Interactions via intrinsically disordered regions: what kind of motifs?

Proteins containing intrinsically disordered (ID) regions are widespread in eukaryotic organisms and are mostly utilized in regulatory processes. ID regions can mediate binary interactions of proteins or promote organization of large assemblies. Post-translational modifications of ID regions often serve as decision points in signaling pathways. Why Nature distinguished ID proteins in molecular recognition functions? In a simple view, binding of ID regions is accompanied by a large entropic penalty as compared to folded proteins. Even in complexes however, ID regions can preserve their conformational freedom, thereby recruit further partners and perform various functions. What sort of benefits ID regions offer for molecular interactions and which properties are exploited in the corresponding complexes? Here, we review models explaining the recognition mechanisms of ID proteins. Motif-based interactions are central to all proposed scenarios, including prestructured elements, anchoring sites and linear motifs. We aim to extract consensus features of the models, which could be used to predict ID-binding sites for a variety of partners.     

Are structural proteins in insect cuticles dominated by intrinsically disordered regions?

Fifty years ago it was concluded that the highly elastic cuticular protein, resilin, is devoid of secondary structure and that the peptide chains are randomly coiled and easily and reversibly deformed. These properties indicate that resilin is an intrinsically disordered protein and suggest that also other cuticular proteins may contain disordered regions. Amino acid sequences are now available for cuticular proteins from many insect species, and several programs have been developed to predict the probability for a given protein to contain disordered regions.The present paper describes the results obtained when the predictors are applied to various types of cuticular proteins from several insects. The results suggest that most cuticular proteins contain shorter or longer disordered regions, and the possible functions for such regions are briefly discussed.     

Predicting molecular properties of α-synuclein using force fields for intrinsically disordered proteins

Independent force field validation is an essential practice to keep track of developments and for performing meaningful Molecular Dynamics simulations. In this work, atomistic force fields for intrinsically disordered proteins (IDP) are tested by simulating the archetypical IDP α-synuclein in solution for 2.5 μs. Four combinations of protein and water force fields were tested: ff19SB/OPC , ff19SB/TIP4P-D , ff03CMAP/TIP4P-D , and a99SB-disp/TIP4P-disp , with four independent repeat simulations for each combination. We compare our simulations to the results of a 73 μs simulation using the a99SB-disp/TIP4P-disp combination, provided by D. E. Shaw Research. From the trajectories, we predict a range of experimental observations of α-synuclein and compare them to literature data. This includes protein radius of gyration and hydration, intramolecular distances, NMR chemical shifts, and ³J-couplings. Both ff19SB/TIP4P-D and a99SB-disp/TIP4P-disp produce extended conformational ensembles of α-synuclein that agree well with experimental radius of gyration and intramolecular distances while a99SB-disp/TIP4P-disp reproduces a balanced α-synuclein secondary structure content. It was found that ff19SB/OPC and ff03CMAP/TIP4P-D produce overly compact conformational ensembles and show discrepancies in the secondary structure content compared to the experimental data.     

Is there nascent structure in the intrinsically disordered region of troponin I?

In striated muscle, the binding of calcium to troponin C (TnC) results in the removal of the C‐terminal region of the inhibitory protein troponin I (TnI) from actin. While structural studies of the muscle system have been successful in determining the overall organization of most of the components involved in force generation at the atomic level, the structure and dynamics of the C‐terminal region of TnI remains controversial. This domain of TnI is highly flexible, and it has been proposed that this intrinsically disordered region (IDR) regulates contraction via a “fly‐casting” mechanism. Different structures have been presented for this region using different methodologies: a single α‐helix, a “mobile domain” containing a small β‐sheet, an unstructured region, and a two helix segment. To investigate whether this IDR has in fact any nascent structure, we have constructed a skeletal TnC‐TnI chimera that contains the N‐domain of TnC (1–90), a short linker (GGAGG), and the C‐terminal region of TnI (97–182) and have acquired ¹⁵N NMR relaxation data for this chimera. We compare the experimental relaxation parameters with those calculated from molecular dynamic simulations using four models based upon the structural studies. Our experimental results suggest that the C‐terminal region of TnI does not contain any defined secondary structure, supporting the “fly‐casting” mechanism. We interpret the presence of a “plateau” in the ¹⁵N NMR relaxation data as being an intrinsic property of IDRs. We also identified a more rigid adjacent region of TnI that has implications for muscle performance under ischemic conditions. Proteins 2011. © 2011 Wiley‐Liss, Inc.     

