Intrinsically disordered inhibitor of glutamine synthetase is a functional protein with random‐coil‐like pKa values

The sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT) in cyanobacteria allows the incorporation of ammonium into carbon skeletons. In the cyanobacterium Synechocystis sp. PCC 6803, the activity of GS is modulated by the interaction with proteins, which include a 65‐residue‐long intrinsically disordered protein (IDP), the inactivating factor IF7. This interaction is regulated by the presence of charged residues in both IF7 and GS. To understand how charged amino acids can affect the binding of an IDP with its target and to provide clues on electrostatic interactions in disordered states of proteins, we measured the pK_a values of all IF7 acidic groups (Glu32, Glu36, Glu38, Asp40, Asp58, and Ser65, the backbone C‐terminus) at 100 mM NaCl concentration, by using NMR spectroscopy. We also obtained solution structures of IF7 through molecular dynamics simulation, validated them on the basis of previous experiments, and used them to obtain theoretical estimates of the pK_a values. Titration values for the two Asp and three Glu residues of IF7 were similar to those reported for random‐coil models, suggesting the lack of electrostatic interactions around these residues. Furthermore, our results suggest the presence of helical structure at the N‐terminus of the protein and of conformational changes at acidic pH values. The overall experimental and in silico findings suggest that local interactions and conformational equilibria do not play a role in determining the electrostatic features of the acidic residues of IF7.     

Microtubule-associated protein (MAP) 4 interacts with microtubules in an intrinsically disordered manner

We previously used nuclear magnetic resonance (NMR) to analyze the structure of a synthetic tricosapeptide corresponding to an active site of microtubule-associated protein 4 (MAP4). To further the structural analysis, we have constructed a minimal active domain fragment of MAP4, encompassing the entire active site, and obtained its NMR spectra. The secondary structure prediction using partially assigned NMR data suggested that the fragment is largely unfolded. Two other independent techniques also demonstrated its unfolded nature, indicating that MAP4 belongs to the class of intrinsically disordered proteins (IDPs). The NMR spectra of the fragment-microtubule mixture revealed that the fragment binds to the microtubule using multiple binding sites, apparently contradicting our previous quantitative studies. Given that MAP4 is intrinsically disordered, we propose a mechanism in which any one of the binding sites is active at a time, which is one of the typical interaction mechanisms proposed for IDPs.     

The intermembrane space domain of Tim23 is intrinsically disordered with a distinct binding region for presequences

Proteins targeted to the mitochondrial matrix are translocated through the outer and the inner mitochondrial membranes by two protein complexes, the translocase of the outer membrane (TOM) and one of the translocases of the inner membrane (TIM23). The protein Tim23, the core component of TIM23, consists of an N‐terminal, soluble domain in the intermembrane space (IMS) and a C‐terminal domain that forms the import pore across the inner membrane. Before translocation proceeds, precursor proteins are recognized by the N‐terminal domain of Tim23, Tim23N (residues 1–96). By using NMR spectroscopy, we show that Tim23N is a monomeric protein belonging to the family of intrinsically disordered proteins. Titrations of Tim23N with two presequences revealed a distinct binding region of Tim23N formed by residues 71–84. In a charge‐hydropathy plot containing all soluble domains of TOM and TIM23, Tim23N was found to be the only domain with more than 40 residues in the IMS that is predicted to be intrinsically disordered, suggesting that Tim23N might function as hub in the mitochondrial import machinery protein network.     

