Towards Decoding the Sequence-Based Grammar Governing the Functions of Intrinsically Disordered Protein Regions

A substantial portion of the proteome consists of intrinsically disordered regions (IDRs) that do not fold into well-defined 3D structures yet perform numerous biological functions and are associated with a broad range of diseases. It has been a long-standing enigma how different IDRs successfully execute their specific functions. Further putting a spotlight on IDRs are recent discoveries of functionally relevant biomolecular assemblies, which in some cases form through liquid-liquid phase separation. At the molecular level, the formation of biomolecular assemblies is largely driven by weak, multivalent, but selective IDR-IDR interactions. Emerging experimental and computational studies suggest that the primary amino acid sequences of IDRs encode a variety of their interaction behaviors. In this review, we focus on findings and insights that connect sequence-derived features of IDRs to their conformations, propensities to form biomolecular assemblies, selectivity of interaction partners, functions in the context of physiology and disease, and regulation of function. We also discuss directions of future research to facilitate establishing a comprehensive sequence-function paradigm that will eventually allow prediction of selective interactions and specificity of function mediated by IDRs.     

Dynamic Autoinhibition of the HMGB1 Protein via Electrostatic Fuzzy Interactions of Intrinsically Disordered Regions

Highly negatively charged segments containing only aspartate or glutamate residues (“D/E repeats”) are found in many eukaryotic proteins. For example, the C-terminal 30 residues of the HMGB1 protein are entirely D/E repeats. Using nuclear magnetic resonance (NMR), fluorescence, and computational approaches, we investigated how the D/E repeats causes the autoinhibition of HMGB1 against its specific binding to cisplatin-modified DNA. By varying ionic strength in a wide range (40–900 mM), we were able to shift the conformational equilibrium between the autoinhibited and uninhibited states toward either of them to the full extent. This allowed us to determine the macroscopic and microscopic equilibrium constants for the HMGB1 autoinhibition at various ionic strengths. At a macroscopic level, a model involving the autoinhibited and uninhibited states can explain the salt concentration-dependent binding affinity data. Our data at a microscopic level show that the D/E repeats and other parts of HMGB1 undergo electrostatic fuzzy interactions, each of which is weaker than expected from the macroscopic autoinhibitory effect. This discrepancy suggests that the multivalent nature of the fuzzy interactions enables strong autoinhibition at a macroscopic level despite the relatively weak intramolecular interaction at each site. Both experimental and computational data suggest that the D/E repeats interact preferentially with other intrinsically disordered regions (IDRs) of HMGB1. We also found that mutations mimicking post-translational modifications relevant to nuclear export of HMGB1 can moderately modulate DNA-binding affinity, possibly by impacting the autoinhibition. This study illuminates a functional role of the fuzzy interactions of D/E repeats.     

Visualization of intrinsically disordered proteins by high-speed atomic force microscopy

High-speed atomic force microscopy (HS-AFM) is a powerful tool established 13 years ago. This methodology can capture individual protein molecules carrying out functional activities under near-physiological conditions, without chemical labeling, at 2–3 nm lateral and ∼0.1 nm vertical spatial resolution, and at sub-100 ms temporal resolution. Although most biological HS-AFM studies thus far target structured proteins, HS-AFM is also ideally suited to study the dynamics of intrinsically disordered proteins. Here we review some of the dynamic structures and processes of intrinsically disordered proteins that have been unveiled by HS-AFM imaging.     

