An assignment of intrinsically disordered regions of proteins based on NMR structures

Intrinsically disordered proteins (IDPs) do not adopt stable three-dimensional structures in physiological conditions, yet these proteins play crucial roles in biological phenomena. In most cases, intrinsic disorder manifests itself in segments or domains of an IDP, called intrinsically disordered regions (IDRs), but fully disordered IDPs also exist. Although IDRs can be detected as missing residues in protein structures determined by X-ray crystallography, no protocol has been developed to identify IDRs from structures obtained by Nuclear Magnetic Resonance (NMR). Here, we propose a computational method to assign IDRs based on NMR structures. We compared missing residues of X-ray structures with residue-wise deviations of NMR structures for identical proteins, and derived a threshold deviation that gives the best correlation of ordered and disordered regions of both structures. The obtained threshold of 3.2 Å was applied to proteins whose structures were only determined by NMR, and the resulting IDRs were analyzed and compared to those of X-ray structures with no NMR counterpart in terms of sequence length, IDR fraction, protein function, cellular location, and amino acid composition, all of which suggest distinct characteristics. The structural knowledge of IDPs is still inadequate compared with that of structured proteins. Our method can collect and utilize IDRs from structures determined by NMR, potentially enhancing the understanding of IDPs.     

The Balancing Act of Intrinsically Disordered Proteins: Enabling Functional Diversity while Minimizing Promiscuity

Intrinsically disordered proteins (IDPs) or regions (IDRs) perform diverse cellular functions, but are also prone to forming promiscuous and potentially deleterious interactions. We investigate the extent to which the properties of, and content in, IDRs have adapted to enable functional diversity while limiting interference from promiscuous interactions in the crowded cellular environment. Information on protein sequences, their predicted intrinsic disorder, and 3D structure contents is related to data on protein cellular concentrations, gene co-expression, and protein–protein interactions in the well-studied yeast Saccharomyces cerevisiae. Results reveal that both the protein IDR content and the frequency of “sticky” amino acids in IDRs (those more frequently involved in protein interfaces) decrease with increasing protein cellular concentration. This implies that the IDR content and the amino acid composition of IDRs experience negative selection as the protein concentration increases. In the S. cerevisiae protein–protein interaction network, the higher a protein's IDR content, the more frequently it interacts with IDR-containing partners, and the more functionally diverse the partners are. Employing a clustering analysis of Gene Ontology terms, we newly identify ~ 600 putative multifunctional proteins in S. cerevisiae. Strikingly, these proteins are enriched in IDRs and contribute significantly to all the observed trends. In particular, IDRs of multi-functional proteins feature more sticky amino acids than IDRs of their non-multifunctional counterparts, or the surfaces of structured yeast proteins. This property likely affords sufficient binding affinity for the functional interactions, commonly mediated by short IDR segments, thereby counterbalancing the loss in overall IDR conformational entropy upon binding.     

Protein disorder,prion propensities,and self-organizing macromolecular collectives

Eukaryotic cells are partitioned into functionally distinct self-organizing compartments. But while the biogenesis of membrane-surrounded compartments is beginning to be understood, the organizing principles behind large membrane-less structures, such as RNA-containing granules, remain a mystery. Here, we argue that protein disorder is an essential ingredient for the formation of such macromolecular collectives. Intrinsically disordered regions (IDRs) do not fold into a well-defined structure but rather sample a range of conformational states, depending on the local conditions. In addition to being structurally versatile, IDRs promote multivalent and transient interactions. This unique combination of features turns intrinsically disordered proteins into ideal agents to orchestrate the formation of large macromolecular assemblies. The presence of conformationally flexible regions, however, comes at a cost, for many intrinsically disordered proteins are aggregation-prone and cause protein misfolding diseases. This association with disease is particularly strong for IDRs with prion-like amino acid composition. Here, we examine how disease-causing and normal conformations are linked, and discuss the possibility that the dynamic order of the cytoplasm emerges, at least in part, from the collective properties of intrinsically disordered prion-like domains. This article is part of a Special Issue entitled: The emerging dynamic view of proteins: Protein plasticity in allostery, evolution and self-assembly.     