Cellulose synthase ‘class specific regions’ are intrinsically disordered and functionally undifferentiated

Cellulose synthases(CESAs) are glycosyltransferases that catalyze formation of cellulose microfibrils in plant cell walls.Seed plant CESA isoforms cluster in six phylogenetic clades, whose non-interchangeable members play distinct roles within cellulose synthesis complexes(CSCs).A ‘class specific region’(CSR), with higher sequence similarity within versus between functional CESA classes, has been suggested to contribute to specific activities or interactions of different isoforms.We investigated CESA isoform specificity in the moss,Physcomitrella patens(Hedw.) B.S.G.to gain evolutionary insights into CESA structure/function relationships.Like seed plants, P.patens has oligomeric rosette-type CSCs, but the Pp CESAs diverged independently and form a separate CESA clade.We showed that P.patens has two functionally distinct CESAs classes, based on the ability to complement the gametophore-negative phenotype of a ppcesa5 knockout line.Thus, non-interchangeable CESA classes evolved separately in mosses and seed plants.However, testing of chimeric moss CESA genes for complementation demonstrated that functional classspecificity is not determined by the CSR.Sequence analysis and computational modeling showed that the CSR is intrinsically disordered and contains predicted molecular recognition features, consistent with a possible role in CESA oligomerization and explaining the evolution of class-specific sequences without selection for class-specific function.     

Sedimentation velocity of intrinsically disordered proteins: what information can we actually obtain?

Intrinsically disordered proteins are a challenge to characterise structurally because of their innate flexibility. Hydrodynamic methods such as sedimentation velocity analytical ultracentrifugation have been proposed as methods for their characterisation. By examining in detail this assumption we show that although velocity measurements do yield information on gross conformation, the information is restricted to only the weight averaged sedimentation and diffusion coefficients of the conformational ensemble.     

The N-terminus of the intrinsically disordered protein α-synuclein triggers membrane binding and helix folding

Alpha-synuclein (αS) is a 140-amino-acid protein that is involved in a number of neurodegenerative diseases. In Parkinson's disease, the protein is typically encountered in intracellular, high-molecular-weight aggregates. Although αS is abundant in the presynaptic terminals of the central nervous system, its physiological function is still unknown. There is strong evidence for the membrane affinity of the protein. One hypothesis is that lipid-induced binding and helix folding may modulate the fusion of synaptic vesicles with the presynaptic membrane and the ensuing transmitter release. Here we show that membrane recognition of the N-terminus is essential for the cooperative formation of helical domains in the protein. We used circular dichroism spectroscopy and isothermal titration calorimetry to investigate synthetic peptide fragments from different domains of the full-length αS protein. Site-specific truncation and partial cleavage of the full-length protein were employed to further characterize the structural motifs responsible for helix formation and lipid-protein interaction. Unilamellar vesicles of varying net charge and lipid compositions undergoing lateral phase separation or chain melting phase transitions in the vicinity of physiological temperatures served as model membranes. The results suggest that the membrane-induced helical folding of the first 25 residues may be driven simultaneously by electrostatic attraction and by a change in lipid ordering. Our findings highlight the significance of the αS N-terminus for folding nucleation, and provide a framework for elucidating the role of lipid-induced conformational transitions of the protein within its intracellular milieu.     

