Intrinsically Disordered Tardigrade Proteins Self-Assemble into Fibrous Gels in Response to Environmental Stress.

Tardigrades are remarkable for their ability to survive harsh stress conditions as diverse as extreme temperature and desiccation. The molecular mechanisms that confer this unusual resistance to physical stress remain unknown. Recently, tardigrade-unique intrinsically disordered proteins have been shown to play an essential role in tardigrade anhydrobiosis. Here, we characterize the conformational and physical behaviour of CAHS-8 from Hypsibius exemplaris. NMR spectroscopy reveals that the protein comprises an extended central helical domain flanked by disordered termini. Upon concentration, the protein is shown to successively form oligomers, long fibres, and finally gels constituted of fibres in a strongly temperature-dependent manner. The helical domain forms the core of the fibrillar structure, with the disordered termini remaining highly dynamic within the gel. Soluble proteins can be encapsulated within cavities in the gel, maintaining their functional form. The ability to reversibly form fibrous gels may be associated with the enhanced protective properties of these proteins.

Phosphorylation of a Human Microprotein Promotes Dissociation of Biomolecular Condensates.

Proteogenomic identification of translated small open reading frames in humans has revealed thousands of microproteins, or polypeptides of fewer than 100 amino acids, that were previously invisible to geneticists. Hundreds of microproteins have been shown to be essential for cell growth and proliferation, and many regulate macromolecular complexes. However, the vast majority of microproteins remain functionally uncharacterized, and many lack secondary structure and exhibit limited evolutionary conservation. One such intrinsically disordered microprotein is NBDY, a 68-amino acid component of membraneless organelles known as P-bodies. In this work, we show that NBDY can undergo liquid-liquid phase separation, a biophysical process thought to underlie the formation of membraneless organelles, in the presence of RNA in vitro. Phosphorylation of NBDY drives liquid phase remixing in vitro and macroscopic P-body dissociation in cells undergoing growth factor signaling and cell division. These results suggest that NBDY phosphorylation enables regulation of P-body dynamics during cell proliferation and, more broadly, that intrinsically disordered microproteins may contribute to liquid-liquid phase separation and remixing behavior to affect cellular processes.

Molecular Details of a Coupled Binding and Folding Reaction between the Amyloid Precursor Protein and a Folded Domain.

Intrinsically disordered regions in proteins often function as binding motifs in protein-protein interactions. The mechanistic aspects and molecular details of such coupled binding and folding reactions, which involve formation of multiple noncovalent bonds, have been broadly studied theoretically, but experimental data are scarce. Here, using a combination of protein semisynthesis to incorporate phosphorylated amino acids, backbone amide-to-ester modifications, side chain substitutions, and binding kinetics, we examined the interaction between the intrinsically disordered motif of amyloid precursor protein (APP) and the phosphotyrosine binding (PTB) domain of Mint2. We show that the interaction is regulated by a self-inhibitory segment of the PTB domain previously termed ARM. The helical ARM linker decreases the association rate constant 30-fold through a fast pre-equilibrium between an open and a closed state. Extensive side chain substitutions combined with kinetic experiments demonstrate that the rate-limiting transition state for the binding reaction is governed by native and non-native hydrophobic interactions and hydrogen bonds. Hydrophobic interactions were found to be particularly important during crossing of the transition state barrier. Furthermore, linear free energy relationships show that the overall coupled binding and folding reaction involves cooperative formation of interactions with roughly 30% native contacts formed at the transition state. Our data support an emerging picture of coupled binding and folding reactions following overall chemical principles similar to those of folding of globular protein domains but with greater malleability of ground and transition states.

Atomic Resolution Map of Hierarchical Self-Assembly for an Amyloidogenic Protein Probed through Thermal 15N-R2 Correlation Matrices.

Soluble oligomers formed by amyloidogenic intrinsically disordered proteins are some of the most cytotoxic species linked to neurodegeneration. Due to the transient and heterogeneous nature of such oligomeric intermediates, the underlying self-association events often remain elusive. NMR relaxation measurements sensitive to zero-frequency spectral densities (J(0)), such as the 15N - R2 rates, are ideally suited to map sites of self-association at atomic resolution without the need of exogenous labels. Such experiments exploit the dynamic exchange between NMR visible monomers and slowly tumbling oligomers. However,15N - R2 rates are also sensitive to intrinsic monomer dynamics, and it is often difficult to discern these contributions from those arising from exchange with oligomers. Another challenge pertains to defining a hierarchy of self-association. Here, using the archetypical amyloidogenic protein alpha synuclein (αS), we show that the temperature-dependence of 15N - R2 effectively identifies self-association sites with reduced bias from internal dynamics. The key signature of the residues involved in self-association is a nonlinear temperature-dependence of 15N - R2 with a positive ΔR2/ΔT slope. These two hallmarks are systematically probed through a thermal R2 correlation matrix, from which the network of residues involved in self-association as well as the hierarchy of αS self-association sites is extracted through agglomerative clustering. We find that aggregation is initiated by residues within the NAC region that is solvent inaccessible in αS fibrils and eventually extends to the N-terminal segment harboring familial PD mutations. These hierarchical self-association maps help dissect the essential drivers of oligomerization and reveal how amyloid inhibitors affect oligomer formation.