The cytoplasmic domain of the chloride channel ClC-0: structural and dynamic characterization of flexible regions

Eukaryotic members of the ClC family of chloride channels and transporters are composed of a transmembrane ion transport domain followed by a cytoplasmic domain, which is believed to be involved in the modulation of ClC function. In some family members this putative regulatory domain contains next to a well-folded structured part, long sequence stretches with low sequence complexity. These regions, a 96 residue long linker connecting two structured sub-domains, and 35 residues on the C teminus of the domain were found disordered in a recent crystal structure of this domain in ClC-0. Both regions have a large influence on the modulation of channel function in closely related family members. Here we describe a NMR study to characterize the structural and dynamic properties of these putatively unstructured stretches. Our study reveals that the two regions indeed show large conformational flexibility with dynamics on the nanosecond timescale. However, small islands of secondary structure are found interdispersed between the unfolded regions. This study characterizes for the first time the biophysical properties of these protein segments, which may become important for the understanding of novel regulatory mechanisms within the ClC family.     

Conservation of Flexible Residue Clusters among Structural and Functional Enzyme Homologues

Conformational flexibility between structural ensembles is an essential component of enzyme function. Although the broad dynamical landscape of proteins is known to promote a number of functional events on multiple time scales, it is yet unknown whether structural and functional enzyme homologues rely on the same concerted residue motions to perform their catalytic function. It is hypothesized that networks of contiguous and flexible residue motions occurring on the biologically relevant millisecond time scale evolved to promote and/or preserve optimal enzyme catalysis. In this study, we use a combination of NMR relaxation dispersion, model-free analysis, and ligand titration experiments to successfully capture and compare the role of conformational flexibility between two structural homologues of the pancreatic ribonuclease family: RNase A and eosinophil cationic protein (or RNase 3). In addition to conserving the same catalytic residues and structural fold, both homologues show similar yet functionally distinct clusters of millisecond dynamics, suggesting that conformational flexibility can be conserved among analogous protein folds displaying low sequence identity. Our work shows that the reduced conformational flexibility of eosinophil cationic protein can be dynamically and functionally reproduced in the RNase A scaffold upon creation of a chimeric hybrid between the two proteins. These results support the hypothesis that conformational flexibility is partly required for catalytic function in homologous enzyme folds, further highlighting the importance of dynamic residue sectors in the structural organization of proteins.     

Intrinsic Disorder in the BK Channel and Its Interactome

The large-conductance Ca²⁺-activated K⁺ (BK) channel is broadly expressed in various mammalian cells and tissues such as neurons, skeletal and smooth muscles, exocrine cells, and sensory cells of the inner ear. Previous studies suggest that BK channels are promiscuous binders involved in a multitude of protein-protein interactions. To gain a better understanding of the potential mechanisms underlying BK interactions, we analyzed the abundance, distribution, and potential mechanisms of intrinsic disorder in 27 BK channel variants from mouse cochlea, 104 previously reported BK-associated proteins (BKAPS) from cytoplasmic and membrane/cytoskeletal regions, plus BK β- and γ-subunits. Disorder was evaluated using the MFDp algorithm, which is a consensus-based predictor that provides a strong and competitive predictive quality and PONDR, which can determine long intrinsically disordered regions (IDRs). Disorder-based binding sites or molecular recognition features (MoRFs) were found using MoRFpred and ANCHOR. BKAP functions were categorized based on Gene Ontology (GO) terms. The analyses revealed that the BK variants contain a number of IDRs. Intrinsic disorder is also common in BKAPs, of which ∼5% are completely disordered. However, intrinsic disorder is very differently distributed within BK and its partners. Approximately 65% of the disordered segments in BK channels are long (IDRs) (>50 residues), whereas >60% of the disordered segments in BKAPs are short IDRs that range in length from 4 to 30 residues. Both α and γ subunits showed various amounts of disorder as did hub proteins of the BK interactome. Our analyses suggest that intrinsic disorder is important for the function of BK and its BKAPs. Long IDRs in BK are engaged in protein-protein and protein-ligand interactions, contain multiple post-translational modification sites, and are subjected to alternative splicing. The disordered structure of BK and its BKAPs suggests one of the underlying mechanisms of their interaction.     

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.     

Do sequence neighbours of intrinsically disordered regions promote structural flexibility in intrinsically disordered proteins?