Segmental conformational disorder and dynamics in the intrinsically disordered protein α-synuclein and its chain length dependence

Conformational ensembles of fully disordered natural polypeptides represent the starting point of protein refolding initiated by transfer to folding conditions. Thus, understanding the transient properties and dimensions of such peptides under folding conditions is a necessary step in the understanding of their subsequent folding behavior. Such ensembles can also undergo alternative folding and form amyloid structures, which are involved in many neurological degenerative diseases. Here, we performed a structural study of this initial state using time-resolved fluorescence resonance energy transfer analysis of a series of eight partially overlapping double-labeled chain segments of the N-terminal and NAC domains of the α-synuclein molecule. The distributions of end-to-end distance and segmental intramolecular diffusion coefficients were simultaneously determined for eight labeled chain segments. We used the coefficient of variation, C_v, as a measure of the conformational heterogeneity (i.e., structural disorder). With the exception of two segments, the C_vs were characteristic of a fully disordered state of the chain. Subtle deviations from this behavior at the segment labeled in the NAC domain and the segment at the N termini reflected subtle conformational bias that might be related to the initiation of transition to amyloid aggregates. The chain length dependence of the mean segmental end-to-end distance followed a power law as predicted by Flory, but the dependence was steeper than previously predicted, probably due to the contribution of the excluded volume effect, which is more dominant for shorter-chain segments. The observed intramolecular diffusion coefficients (< 10 to ∼ 25 ?²/ns) are only an order of magnitude lower than the common diffusion coefficients of low molecular weight probes. This diffusion coefficient increased with chain length, probably due to the cumulative contributions of minor bond rotations along the chain. These results gave us a reference both for characteristics of a natural unfolded polypeptide at the moment of initiation of folding and for detection of possible initiation sites of the amyloid transition.     

Temperature-dependent structural changes in intrinsically disordered proteins: Formation of α-helices or loss of polyproline II?

Structural characterization of intrinsically disordered proteins (IDPs) is mandatory for deciphering their potential unique physical and biological properties. A large number of circular dichroism (CD) studies have demonstrated that a structural change takes place in IDPs with increasing temperature, which most likely reflects formation of transient α-helices or loss of polyproline II (PPII) content. Using three IDPs, ACTR, NHE1, and Spd1, we show that the temperature-induced structural change is common among IDPs and is accompanied by a contraction of the conformational ensemble. This phenomenon was explored at residue resolution by multidimensional NMR spectroscopy. Intrinsic chemical shift referencing allowed us to identify regions of transiently formed helices and their temperature-dependent changes in helicity. All helical regions were found to lose rather than gain helical structures with increasing temperature, and accordingly these were not responsible for the change in the CD spectra. In contrast, the nonhelical regions exhibited a general temperature-dependent structural change that was independent of long-range interactions. The temperature-dependent CD spectroscopic signature of IDPs that has been amply documented can be rationalized to represent redistribution of the statistical coil involving a general loss of PPII conformations.     

Effect of polyols on the structure and aggregation of recombinant human γ-Synuclein,an intrinsically disordered protein

Polyol osmolytes accumulated in cells under stress are known to promote stability in globular proteins with respect to their increasing hydroxyl groups but their effect on the structure, stability and aggregation of intrinsically disordered proteins (IDPs) is still elusive. The lack of a natively folded structure in intrinsically disordered proteins under physiological conditions results in their aggregation and fibrillation that gives rise to a number of diseases. We have investigated the effect of a series of polyols, ethylene glycol (EG), glycerol, erythritol, xylitol and sorbitol on the fibrillation pathway of recombinant human γ-Synuclein, used as a model, for an IDP known to form fibrils that play a role in neurodegeneration and cancer. With an increase in the number of –OH groups in polyols except EG, we observe a decrease in lag time for fibrillation at equimolar concentrations, suggesting stronger preferential exclusion of polyols that promotes γ-Syn self-association and oligomerization. The polyols act early during nucleation and their diverse effect on the rate of fibrillation suggests the role of favourable solvent-side chain interactions. With increasing –OH group, polyols stabilize the natively unfolded conformation of γ-Syn under non-fibrillating conditions and delay the structural transition to characteristic β-sheet structure by forming an α-helical intermediate during fibrillation. The results, overall suggest that the effect of osmolytes on IDPs is much more complex than their effect on globular protein stability and aggregation and a fine balance between the dominant unfavourable osmolyte-peptide backbone and favourable osmolyte-charged side chain interactions would govern their stability and aggregation properties.     