Interaction of the p53 DNA-binding domain with its n-terminal extension modulates the stability of the p53 tetramer

The tetrameric tumor suppressor p53 plays a pivotal role in the control of the cell cycle and provides a paradigm for an emerging class of oligomeric, multidomain proteins with structured and intrinsically disordered regions. Many of its biophysical and functional properties have been extrapolated from truncated variants, yet the exact structural and functional role of certain segments of the protein is unclear. We found from NMR and X-ray crystallography that the DNA-binding domain (DBD) of human p53, usually defined as residues 94-292, extends beyond these domain boundaries. Trp91, in the hinge region between the disordered proline-rich N-terminal domain and the DBD, folds back onto the latter and has a cation-π interaction with Arg174. These additional interactions increase the melting temperature of the DBD by up to 2 °C and inhibit aggregation of the p53 tetramer. They also modulate the dissociation of the p53 tetramer. The absence of the Trp91/Arg174 packing presumably allows nonnative DBD-DBD interactions that both nucleate aggregation and stabilize the interface. These data have important implications for studies of multidomain proteins in general, highlighting the fact that weak ordered-disordered domain interactions can modulate the properties of proteins of complex structure.     

Structural characterization of an intrinsically unfolded mini-HBX protein from hepatitis B virus

The hepatitis B virus x protein (HBX) is expressed in HBVinfected liver cells and can interact with a wide range of cellular proteins. In order to understand such promiscuous behavior of HBX we expressed a truncated mini-HBX protein (named Tr-HBX) (residues 18-142) with 5 Cys → Ser mutations and characterized its structural features using circular dichroism (CD) spectropolarimetry, NMR spectroscopy as well as bioinformatics tools for predicting disorder in intrinsically unstructured proteins (IUPs). The secondary structural content of Tr-HBX from CD data suggests that Tr-HBX is only partially folded. The protein disorder prediction by IUPred reveals that the unstructured region encompasses its N-terminal ～30 residues of Tr-HBX. A two-dimensional (1)H-(15)N HSQC NMR spectrum exhibits fewer number of resonances than expected, suggesting that Tr-HBX is a hybrid type IUP where its folded C-terminal half coexists with a disordered N-terminal region. Many IUPs are known to be capable of having promiscuous interactions with a multitude of target proteins. Therefore the intrinsically disordered nature of Tr-HBX revealed in this study provides a partial structural basis for the promiscuous structure-function behavior of HBX.     

Network representation of protein interactions—Experimental results

A graph theoretical analysis of nuclear magnetic resonance (NMR) data of six different protein interactions has been presented. The representation of the protein interaction data as a graph or network reveals that all of the studied interactions are based on a common functional concept. They all involve a single densely packed hub of functionally correlated residues that mediate the ligand binding events. This is found independent of the kind of protein (folded or unfolded) or ligand (protein, polymer or small molecule). Furthermore, the power of the graph analysis is demonstrated at the examples of the Calmodulin (CaM)/Calcium and the Cold Shock Protein A (CspA)/RNA interaction. The presented approach enables the precise determination of multiple binding sites for the respective ligand molecules.     

Purification and reconstitution of the connexin43 carboxyl terminus attached to the 4th transmembrane domain in detergent micelles

In recent years, reports have identified that many eukaryotic proteins contain disordered regions spanning greater than 30 consecutive residues in length. In particular, a number of these intrinsically disordered regions occur in the cytoplasmic segments of plasma membrane proteins. These intrinsically disordered regions play important roles in cell signaling events, as they are sites for protein–protein interactions and phosphorylation. Unfortunately, in many crystallographic studies of membrane proteins, these domains are removed because they hinder the crystallization process. Therefore, a purification procedure was developed to enable the biophysical and structural characterization of these intrinsically disordered regions while still associated with the lipid environment. The carboxyl terminal domain from the gap junction protein connexin43 attached to the 4th transmembrane domain (TM4-Cx43CT) was used as a model system (residues G178-I382). The purification was optimized for structural analysis by nuclear magnetic resonance (NMR) because this method is well suited for small membrane proteins and proteins that lack a well-structured three-dimensional fold. The TM4-Cx43CT was purified to homogeneity with a yield of 6 mg/L from C41(DE3) bacterial cells, reconstituted in the anionic detergent 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)], and analyzed by circular dichroism and NMR to demonstrate that the TM4-Cx43CT was properly folded into a functional conformation by its ability to form α-helical structure and associate with a known binding partner, the c-Src SH3 domain, respectively.     

