FUS) droplets using single-molecule fluorescence microscopy in comparison with p53 droplets. Unlike p53 droplets, disordered guest proteins were recruited into FUS droplets more efficiently than folded guest proteins, suggesting physical exclusion of the folded proteins from the small voids of the droplet. The recruitment did not appear to depend on the physical parameters (electrostatic or cation-π) of guests, implying that molecular size exclusion limits intermolecular interaction-assisted uptake. The diffusion of disordered guest proteins was comparable to that of the host FUS, whereas that of folded proteins varied widely, similar to the results for host p53. The scaling exponent of diffusion highlights the molecular sieving of large folded proteins in droplets. Finally, we proposed a molecular recruitment and diffusion model for guest proteins in FUS droplets.

Characterization of design grammar of peptides for regulating liquid droplets and aggregates of FUS.

Liquid droplets of aggregation-prone proteins, which become hydrogels or form amyloid fibrils, are a potential target for drug discovery. In this study, we proposed an experiment-guided protocol for characterizing the design grammar of peptides that can regulate droplet formation and aggregation. The protocol essentially involves investigation of 19 amino acid additives and polymerization of the identified amino acids. As a proof of concept, we applied this protocol to fused in sarcoma (FUS). First, we evaluated 19 amino acid additives for an FUS solution and identified Arg and Tyr as suppressors of droplet formation. Molecular dynamics simulations suggested that the Arg additive interacts with specific residues of FUS, thereby inhibiting the cation-π and electrostatic interactions between the FUS molecules. Second, we observed that Arg polymers promote FUS droplet formation, unlike Arg monomers, by bridging the FUS molecules. Third, we found that the Arg additive suppressed solid aggregate formation of FUS, while Arg polymer enhanced it. Finally, we observed that amyloid-forming peptides induced the conversion of FUS droplets to solid aggregates of FUS. The developed protocol could be used for the primary design of peptides controlling liquid droplets and aggregates of proteins.

Molecular principles of recruitment and dynamics of guest proteins in liquid droplets.

Despite the continuous discovery of host and guest proteins in membraneless organelles, complex host-guest interactions hinder the understanding of the molecular grammar governing liquid-liquid phase separation. In this study, we characterized the localization and dynamic properties of guest proteins in liquid droplets using single-molecule fluorescence microscopy. Eighteen guest proteins of different sizes, structures, and oligomeric states were examined in host p53 liquid droplets. Recruitment did not significantly depend on the structural properties of the guest proteins, but was moderately correlated with their length, total charge, and number of R and Y residues. In contrast, the diffusion of disordered guest proteins was comparable to that of host p53, whereas that of folded proteins varied widely. Molecular dynamics simulations suggest that folded proteins diffuse within the voids of the liquid droplet while interacting weakly with neighboring host proteins, whereas disordered proteins adapt their structures to form tight interactions with the host proteins. Our study provides insights into the key molecular principles of the localization and dynamics of guest proteins in liquid droplets.

Liquid-like droplet formation by tumor suppressor p53 induced by multivalent electrostatic interactions between two disordered domains.

Early in vivo studies demonstrated the involvement of a tumor-suppressing transcription factor, p53, into cellular droplets such as Cajal and promyelocytic leukemia protein bodies, suggesting that the liquid-liquid phase separation (LLPS) might be involved in the cellular functions of p53. To examine this possibility, we conducted extensive investigations on the droplet formation of p53 in vitro. First, p53 itself was found to form liquid-like droplets at neutral and slightly acidic pH and at low salt concentrations. Truncated p53 mutants modulated droplet formation, suggesting the importance of multivalent electrostatic interactions among the N-terminal and C-terminal domains. Second, FRET efficiency measurements for the dimer mutants of p53 revealed that distances between the core domains and between the C-terminal domains were modulated in an opposite manner within the droplets. Third, the molecular crowding agents were found to promote droplet formation, whereas ssDNA, dsDNA, and ATP, to suppress it. Finally, the p53 mutant mimicking posttranslational phosphorylation did not form the droplets. We conclude that p53 itself has a potential to form droplets that can be controlled by cellular molecules and by posttranslational modifications, suggesting that LLPS might be involved in p53 function.

