Spatiotemporal modulations in heterotypic condensates of prion and α-synuclein control phase transitions and amyloid conversion

Abstract

Biomolecular condensation via liquid-liquid phase separation of proteins and nucleic acids is associated with a range of critical cellular functions and neurodegenerative diseases. Here, we demonstrate that complex coacervation of the prion protein and α-synuclein within narrow stoichiometry results in the formation of highly dynamic, reversible, thermo-responsive liquid droplets via domain-specific electrostatic interactions between the positively-charged intrinsically disordered N-terminal segment of prion and the acidic C-terminal tail of α-synuclein. The addition of RNA to these coacervates yields multiphasic, vesicle-like, hollow condensates. Picosecond time-resolved measurements revealed the presence of transient electrostatic nanoclusters that are stable on the nanosecond timescale and can undergo breaking-and-making of interactions on slower timescales giving rise to a liquid-like behavior in the mesoscopic regime. The liquid-to-solid transition drives a rapid conversion of complex coacervates into heterotypic amyloids. Our results suggest that synergistic prion-α-synuclein interactions within condensates provide mechanistic underpinnings of their physiological role and overlapping neuropathological features.

Molecular Origin of Internal Friction in Intrinsically Disordered Proteins

“…This work revealed a characteristic rotational time scale (∼1.4 ns) involving collective, short-range backbone dihedral rotations of the disordered polypeptide chain. 4 This work showed that complex coacervation of the prion protein and αsynuclein results in the formation of liquid droplets that contain heterotypic electrostatic nanoclusters detected by fluorescence depolarization kinetics.…”

“…Therefore, deciphering the conformational and dynamic properties of disordered proteins in their droplet states will be of interest to connect the mesoscopic description to the microscopic level molecular details of the polypeptide backbones. Picosecond time-resolved fluorescence studies from our group directly demonstrated the rapid large-amplitude segmental fluctuations in the extended IDP chains that govern the making and breaking of noncovalent intermolecular interactions controlling the fluidity of the droplet interior . In our recent work, we have shown that domain-specific electrostatic contact formation between the intrinsically disordered N-terminal domain of the prion protein and α-syn at a narrow stoichiometry regime leads to the formation of highly mobile liquid droplets with a dynamic internal organization .…”

“…Biomolecular condensates are formed via phase separation of proteins containing low-complexity regions, prion-like domains, oligopeptide repeat regions, and multivalent interaction domains. ,,− Phase-separated droplets are mesoscopic, dynamic, liquid-like, nonstoichiometric, intracellular supramolecular assemblies and have the unique ability to control the cellular organization, compartmentalization, and biochemical processes. These liquid-like assemblies are implicated in a myriad of cellular functions, whereas liquid-to-gel and liquid-to-solid transitions are linked to deadly neurodegenerative diseases .…”

“…The dynamic personality of proteins has been of prime interest due to its crucial roles in protein folding and dynamics occurring on a wide range of time scales ranging from nanoseconds to seconds in an aqueous environment. − These fundamental dynamical features of highly flexible and rapidly fluctuating intrinsically disordered proteins (IDPs) are believed to govern the behavior of this class of proteins in their free monomeric or binding-induced folded forms as well as in their assembled states including phase-separated liquid droplets and amyloid aggregates. , Protein dynamics involves complex, diffusional, thermal energy-driven processes along a single reaction coordinate on a free-energy surface. − Addressing this particular diffusional one-dimensional problem has held the theoretical base of the well-known Kramers rate theory (1940), which describes diffusive dynamics of proteins in the high-friction-limit. ,− Kramers theorized that a particle, subject to friction, is driven across a potential barrier by thermal fluctuations (Figure A). This theory predicts that the average time for barrier crossing (τ) is directly proportional to the frictional drag parameter (γ) experienced by the reactant molecules near the barrier top considering each possible energy fluctuation or dissipation that influences the rate of protein folding or other conformational changes , (Figure A).…”

“…1−3 These fundamental dynamical features of highly flexible and rapidly fluctuating intrinsically disordered proteins (IDPs) are believed to govern the behavior of this class of proteins in their free monomeric or binding-induced folded forms as well as in their assembled states including phase-separated liquid droplets and amyloid aggregates. 4,5 Protein dynamics involves complex, diffusional, thermal energy-driven processes along a single reaction coordinate on a free-energy surface. 6−12 Addressing this particular diffusional one-dimensional problem has held the theoretical base of the well-known Kramers rate theory (1940), which describes diffusive dynamics of proteins in the high-friction-limit.…”