Nucleoporins’ exclusive amino acid sequence features regulate their transient interaction with and selectivity of cargo complexes in the nuclear pore

Nucleocytoplasmic traffic of nucleic acids and proteins across the nuclear envelop via the nuclear pore complexes (NPCs) is vital for eukaryotic cells. NPCs screen transported macromolecules based on their morphology and surface chemistry. This selective nature of the NPC-mediated traffic is essential for regulating the fundamental functions of the nucleus, such as gene regulation, protein synthesis, and mechanotransduction. Despite the fundamental role of the NPC in cell and nuclear biology, the detailed mechanisms underlying how the NPC works have remained largely unknown. The critical components of NPCs enabling their selective barrier function are the natively unfolded phenylalanine- and glycine-rich proteins called “FG-nucleoporins” (FG Nups). These intrinsically disordered proteins are tethered to the inner wall of the NPC, and together form a highly dynamic polymeric meshwork whose physicochemical conformation has been the subject of intense debate. We observed that specific sequence features (called largest positive like-charge regions, or lpLCRs), characterized by extended subsequences that only possess positively charged amino acids, significantly affect the conformation of FG Nups inside the NPC. Here we investigate how the presence of lpLCRs affects the interactions between FG Nups and their interactions with the cargo complex. We combine coarse-grained molecular dynamics simulations with time-resolved force distribution analysis to disordered proteins to explore the behavior of the system. Our results suggest that the number of charged residues in the lpLCR domain directly governs the average distance between Phe residues and the intensity of interaction between them. As a result, the number of charged residues within lpLCR determines the balance between the hydrophobic interaction and the electrostatic repulsion and governs how dense and disordered the hydrophobic network formed by FG Nups is. Moreover, changing the number of charged residues in an lpLCR domain can interfere with ultrafast and transient interactions between FG Nups and the cargo complex.     

Combining prediction, computation and experiment for the characterization of protein disorder

Several computational and experimental methods exist for identifying disordered residues within proteins. Computational algorithms can now identify these disordered sequences and predict their occurrence within genomes with relatively high accuracy. Recent advances in NMR and mass spectroscopy permit faster and more detailed studies of disordered states at atomic resolutions. Combining prediction, computation and experimentation is proposed to accelerate and enhance the characterization of intrinsically disordered protein.     

Abundance of intrinsic disorder in protein associated with cardiovascular disease

Evidence that many protein regions and even entire proteins lacking stable tertiary and/or secondary structure in solution (i.e., intrinsically disordered proteins) might be involved in protein-protein interactions, regulation, recognition, and signal transduction is rapidly accumulating. These signaling proteins play a crucial role in the development of several pathological conditions, including cancer. To test a hypothesis that intrinsic disorder is also abundant in cardiovascular disease (CVD), a data set of 487 CVD-related proteins was extracted from SWISS-PROT. CVD-related proteins are depleted in major order-promoting residues (Trp, Phe, Tyr, Ile, and Val) and enriched in some disorder-promoting residues (Arg, Gln, Ser, Pro, and Glu). The application of a neural network predictor of natural disordered regions (PONDR VL-XT) together with cumulative distribution function (CDF) analysis, charge-hydropathy plot (CH plot) analysis, and alpha-helical molecular recognition feature (alpha-MoRF) indicator revealed that CVD-related proteins are enriched in intrinsic disorder. In fact, the percentage of proteins with 30 or more consecutive residues predicted by PONDR VL-XT to be disordered was 57 +/- 4% for CVD-associated proteins. This value is close that described earlier for signaling proteins (66 +/- 6%) and is significantly larger than the content of intrinsic disorder in eukaryotic proteins from SWISS-PROT (47 +/- 4%) and in nonhomologous protein segments with a well-defined three-dimensional structure (13 +/- 4%). Furthermore, CDF and CH-plot analyses revealed that 120 and 36 CVD-related proteins, respectively, are wholly disordered. This high level of intrinsic disorder could be important for the function of CVD-related proteins and for the control and regulation of processes associated with cardiovascular disease. In agreement with this hypothesis, 198 alpha-MoRFs were predicted in 101 proteins from the CVD data set. A comparison of disorder predictions with the experimental structural and functional data for a subset of the CVD-associated proteins indicated good agreement between predictions and observations. Thus, our data suggest that intrinsically disordered proteins might play key roles in cardiovascular disease.     