FapA is an Intrinsically Disordered Chaperone for Pseudomonas Functional Amyloid FapC

Bacterial functional amyloids contribute to biofilm development by bacteria and provide protection from the immune system and prevent antibiotic treatment. Strategies to target amyloid formation and interrupt biofilm formation have attracted recent interest due to their antimicrobial potential. Functional amyloid in Pseudomonas (Fap) includes FapC as the major component of the fibril while FapB is a minor component suggested to function as a nucleator of FapC. The system also includes the small periplasmic protein FapA, which has been shown to regulate fibril composition and morphology. The interplay between these three components is central in Fap fibril biogenesis. Here we present a comprehensive biophysical and spectroscopy analysis of FapA, FapB and FapC and provide insight into their molecular interactions. We show that all three proteins are primarily disordered with some regions with structural propensities for α-helix and β-sheet. FapA inhibits FapC fibrillation by targeting the nucleation step, whereas for FapB the elongation step is modulated. Furthermore, FapA alters the morphology of FapC (more than FapB) fibrils. Complex formation is observed between FapA and FapC, but not between FapA and FapB, and likely involves the N-terminus of FapA. We conclude that FapA is an intrinsically disordered chaperone for FapC that guards against fibrillation within the periplasm. This new understanding of a natural protective mechanism of Pseudomonas against amyloid formations can serve as inspiration for strategies blocking biofilm formation in infections.     

Folding Steps in the Fibrillation of Functional Amyloid: Denaturant Sensitivity Reveals Common Features in Nucleation and Elongation

Functional bacterial amyloids (FuBA) are intrinsically disordered proteins (IDPs) which rapidly and efficiently aggregate, forming extremely stable fibrils. The conversion from IDP to amyloid is evolutionarily optimized and likely couples folding to association. Many FuBA contain several imperfect repeat sequences which contribute to the stability of mature FuBA fibrils. Aggregation can be considered an intermolecular extension of the process of intramolecular protein folding which has traditionally been studied using chemical denaturants. Here we employ denaturants to investigate folding steps during fibrillation of CsgA and FapC. We quantify protein compactification (i.e. the extent of burial of otherwise exposed surface area upon association of proteins) during different stages of fibrillation based on the dependence of fibrillation rate constants on the denaturant concentration (m-values) determined from fibrillation curves. For both proteins, urea mainly affects nucleation and elongation (not fragmentation), consistent with the fact that these steps involve both intra- and intermolecular association. The two steps have similar m-values, indicating that activation steps in nucleation and elongation involve the same level of folding. Surprisingly, deletion of two or three repeats from FapC leads to larger m-values (i.e. higher compactification) during the activation step of fibril growth. This observation is extended by SAXS analysis of the fibrils which indicates that weakening of the amyloidogenic core caused by repeat deletions causes a larger portion of normally unstructured regions of the protein to be included into the amyloid backbone. We conclude that the sensitivity of fibrillation to denaturants can provide useful insight into molecular mechanisms of aggregation.     

Negatively Charged Disordered Regions are Prevalent and Functionally Important Across Proteomes

Intrinsically disordered regions (IDRs) of proteins are often characterized by a high fraction of charged residues, but differ in their overall net charge and in the organization of the charged residues. The function-encoding information stored via IDR charge composition and organization remains elusive. Here, we aim to decipher the sequence–function relationship in IDRs by presenting a comprehensive bioinformatic analysis of the charge properties of IDRs in the human, mouse, and yeast proteomes. About 50% of the proteins comprise at least a single IDR, which is either positively or negatively charged. Highly negatively charged IDRs are longer and possess greater net charge per residue compared with highly positively charged IDRs. A striking difference between positively and negatively charged IDRs is the characteristics of the repeated units, specifically, of consecutive Lys or Arg residues (K/R repeats) and Asp or Glu (D/E repeats) residues. D/E repeats are found to be about five times longer than K/R repeats, with the longest found containing 49 residues. Long stretches of consecutive D and E are found to be more prevalent in nucleic acid-related proteins. They are less common in prokaryotes, and in eukaryotes their abundance increases with genome size. The functional role of D/E repeats and the profound differences between them and K/R repeats are discussed.     