Quantum Chemical Calculations of NMR Chemical Shifts in Phosphorylated Intrinsically Disordered Proteins.

Quantum mechanics (QM) calculations are applied to examine 1H, 13C, 15N, and 31P chemical shifts of two phosphorylation sites in an intrinsically disordered protein region. The QM calculations employ a combination of (1) structural ensembles generated by molecular dynamics, (2) a fragmentation technique based on the adjustable density matrix assembler, and (3) density functional methods. The combined computational approach is used to obtain chemical shifts (i) in the S19 and S40 residues of the nonphosphorylated and (ii) in the pS19 and pS40 residues of the doubly phosphorylated human tyrosine hydroxylase 1 as the system of interest. We study the effects of conformational averaging and explicit solvent sampling as well as the effects of phosphorylation on the computed chemical shifts. Good to great quantitative agreement with the experiment is achieved for all nuclei, provided that the systematic error cancellation is optimized by the choice of a suitable NMR standard. The effect of the standard reference on the computed 15N and 31P chemical shifts is demonstrated by employing three different referencing methods. Error bars associated with the statistical averaging of the computed 31P chemical shifts are larger than the difference between the 31P chemical shift of pS19 and pS40. The sequence trend of 31P shifts therefore could not be reliably reproduced. On the contrary, the calculations correctly predict the change of the 13C chemical shift for CB induced by the phosphorylation of the serine residues. The present work demonstrates that QM calculations coupled with molecular dynamics simulations and fragmentation techniques can be used as an alternative to empirical prediction tools in the structure characterization of intrinsically disordered proteins.

Intrinsic Disorder-Based Design of Stable Globular Proteins.

Directed stabilization of globular proteins via substitution of a minimal number of amino acid residues is one of the most complicated experimental tasks. This work summarizes our research on the effect of amino acid substitutions on the protein stability utilizing the outputs of the analysis of intrinsic disorder predisposition of target proteins. This allowed us to formulate the basis of one of the possible approaches to the stabilization of globular proteins. The idea is quite simple. To stabilize a protein as a whole, one needs to find its "weakest spot" and stabilize it, but the question is how this weak spot can be found in a query protein. Our approach is based on the utilization of the computational tools for the per-residue evaluation of intrinsic disorder predisposition to search for the "weakest spot" of a query protein (i.e., the region(s) with the highest local predisposition for intrinsic disorder). When such "weakest spot" is found, it can be stabilized through a limited number of point mutations by introducing order-promoting residues at hot spots, thereby increasing structural stability of a protein as a whole. Using this approach, we were able to obtain stable mutant forms of several globular proteins, such as Gαo, GFP, ribosome protein L1, and circular permutant of apical domain of GroEL.

An Intrinsically Disordered Peptide-Peptide Stapler for Highly Efficient Protein Ligation Both in Vivo and in Vitro.

Herein, we report an intrinsically disordered protein SpyStapler that can catalyze the isopeptide bond formation between two peptide tags, that is, SpyTag and BDTag, both in vitro and in vivo. SpyStapler and BDTag are developed by splitting SpyCatcher-the cognate protein partner of SpyTag-at the more solvent exposed second loop region. Regardless of their locations in protein constructs, SpyStapler enables efficient covalent coupling of SpyTag and BDTag under a variety of mild conditions in vitro (yield ∼80%). Co-expression of SpyStapler with telechelic dihydrofolate reductase (DHFR) bearing a SpyTag at N-terminus and a BDTag at C-terminus leads to direct cellular synthesis of a circular DHFR. Mechanistic studies involving circular dichroism and nuclear magnetic resonance spectrometry reveal that SpyStapler alone is disordered in solution and forms a stable folded structure ( Tm ∼ 55 °C) in the presence of both SpyTag and BDTag upon isopeptide bonding. No ordered structure can be formed in the absence of either tag. The catalytically inactive SpyStapler-EQ mutant cannot form a stable physical complex with SpyTag and BDTag, but it can fold into ordered structure in the presence of the ligated product (SpyTag-BDTag). It suggests that the isopeptide bond is important in stabilizing the complex. Given its efficiency, resilience, and robustness, SpyStapler provides new opportunities for bioconjugation and creation of complex protein architectures.

Calcium-Mediated Allostery of the EGF Fold.