N-terminal acetylation of α-synuclein induces increased transient helical propensity and decreased aggregation rates in the intrinsically disordered monomer

The conformational properties of soluble α-synuclein, the primary protein found in patients with Parkinson's disease, are thought to play a key role in the structural transition to amyloid fibrils. In this work, we report that recombinant 100% N-terminal acetylated α-synuclein purified under mild physiological conditions presents as a primarily monomeric protein, and that the N-terminal acetyl group affects the transient secondary structure and fibril assembly rates of the protein. Residue-specific NMR chemical shift analysis indicates substantial increase in transient helical propensity in the first 9 N-terminal residues, as well as smaller long-range changes in residues 28-31, 43-46, and 50-66: regions in which the three familial mutations currently known to be causative of early onset disease are found. In addition, we show that the N-terminal acetylated protein forms fibrils that are morphologically similar to those formed from nonacetylated α-synuclein, but that their growth rates are slower. Our results highlight that N-terminal acetylation does not form significant numbers of dimers, tetramers, or higher molecular weight species, but does alter the conformational distributions of monomeric α-synuclein species in regions known to be important in metal binding, in association with membranes, and in regions known to affect fibril formation rates.     

Cyclization of the intrinsically disordered α1S dihydropyridine receptor II-III loop enhances secondary structure and in vitro function

A key component of excitation contraction (EC) coupling in skeletal muscle is the cytoplasmic linker (II-III loop) between the second and third transmembrane repeats of the α(1S) subunit of the dihydropyridine receptor (DHPR). The II-III loop has been previously examined in vitro using a linear II-III loop with unrestrained N- and C-terminal ends. To better reproduce the loop structure in its native environment (tethered to the DHPR transmembrane domains), we have joined the N and C termini using intein-mediated technology. Circular dichroism and NMR spectroscopy revealed a structural shift in the cyclized loop toward a protein with increased α-helical and β-strand structure in a region of the loop implicated in its in vitro function and also in a critical region for EC coupling. The affinity of binding of the II-III loop binding to the SPRY2 domain of the skeletal ryanodine receptor (RyR1) increased 4-fold, and its ability to activate RyR1 channels in lipid bilayers was enhanced 3-fold by cyclization. These functional changes were predicted consequences of the structural enhancement. We suggest that tethering the N and C termini stabilized secondary structural elements in the DHPR II-III loop and may reflect structural and dynamic characteristics of the loop that are inherent in EC coupling.     

A maximum entropy approach to the study of residue-specific backbone angle distributions in α-synuclein,an intrinsically disordered protein

α-Synuclein is an intrinsically disordered protein of 140 residues that switches to an α-helical conformation upon binding phospholipid membranes. We characterize its residue-specific backbone structure in free solution with a novel maximum entropy procedure that integrates an extensive set of NMR data. These data include intraresidue and sequential H^N–H^α and H^N–H^N NOEs, values for ³J_HNHα, ¹J_HαCα, ²J_CαN, and ¹J_CαN, as well as chemical shifts of ¹⁵N, ¹³C^α, and ¹³C′ nuclei, which are sensitive to backbone torsion angles. Distributions of these torsion angles were identified that yield best agreement to the experimental data, while using an entropy term to minimize the deviation from statistical distributions seen in a large protein coil library. Results indicate that although at the individual residue level considerable deviations from the coil library distribution are seen, on average the fitted distributions agree fairly well with this library, yielding a moderate population (20–30%) of the PP_II region and a somewhat higher population of the potentially aggregation-prone β region (20–40%) than seen in the database. A generally lower population of the α_R region (10–20%) is found. Analysis of ¹H–¹H NOE data required consideration of the considerable backbone diffusion anisotropy of a disordered protein.     