Rational design using sequence information only produces a peptide that binds to the intrinsically disordered region of p53.

Intrinsically disordered regions (IDRs) of proteins are involved in many diseases. The rational drug design against disease-mediating proteins is often based on the 3D structure; however, the flexible structure of IDRs hinders the use of such structure-based design methods. Here, we developed a rational design method to obtain a peptide that can bind an IDR using only sequence information based on the statistical contact energy of amino acid pairs. We applied the method to the disordered C-terminal domain of the tumor suppressor p53. Titration experiments revealed that one of the designed peptides, DP6, has a druggable affinity of ~1 µM to the p53 C-terminal domain. NMR spectroscopy and molecular dynamics simulation revealed that DP6 selectively binds to the vicinity of the target sequence in the C-terminal domain of p53. DP6 inhibits the nonspecific DNA binding of a tetrameric form of the p53 C-terminal domain, but does not significantly affect the specific DNA binding of a tetrameric form of the p53 core domain. Single-molecule measurements revealed that DP6 retards the 1D sliding of p53 along DNA, implying modulation of the target searching of p53. Statistical potential-based design may be useful in designing peptides that target IDRs for therapeutic purposes.

High Free-Energy Barrier of 1D Diffusion Along DNA by Architectural DNA-Binding Proteins.

Architectural DNA-binding proteins function to regulate diverse DNA reactions and have the defining property of significantly changing DNA conformation. Although the 1D movement along DNA by other types of DNA-binding proteins has been visualized, the mobility of architectural DNA-binding proteins on DNA remains unknown. Here, we applied single-molecule fluorescence imaging on arrays of extended DNA molecules to probe the binding dynamics of three structurally distinct architectural DNA-binding proteins: Nhp6A, HU, and Fis. Each of these proteins was observed to move along DNA, and the salt concentration independence of the 1D diffusion implies sliding with continuous contact to DNA. Nhp6A and HU exhibit a single sliding mode, whereas Fis exhibits two sliding modes. Based on comparison of the diffusion coefficients and sizes of many DNA binding proteins, the architectural proteins are categorized into a new group distinguished by an unusually high free-energy barrier for 1D diffusion. The higher free-energy barrier for 1D diffusion by architectural proteins can be attributed to the large DNA conformational changes that accompany binding and impede rotation-coupled movement along the DNA grooves.

One-Dimensional Sliding of p53 Along DNA Is Accelerated in the Presence of Ca(2+) or Mg(2+) at Millimolar Concentrations.

One-dimensional (1D) sliding of the tumor suppressor p53 along DNA is an essential dynamics required for its efficient search for the binding sites in the genome. To address how the search process of p53 is affected by the changes in the concentration of Mg(2+) and Ca(2+) after the cell damages, we investigated its sliding dynamics at different concentrations of the divalent cations. The 1D sliding trajectories of p53 along the stretched DNA were measured by using single-molecule fluorescence microscopy. The averaged diffusion coefficient calculated from the mean square displacement of p53 on DNA increased significantly at the higher concentration of Mg(2+) or Ca(2+), indicating that the divalent cations accelerate the sliding likely by weakening the DNA-p53 interaction. In addition, two distributions were identified in the displacement of the observed trajectories of p53, demonstrating the presence of the fast and slow sliding modes having large and small diffusion coefficients, respectively. A coreless mutant of p53, in which the core domain was deleted, showed only a single mode whose diffusion coefficient is about twice that of the fast mode for the full-length p53. Thus, the two modes are likely the result of the tight and loose interactions between the core domain of p53 and DNA. These results demonstrated clearly that the 1D sliding dynamics of p53 is strongly dependent on the concentration of Mg(2+) and Ca(2+), which maintains the search distance of p53 along DNA in cells that lost homeostatic control of the divalent cations.