AlphaFold and the amyloid landscape

Protein aggregation is a widespread phenomenon with important implications in many scientific areas. Although amyloid formation is typically considered as detrimental, functional amyloids that perform physiological roles have been identified in all kingdoms of life. Despite their functional and pathological relevance, the structural details of the majority of molecular species involved in the amyloidogenic process remains elusive. Here, we explore the application of AlphaFold, a highly accurate protein structure predictor, in the field of protein aggregation. While we envision a straightforward application of AlphaFold in assisting the design of globular proteins with improved solubility for biomedical and industrial purposes, the use of this algorithm for predicting the structure of aggregated species seems far from trivial. First, in amyloid diseases, the presence of multiple amyloid polymorphs and the heterogeneity of aggregation intermediates challenges the “one sequence, one structure” paradigm, inherent to sequence-based predictions. Second, aberrant aggregation is not the subject of positive selective pressure, precluding the use of evolutionary-based approaches, which are the core of the AlphaFold pipeline. Instead, amyloid polymorphism seems to be constrained by the need for a defined structure-activity relationship in functional amyloids. They may thus provide a starting point for the application of AlphaFold in the amyloid landscape.     

Microsecond Dynamics During the Binding-induced Folding of an Intrinsically Disordered Protein

Tau is an intrinsically disordered protein implicated in many neurodegenerative diseases. The repeat domain fragment of tau, tau-K18, is known to undergo a disorder to order transition in the presence of lipid micelles and vesicles, in which helices form in each of the repeat domains. Here, the mechanism of helical structure formation, induced by a phospholipid mimetic, sodium dodecyl sulfate (SDS) at sub-micellar concentrations, has been studied using multiple biophysical probes. A study of the conformational dynamics of the disordered state, using photoinduced electron transfer coupled to fluorescence correlation spectroscopy (PET-FCS) has indicated the presence of an intermediate state, I, in equilibrium with the unfolded state, U. The cooperative binding of the ligand (L), SDS, to I has been shown to induce the formation of a compact, helical intermediate (IL₅) within the dead time (∼37 µs) of a continuous flow mixer. Quantitative analysis of the PET-FCS data and the ensemble microsecond kinetic data, suggests that the mechanism of induction of helical structure can be described by a U ↔ I ↔ IL₅ ↔ FL₅ mechanism, in which the final helical state, FL₅, forms from IL₅ with a time constant of 50–200 µs. Finally, it has been shown that the helical conformation is an aggregation-competent state that can directly form amyloid fibrils.     

Transient Tertiary Structures of Disordered Dynein Intermediate Chain Regulate its Interactions with Multiple Partners

The N-terminal domain of dynein intermediate chain (N–IC) is central to the cytoplasmic dynein ‘cargo attachment subcomplex’ and regulation of motor activity. It is a prototypical intrinsically disordered protein (IDP), serving as a primarily disordered polybivalent molecular scaffold for numerous binding partners, including three dimeric dynein light chains and coiled coil domains of dynein partners dynactin p150^Glued and NudE. At the very N-terminus, a 40 amino acid single alpha helix (SAH) forms the major binding site for both p150^Glued and NudE, while a shorter nascent helix (H2) separated from SAH by a disordered linker, is necessary for tight binding to dynactin p150^Glued but not to NudE. Here we demonstrate that transient tertiary interactions in this highly dynamic protein underlie the differences in its interactions with p150^Glued and NudE. NMR paramagnetic relaxation enhancement experiments and restrained molecular dynamics simulations identify interactions between the two non-contiguous SAH and H2 helical regions, the extent of which correlates with the length and stability of H2, showing clearly that tertiary and secondary structure formation are coupled in IDPs. These interactions are significantly attenuated when N–IC is bound to NudE, suggesting that NudE binding shifts the conformational ensemble to one that is more extended and with less structure in H2. While the intrinsic disorder and flexibility in N–IC modulate its ability to serve as a binding platform for numerous partners, deviations of this protein from random-coil behavior provide a process for regulating these binding interactions and potentially the dynein motor.     