Flexible nets. The roles of intrinsic disorder in protein interaction networks

Proteins participate in complex sets of interactions that represent the mechanistic foundation for much of the physiology and function of the cell. These protein-protein interactions are organized into exquisitely complex networks. The architecture of protein-protein interaction networks was recently proposed to be scale-free, with most of the proteins having only one or two connections but with relatively fewer 'hubs' possessing tens, hundreds or more links. The high level of hub connectivity must somehow be reflected in protein structure. What structural quality of hub proteins enables them to interact with large numbers of diverse targets? One possibility would be to employ binding regions that have the ability to bind multiple, structurally diverse partners. This trait can be imparted by the incorporation of intrinsic disorder in one or both partners. To illustrate the value of such contributions, this review examines the roles of intrinsic disorder in protein network architecture. We show that there are three general ways that intrinsic disorder can contribute: First, intrinsic disorder can serve as the structural basis for hub protein promiscuity; secondly, intrinsically disordered proteins can bind to structured hub proteins; and thirdly, intrinsic disorder can provide flexible linkers between functional domains with the linkers enabling mechanisms that facilitate binding diversity. An important research direction will be to determine what fraction of protein-protein interaction in regulatory networks relies on intrinsic disorder.     

Interaction between Intrinsically Disordered Proteins Frequently Occurs in a Human Protein-Protein Interaction Network

Intrinsic protein disorder is a widespread phenomenon characterised by a lack of stable three-dimensional structures and is considered to play an important role in protein-protein interactions (PPIs). This study examined the genome-wide preference of disorder in PPIs by using exhaustive disorder prediction in human PPIs. We categorised the PPIs into three types (interaction between disordered proteins, interaction between structured proteins, and interaction between a disordered protein and a structured protein) with regard to the flexibility of molecular recognition and compared these three interaction types in an existing human PPI network with those in a randomised network. Although the structured regions were expected to become the identifiers for binding recognition, this comparative analysis revealed unexpected results. The occurrence of interactions between disordered proteins was significantly frequent, and that between a disordered protein and a structured protein was significantly infrequent. We found that this propensity was much stronger in interactions between nonhub proteins. We also analysed the interaction types from a functional standpoint by using GO, which revealed that the interaction between disordered proteins frequently occurred in cellular processes, regulation, and metabolic processes. The number of interactions, especially in metabolic processes between disordered proteins, was 1.8 times as large as that in the randomised network. Another analysis conducted by using KEGG pathways provided results where several signaling pathways and disease-related pathways included many interactions between disordered proteins. All of these analyses suggest that human PPIs preferably occur between disordered proteins and that the flexibility of the interacting protein pairs may play an important role in human PPI networks.     

Close encounters of the third kind: disordered domains and the interactions of proteins

Protein–protein interactions are thought to be mediated by domains, which are autonomous folding units of proteins. Recently, a second type of interaction has been suggested, mediated by short segments termed linear motifs, which are related to recognition elements of intrinsically disordered regions. Here, we propose a third kind of protein–protein recognition mechanism, mediated by disordered regions longer than 20–30 residues. Bioinformatics predictions and well‐characterized examples, such as the kinase‐inhibitory domain of Cdk inhibitors and the Wiskott–Aldrich syndrome protein (WASP)‐homology domain 2 of actin‐binding proteins, show that these disordered regions conform to the definition of domains rather than motifs, i.e., they represent functional, evolutionary, and structural units. Their functions are distinct from those of short motifs and ordered domains, and establish a third kind of interaction principle. With these points, we argue that these long disordered regions should be recognized as a distinct class of biologically functional protein domains.     

