Molecular interaction and molecular structure for metastability
In accordance with the metastable free energy state, IDPs, as the
scaffold protein of MLOs, often harbor transient and weak molecular
interactions 34. The low complexity domains(LCDs), as the domains that mediate the LLPS of IDPs, are largely
enriched with charged, polar and aromatic residues whilst commonly
devoid of hydrophobic residues 22. Weak, multivalent
and non-specific interactions, including electrostatic, pi–pi,
cation–pi and dipole–dipole interactions (between polar amino acids),
are prevalent among residues in LCDs (Figure 2A )36. The long-range electrostatic interactions among
charged blocks may facilitate the initiation of LLPS, while short range
interactions, including pi–pi, cation–pi and dipole–dipole
interactions, may mediate the multivalent contacts among weakly
interacting motifs 34. Among these molecular
interactions, cation–pi interactions are considered as the strongest,
with the free energy of binding (\(\Delta G_{\text{bind}}\)) around
–3.6 kcal/mol 57. This magnitude is lower than the
average \(\Delta G_{\text{bind}}\) per residue implicated in the
formation of Aβ17–42 amyloids-β protofibrils (–19.3
kcal/mol) 58, thus suggesting molecular interactions
driving the formation of MLOs are much weaker than amyloid plaques.
Compared with static amyloids 34, LCDs harbor
transient interactions among residues with higher dynamics. This can be
quantified by fluorescence recovery after photobleaching (FRAP),
commonly showing a half time of recovery (\(t_{\frac{1}{2}}\)) on the
order of seconds (normalizing the diameter of bleaching spot to 1 μm)34. Efforts have been made to shed light on the
possible reason of liquid phase formation from the molecular level,
including theory of amyloids-β fibril formation 59,
multivalent domain interaction network model 60 and
theory of polymer physics 22.
IDPs lack a stable and well-defined 3D molecular structure61–63. IDPs are always devoid of stable tertiary
structures under physiological conditions, albeit collapsed IDPs could
harbor some stable secondary structure elements 64.
The lack of stable structure can be possibly considered as one common
and crucial feature for IDPs to form metastable MLOs65. The unstable conformation allows the flexibility
of IDPs as major scaffold constituents, which may contribute to the
physical fluidity of MLOs 65. The unstable
conformation allows formation of the weak and multivalent interactions,
which is a common hallmark for the interactions that contribute to LLPS3. The IDPs harbor ‘stickers-and-spacers’ structural
features, wherein modules provide attractive interactions are considered
as ‘stickers’, and flexible linkers provide no significant attractive
interactions are considered as ‘spacers’. The unstable structure with
‘stickers-and-spacers’ features allows the multivalent presence of PTM
sites 66, whilst PTMs can efficiently alter the
stability of MLOs 53.
Modular interaction domains connected by disordered linkers can mediate
multivalent interactions that drive LLPS. Rosen et al. reported
the LLPS of multivalent signaling proteins. Neural Wiskott–Aldrich
syndrome protein (N-WASP), the actin-regulatory protein, interact with
its established biological partners NCK and phosphorylated nephrin1 to
form LLPS, wherein NCK contain three SH3 domains that can bind to the
six proline-rich motif (PRM) ligands of N-WASP 67.
Similar multivalent system were also reported in T cell receptor
signaling pathway 68, nucleophosmin (NPM1) interacting
with proteins comprising arginine-rich linear motifs and ribosomal RNA69 and pair of polySUMO–polySIM interacting
multivalent scaffold proteins 70.
Weakly interacting motifs are prevalent in LCDs to mediate LLPS. It has
been widely known that tightly self-complementing ‘steric
zipper ’ structure forms solid-like amyloid-β plaques with hydrophobic
interfaces and high stability 71–73. By contrast,
IDPs largely host motifs that can form thermodynamically metastable
‘kinked β sheets ’ 74–77 molecular structure,i.e. , the archetypical [G/S]Y[G/S] motifs of FUS protein.
These motifs can form close interactions as quantified by the structural
complementarity (\(S_{c}\)) (Table 1 ). However, side chains
cannot interdigitate across the β-sheet interface owing to the
prevention of kinks. They thus harbor smaller buried solvent-accessible
surface area (\(A_{b}\)) and more hydrophilic interfaces, thus
exhibiting much lower stability 74 (Figure 2Band Table 1 ). This is exemplified by metastable interaction
motifs in LCDs of FUS 55,78–82, Tau83, TDP-43 84 and hnRNP17,76,85 proteins. Specifically, short associative
peptide motifs within LCDs can form metastable fibrils in vitro ,
whilst exhibiting melting behavior in response to mild heating, which is
distinctive from stable amyloid fibrils (Table 2 ).Besides
kinked β sheets, other interaction motifs that can mediate multivalent
interactions were also reported, including repeated [F/R]G and
G[F/R] pair motifs of Ddx4 proteins 53,
α-helix-forming 321AMMAAAQAAL330motif of TDP-43 proteins 86, VPGXG (X is a guest
residue except proline that can modulate phase behaviour) motifs of
elastin-like proteins 87 and GHGLY motif of
histidine-rich squid beak proteins 52. In addition,
specific motifs may hinder LLPS, i.e. , FGDF can bind to G3BPs to
block the formation of stress granules 88.
The metastable molecular structures and phase behavior of IDPs can be
drastically altered simply by mutations17,55,76,80,84,85,89,90 or PTMs55,82,84 on one single residue. For example, The
phosphorylation of FUS protein by kinase at the Ser42 site drastically
altered the molecular interactions of LCDs, haltering the formation of
metastable fibrils and LLPS formation 55. This
prominent alteration of phase behavior can be attributed to disruption
of metastable kinked structure. The Ser42 site is the primary
phosphorylation site by DNA-dependent protein kinase (DNA-PK)91. The phosphorylation at Ser42 can significantly
disrupt the hydrogen bonds between Ser42 and Tyr38, interfere with the
interaction of mating sheet and destabilize the RAC1 interacting motif,
thereby modulating the ability of FUS to undergo LLPS. Additionally, the
mutation of Ser42 to Asp (S42D) can also remarkably depress the LLPS of
LCD of FUS protein, decreasing the critical temperature of LLPS by 5 °C,
as the mutation S42D is a change that mimics serine phosphorylation55.
There are two major types of phase behavior for a biological LLPS system
of interest, namely, the entropy-driven LCST phase behavior and
enthalpy-driven UCST phase behavior 24. How is the
type of phase behavior encoded in motifs of protein sequences? Chilkotiet al. synthesized artificial IDP-like polymers harboring several
tens of repeats of short peptide motifs 87. They found
that motifs with low-charge content and high hydrophobicity feature tend
to engender IDP-like polymer with LCST behaviour, which is reminiscent
of tropoelastins. By comparison, motifs with high-charge content and low
hydrophobicity feature tend to engender IDP-like polymer with UCST
behaviour, which is reminiscent of the dual UCST and LCST behaviour at
extremes of temperature of resilin. Furthermore, Chilkoti et al.found that hysteresis behavior can also be encoded and tuned at the
motif level by the precise position of an amino acid within a motif, as
well as at the macromolecule level by chain length 92.
The unique molecular interaction and molecular structure allow
metastable MLOs with unique properties, including liquidity, high
dynamics and environmental responsiveness. Learning from nature, the
responsible domains and motifs of IDPs have also been exploited as
building blocks to design bio-inspired
materials93–95, which have been reviewed elsewhere96.