Dynamic Refolding of OxyS sRNA by the Hfq RNA Chaperone

The Sm protein Hfq chaperones small non-coding RNAs (sRNAs) in bacteria, facilitating sRNA regulation of target mRNAs. Hfq acts in part by remodeling the sRNA and mRNA structures, yet the basis for this remodeling activity is not understood. To understand how Hfq remodels RNA, we used single-molecule Förster resonance energy transfer (smFRET) to monitor conformational changes in OxyS sRNA upon Hfq binding. The results show that E. coli Hfq first compacts OxyS, bringing its 5′ and 3 ends together. Next, Hfq destabilizes an internal stem-loop in OxyS, allowing the RNA to adopt a more open conformation that is stabilized by a conserved arginine on the rim of Hfq. The frequency of transitions between compact and open conformations depend on interactions with Hfqs flexible C-terminal domain (CTD), being more rapid when the CTD is deleted, and slower when OxyS is bound to Caulobacter crescentus Hfq, which has a shorter and more stable CTD than E. coli Hfq. We propose that the CTDs gate transitions between OxyS conformations that are stabilized by interaction with one or more arginines. These results suggest a general model for how basic residues and intrinsically disordered regions of RNA chaperones act together to refold RNA.     

EGCG has Dual and Opposing Effects on the N-terminal Region of Self-associating α-synuclein Oligomers

Oligomers of the protein α-synuclein (α-syn) are thought to be a major toxic species in Parkinson’s disease, particularly through their ability to permeabilize cell membranes. The green tea polyphenol epigallocatechin gallate (EGCG) has been found to reduce this ability. We have analyzed α-syn oligomer dynamics and interconversion by H/D exchange monitored by mass spectrometry (HDX-MS). Our results show that the two oligomers OI and OII co-exist in equilibrium; OI is a multimer of OII and its dissociation can be followed by HDX-MS by virtue of the correlated exchange of the N-terminal region. Urea destabilizes the α-syn oligomers, dissociating OI to OII and monomers. Oligomers exposed to EGCG undergo Met oxidation. Intriguingly, EGCG induces an oxidation-dependent effect on the structure of the N-terminal region. For the non-oxidized N-terminal region, EGCG increases the stability of the folded structure as measured by a higher level of protection against H/D exchange. In contrast, protection is clearly abrogated in the Met oxidized N-terminal region. Having a non-oxidized and disordered N-terminal region is known to be essential for efficient membrane binding. Therefore, our results suggest that the combined effect of a structural stabilization of the non-oxidized N-terminal region and the presence of a disordered oxidized N-terminal region renders the oligomers less cytotoxic by decreasing the ability of the N-terminal region to bind to cell membranes and facilitate their permeabilization.     

Membrane Binding and Homodimerization of Atg16 Via Two Distinct Protein Regions is Essential for Autophagy in Yeast

Macroautophagy is a bulk degradation mechanism in eukaryotic cells. Efficiency of an essential step of this process in yeast, Atg8 lipidation, relies on the presence of Atg16, a subunit of the Atg12–Atg5-Atg16 complex acting as the E3-like enzyme in the ubiquitination-like reaction. A current view on the functional structure of Atg16 in the yeast S. cerevisiae comes from the two crystal structures that reveal the Atg5-interacting α-helix linked via a flexible linker to another α-helix of Atg16, which then assembles into a homodimer. This view does not explain the results of previous in vitro studies revealing Atg16-dependent deformations of membranes and liposome-binding of the Atg12–Atg5 conjugate upon addition of Atg16. Here we show that Atg16 acts as both a homodimerizing and peripheral membrane-binding polypeptide. These two characteristics are imposed by the two distinct regions that are disordered in the nascent protein. Atg16 binds to membranes in vivo via the amphipathic α-helix (amino acid residues 113–131) that has a coiled-coil-like propensity and a strong hydrophobic face for insertion into the membrane. The other protein region (residues 64–99) possesses a coiled-coil propensity, but not amphipathicity, and is dispensable for membrane anchoring of Atg16. This region acts as a Leu-zipper essential for formation of the Atg16 homodimer. Mutagenic disruption in either of these two distinct domains renders Atg16 proteins that, in contrast to wild type, completely fail to rescue the autophagy-defective phenotype of atg16Δ cells. Together, the results of this study yield a model for the molecular mechanism of Atg16 function in macroautophagy.     