Binding of intrinsically disordered proteins is not necessarily accompanied by a structural transition to a folded form

There are a large number of protein domains and even entire proteins, lacking ordered structure under physiological conditions. Intriguingly, a highly flexible, random coil-like conformation is the native and functional state for many proteins known to be involved in cell signaling. An example is a key component of immune signaling, the cytoplasmic region of the T cell receptor zeta subunit. This domain exhibits specific dimerization that is distinct from non-specific aggregation behavior seen in many systems. In this work, we use diffusion and chemical shift mapping NMR data to show that the protein does not undergo a transition between disordered and ordered states upon dimerization. This finding opposes the generally accepted view on the behavior of intrinsically disordered proteins, provides evidence for the existence of specific dimerization interactions for intrinsically disordered protein species and opens a new line of research in this new and quickly developing field.     

Choice of Force Field for Proteins Containing Structured and Intrinsically Disordered Regions

Biomolecular force fields optimized for globular proteins fail to properly reproduce properties of intrinsically disordered proteins. In particular, parameters of the water model need to be modified to improve applicability of the force fields to both ordered and disordered proteins. Here, we compared performance of force fields recommended for intrinsically disordered proteins in molecular dynamics simulations of three proteins differing in the content of ordered and disordered regions (two proteins consisting of a well-structured domain and of a disordered region with and without a transient helical motif and one disordered protein containing a region of increased helical propensity). The obtained molecular dynamics trajectories were used to predict measurable parameters, including radii of gyration of the proteins and chemical shifts, residual dipolar couplings, paramagnetic relaxation enhancement, and NMR relaxation data of their individual residues. The predicted quantities were compared with experimental data obtained within this study or published previously. The results showed that the NMR relaxation parameters, rarely used for benchmarking, are particularly sensitive to the choice of force-field parameters, especially those defining the water model. Interestingly, the TIP3P water model, leading to an artificial structural collapse, also resulted in unrealistic relaxation properties. The TIP4P-D water model, combined with three biomolecular force-field parameters for the protein part, significantly improved reliability of the simulations. Additional analysis revealed only one particular force field capable of retaining the transient helical motif observed in NMR experiments. The benchmarking protocol used in our study, being more sensitive to imperfections than the commonly used tests, is well suited to evaluate the performance of newly developed force fields.     

On the Importance of Polar Interactions for Complexes Containing Intrinsically Disordered Proteins

There is a growing recognition for the importance of proteins with large intrinsically disordered (ID) segments in cell signaling and regulation. ID segments in these proteins often harbor regions that mediate molecular recognition. Coupled folding and binding of the recognition regions has been proposed to confer high specificity to interactions involving ID segments. However, researchers recently questioned the origin of the interaction specificity of ID proteins because of the overrepresentation of hydrophobic residues in their interaction interfaces. Here, we focused on the role of polar and charged residues in interactions mediated by ID segments. Making use of the extended nature of most ID segments when in complex with globular proteins, we first identified large numbers of complexes between globular proteins and ID segments by using radius-of-gyration-based selection criteria. Consistent with previous studies, we found the interfaces of these complexes to be enriched in hydrophobic residues, and that these residues contribute significantly to the stability of the interaction interface. However, our analyses also show that polar interactions play a larger role in these complexes than in structured protein complexes. Computational alanine scanning and salt-bridge analysis indicate that interfaces in ID complexes are highly complementary with respect to electrostatics, more so than interfaces of globular proteins. Follow-up calculations of the electrostatic contributions to the free energy of binding uncovered significantly stronger Coulombic interactions in complexes harbouring ID segments than in structured protein complexes. However, they are counter-balanced by even higher polar-desolvation penalties. We propose that polar interactions are a key contributing factor to the observed high specificity of ID segment-mediated interactions.