Intrinsically disordered proteins (IDPs) are crucial players in various cellular activities. Several experimental and computational analyses have been conducted to study structural pliability and functional potential of IDPs. In spite of active research in past few decades, what induces structural disorder in IDPs and how is still elusive. Many studies testify that sequential and spatial neighbours often play important roles in determining structural and functional behaviour of proteins. Considering this fact, we assessed sequence neighbours of intrinsically disordered regions (IDRs) to understand if they have any role to play in inducing structural flexibility in IDPs. Our analysis includes 97% eukaryotic IDPs and 3% from bacteria and viruses. Physicochemical and structural parameters including amino acid propensity, hydrophobicity, secondary structure propensity, relative solvent accessibility, B-factor and atomic packing density are used to characterise the neighbouring residues of IDRs (NRIs). We show that NRIs exhibit a unique nature, which makes them stand out from both ordered and disordered residues. They show correlative occurrences of residue pairs like Ser-Thr and Gln-Asn, indicating their tendency to avoid strong biases of order or disorder promoting amino acids. We also find differential preferences of amino acids between N- and C-terminal neighbours, which might indicate a plausible directional effect on the dynamics of adjacent IDRs. We designed an efficient prediction tool using Random Forest to distinguish the NRIs from the ordered residues. Our findings will contribute to understand the behaviour of IDPs, and may provide potential lead in deciphering the role of IDRs in protein folding and assembly.     

Targeting disordered-structured domain interactions in Galectin-3 based on NMR and enhanced MD

Intrinsically disordered regions (IDRs) are common and important functional domains in many proteins. However, IDRs are difficult to target for drug development due to the lack of defined structures that would facilitate the identification of possible drug-binding pockets. Galectin-3 is a carbohydrate-binding protein of which overexpression has been implicated in a wide variety of disorders, including cancer and inflammation. Apart from its carbohydrate-recognition/binding domain (CRD), Galectin-3 also contains a functionally important disordered N-terminal domain (NTD) that contacts the C-terminal domain (CTD) and could be a target for drug development. To overcome challenges involved in inhibitor design due to lack of structure and the highly dynamic nature of the NTD, we used a protocol combining nuclear magnetic resonance data from recombinant Galectin-3 with accelerated molecular dynamics (MD) simulations. This approach identified a pocket in the CTD with which the NTD makes frequent contact. In accordance with this model, mutation of residues L131 and L203 in this pocket caused loss of Galectin-3 agglutination ability, signifying the functional relevance of the cavity. In silico screening was used to design candidate inhibitory peptides targeting the newly discovered cavity, and experimental testing of only three of these yielded one peptide that inhibits the agglutination promoted by wild-type Galectin-3. NMR experiments further confirmed that this peptide indeed binds to a cavity in the CTD, not within the actual CRD. Our results show that it is possible to apply a combination of MD simulations and NMR experiments to precisely predict the binding interface of a disordered domain with a structured domain, and furthermore use this predicted interface for designing inhibitors. This procedure can potentially be extended to many other targets in which similar IDR interactions play a vital functional role.     

Markov models of amino acid substitution to study proteins with intrinsically disordered regions

Background

Intrinsically disordered proteins (IDPs) or proteins with disordered regions (IDRs) do not have a well-defined tertiary structure, but perform a multitude of functions, often relying on their native disorder to achieve the binding flexibility through changing to alternative conformations. Intrinsic disorder is frequently found in all three kingdoms of life, and may occur in short stretches or span whole proteins. To date most studies contrasting the differences between ordered and disordered proteins focused on simple summary statistics. Here, we propose an evolutionary approach to study IDPs, and contrast patterns specific to ordered protein regions and the corresponding IDRs.

Results

Two empirical Markov models of amino acid substitutions were estimated, based on a large set of multiple sequence alignments with experimentally verified annotations of disordered regions from the DisProt database of IDPs. We applied new methods to detect differences in Markovian evolution and evolutionary rates between IDRs and the corresponding ordered protein regions. Further, we investigated the distribution of IDPs among functional categories, biochemical pathways and their preponderance to contain tandem repeats.

Conclusions

We find significant differences in the evolution between ordered and disordered regions of proteins. Most importantly we find that disorder promoting amino acids are more conserved in IDRs, indicating that in some cases not only amino acid composition but the specific sequence is important for function. This conjecture is also reinforced by the observation that for of our data set IDRs evolve more slowly than the ordered parts of the proteins, while we still support the common view that IDRs in general evolve more quickly. The improvement in model fit indicates a possible improvement for various types of analyses e.g. de novo disorder prediction using a phylogenetic Hidden Markov Model based on our matrices showed a performance similar to other disorder predictors.