Prediction of Quality-control Degradation Signals in Yeast Proteins

Effective proteome homeostasis is key to cellular and organismal survival, and cells therefore contain efficient quality control systems to monitor and remove potentially toxic misfolded proteins. Such general protein quality control to a large extent relies on the efficient and robust delivery of misfolded or unfolded proteins to the ubiquitin–proteasome system. This is achieved via recognition of so-called degradation motifs—degrons—that are assumed to become exposed as a result of protein misfolding. Despite their importance, the nature and sequence properties of quality-control degrons remain elusive. Here, we have used data from a yeast-based screen of 23,600 17-residue peptides to build a predictor of quality-control degrons. The resulting model, QCDPred (Quality Control Degron Prediction), achieves good accuracy using only the sequence composition of the peptides as input. Our analysis reveals that strong degrons are enriched in hydrophobic amino acids and depleted in negatively charged amino acids, in line with the expectation that they are buried in natively folded proteins. We applied QCDPred to the yeast proteome, enabling us to analyse more widely the potential effects of degrons. As an example, we show a correlation between cellular abundance and degron potential in disordered regions of proteins. Together with recent results on membrane proteins, our work suggest that the recognition of exposed hydrophobic residues is a key and generic mechanism for proteome homeostasis. QCDPred is freely available as open source code and via a web interface.     

Sequence-targeted Peptides Divert Functional Bacterial Amyloid Towards Destabilized Aggregates and Reduce Biofilm Formation

Functional bacterial amyloid provides structural stability in biofilm, making it a promising target for anti-biofilm therapeutics. Fibrils formed by CsgA, the major amyloid component in E. coli are extremely robust and can withstand very harsh conditions. Like other functional amyloids, CsgA contains relatively short aggregation-prone regions (APR) which drive amyloid formation. Here, we demonstrate the use of aggregation-modulating peptides to knock down CsgA protein into aggregates with low stability and altered morphology. Remarkably, these CsgA-peptides also modulate fibrillation of the unrelated functional amyloid protein FapC from Pseudomonas, possibly through recognition of FapC segments with structural and sequence similarity with CsgA. The peptides also reduce the level of biofilm formation in E. coli and P. aeruginosa, demonstrating the potential for selective amyloid targeting to combat bacterial biofilm.     

Extending the Horizon of Homology Detection with Coevolution-based Structure Prediction

Traditional sequence analysis algorithms fail to identify distant homologies when they lie beyond a detection horizon. In this review, we discuss how co-evolution-based contact and distance prediction methods are pushing back this homology detection horizon, thereby yielding new functional insights and experimentally testable hypotheses. Based on correlated substitutions, these methods divine three-dimensional constraints among amino acids in protein sequences that were previously devoid of all annotated domains and repeats. The new algorithms discern hidden structure in an otherwise featureless sequence landscape. Their revelatory impact promises to be as profound as the use, by archaeologists, of ground-penetrating radar to discern long-hidden, subterranean structures. As examples of this, we describe how triplicated structures reflecting longin domains in MON1A-like proteins, or UVR-like repeats in DISC1, emerge from their predicted contact and distance maps. These methods also help to resolve structures that do not conform to a “beads-on-a-string” model of protein domains. In one such example, we describe CFAP298 whose ubiquitin-like domain was previously challenging to perceive owing to a large sequence insertion within it. More generally, the new algorithms permit an easier appreciation of domain families and folds whose evolution involved structural insertion or rearrangement. As we exemplify with α1-antitrypsin, coevolution-based predicted contacts may also yield insights into protein dynamics and conformational change. This new combination of structure prediction (using innovative co-evolution based methods) and homology inference (using more traditional sequence analysis approaches) shows great promise for bringing into view a sea of evolutionary relationships that had hitherto lain far beyond the horizon of homology detection.