The epidermal growth factor (EGF)-like domain is one of the most abundant disulfide-containing domains in nature and is involved in many cellular processes critical to life. Although many EGF-like domains participate in calcium-dependent functions by responding to the local calcium concentration, little is known about how this responsiveness is programmed at the molecular level. Here, we reveal the structural and environmental determinants underpinning the folding of a synthetic analogue of the EGF-A domain (from the low-density lipoprotein receptor). We show that calcium sensitivity is enabled by an allosteric folding pathway, in which calcium binding is connected to the peptide core through local inter-residue interactions. In the absence of calcium, the fold favors disorder because the inherently weak core is insufficient to stabilize the active form, resulting in substantial loss in activity of 2 orders of magnitude. The EGF-A fold, which can freely transition between active and disordered states, is volatile, and we found it to be intolerant of mutations, unlike other disulfide-rich peptides that have been used as stabilizing frameworks. This volatility is beneficial for modularity/plasticity and appears to have evolved for such a purpose, allowing cellular pathways to sense and respond to environmental cues.

Dissecting mechanism of coupled folding and binding of an intrinsically disordered protein by chemical synthesis of conformationally constrained analogues.

Non-canonical α-methyl amino acids were incorporated at various sites in the sequence of intrinsically disordered activation domain from the p160 transcriptional co-activator (ACTR) to facilitate the formation of α-helical structures. Kinetic and thermodynamic data confirm the induced fit mechanism of complex formation between the synthesized ACTR variants and the nuclear co-activator binding domain (NCBD).

Programming molecular self-assembly of intrinsically disordered proteins containing sequences of low complexity.

Dynamic protein-rich intracellular structures that contain phase-separated intrinsically disordered proteins (IDPs) composed of sequences of low complexity (SLC) have been shown to serve a variety of important cellular functions, which include signalling, compartmentalization and stabilization. However, our understanding of these structures and our ability to synthesize models of them have been limited. We present design rules for IDPs possessing SLCs that phase separate into diverse assemblies within droplet microenvironments. Using theoretical analyses, we interpret the phase behaviour of archetypal IDP sequences and demonstrate the rational design of a vast library of multicomponent protein-rich structures that ranges from uniform nano-, meso- and microscale puncta (distinct protein droplets) to multilayered orthogonally phase-separated granular structures. The ability to predict and program IDP-rich assemblies in this fashion offers new insights into (1) genetic-to-molecular-to-macroscale relationships that encode hierarchical IDP assemblies, (2) design rules of such assemblies in cell biology and (3) molecular-level engineering of self-assembled recombinant IDP-rich materials.

Identification of Dynamic Modes in an Intrinsically Disordered Protein Using Temperature-Dependent NMR Relaxation.

The dynamic modes and time scales sampled by intrinsically disordered proteins (IDPs) define their function. Nuclear magnetic resonance (NMR) spin relaxation is probably the most powerful tool for investigating these motions delivering site-specific descriptions of conformational fluctuations from throughout the molecule. Despite the abundance of experimental measurement of relaxation in IDPs, the physical origin of the measured relaxation rates remains poorly understood. Here we measure an extensive range of auto- and cross-correlated spin relaxation rates at multiple magnetic field strengths on the C-terminal domain of the nucleoprotein of Sendai virus, over a large range of temperatures (268-298 K), and combine these data to describe the dynamic behavior of this archetypal IDP. An Arrhenius-type relationship is used to simultaneously analyze up to 61 relaxation rates per amino acid over the entire temperature range, allowing the measurement of local activation energies along the chain, and the assignment of physically distinct dynamic modes. Fast (τ ≤ 50 ps) components report on librational motions, a dominant mode occurs on time scales around 1 ns, apparently reporting on backbone sampling within Ramachandran substates, while a slower component (5-25 ns) reports on segmental dynamics dominated by the chain-like nature of the protein. Extending the study to three protein constructs of different lengths (59, 81, and 124 amino acids) substantiates the assignment of these contributions. The analysis is shown to be remarkably robust, accurately predicting a broad range of relaxation data measured at different magnetic field strengths and temperatures. The ability to delineate intrinsic modes and time scales from NMR spin relaxation will improve our understanding of the behavior and function of IDPs, adding a new and essential dimension to the description of this biologically important and ubiquitous class of proteins.

Design and preparation of single-chain nanocarriers mimicking disordered proteins for combined delivery of dermal bioactive cargos.

Inspired by the multifunctionality of vitamin D-binding protein and the multiple transient-binding behavior of some intrinsically disordered proteins (IDPs), a polymeric platform is designed, prepared, and characterized for combined delivery of dermal protective and anticancer bioactive cargos on the basis of artificial single-chain nano-objects mimicking IDPs. For the first time ever, simultaneous delivery of folic acid or vitamin B9 , and hinokitiol, a relevant natural bioactive compound that exhibits anticancer activity against human malignant melanoma cells, from these multidirectionally self-assembled unimolecular nanocarriers is illustrated.