Deuterium isotope shifts for backbone 1H, 15N and 13C nuclei in intrinsically disordered protein α-synuclein

Intrinsically disordered proteins (IDPs) are abundant in nature and characterization of their potential structural propensities remains a widely pursued but challenging task. Analysis of NMR secondary chemical shifts plays an important role in such studies, but the output of such analyses depends on the accuracy of reference random coil chemical shifts. Although uniform perdeuteration of IDPs can dramatically increase spectral resolution, a feature particularly important for the poorly dispersed IDP spectra, the impact of deuterium isotope shifts on random coil values has not yet been fully characterized. Very precise ²H isotope shift measurements for ¹³C^??, ¹³C^??, ¹³C??, ¹⁵N, and ¹H^N have been obtained by using a mixed sample of protonated and uniformly perdeuterated ??-synuclein, a protein with chemical shifts exceptionally close to random coil values. Decomposition of these isotope shifts into one-bond, two-bond and three-bond effects as well as intra- and sequential residue contributions shows that such an analysis, which ignores conformational dependence, is meaningful but does not fully describe the total isotope shift to within the precision of the measurements. Random coil ²H isotope shifts provide an important starting point for analysis of such shifts in structural terms in folded proteins, where they are known to depend strongly on local geometry.     

Is unphosphorylated Rex,as multifunctional protein of HTLV-1, a fully intrinsically disordered protein? An in silico study

Intracellularlocation of a viral unspliced mRNA in host cell is a crucial factor for normal life of the virus. Rex is a neucleo-cytoplasmic shuffling protein of Human T-cell Leukemia Virus-1(HTLV-1)which has important role in active transport of cargo-containing RNA from nucleus to cytoplasm. Therefore, it plays a crucial role in the disease development by the virus. In spite of its importance, the 3d-structurephosphorylated and unphosphorylated of this protein has not been determined. In this study, first we predicted whether Rex protein is an ordered or disordered protein. In second step protein 3Dstructure of Rex was obtained. The content of disorder-promoting amino acids, flexibility, hydrophobicity, short linear motifs (SLiMs) and protein binding regions and probability of Rex crystallization were calculated by various In Silico methods. The3D models of Rex protein were obtained by various In Silico methods, such as homology modeling, threading and ab initio, including; I-TASSER, LOMETS, SPARSKS, ROBBETA and QUARK servers. By comparing and analyzing Qmean, z-scores and energy levels of selected models, the best structures with highest favored region in Ramachandran plot (higher than 90%) was refined with MODREFINER software. In silico analysis of Rex physicochemical properties and also predicted SLiMs and binding regions sites confirms that unphosphorylated Rex protein in HTLV-1 as Rev protin in HIV is wholly disordered protein belongs to the class of intrinsically disordered proteins with extended disorder (native coils, native pre-molten globules).     

Direct correlation of consecutive C′–N groups in proteins: a method for the assignment of intrinsically disordered proteins

Two novel 3D ¹³C-detected experiments, hNcocaNCO and hnCOcaNCO, are proposed to facilitate the resonance assignment of intrinsically disordered proteins. The experiments correlate the ¹⁵N and ¹³C′ chemical shifts of two consecutive amide moieties without involving other nuclei, thus taking advantage of the good dispersion shown by the ¹⁵N–¹³C′ correlations, even for proteins that lack a well defined tertiary structure. The new pulse sequences were successfully tested using Nupr1, an intrinsically disordered protein of 93 residues.     

Functional dissection of an intrinsically disordered protein: Understanding the roles of different domains of Knr4 protein in protein�Cprotein interactions

Knr4, recently characterized as an intrinsically disordered Saccharomyces cerevisiae protein, participates in cell wall formation and cell cycle regulation. It is constituted of a functional central globular core flanked by a poorly structured N‐terminal and large natively unfolded C‐terminal domains. Up to now, about 30 different proteins have been reported to physically interact with Knr4. Here, we used an in vivo two‐hybrid system approach and an in vitro surface plasmon resonance (BIAcore) technique to compare the interaction level of different Knr4 deletion variants with given protein partners. We demonstrate the indispensability of the N‐terminal domain of Knr4 for the interactions. On the other hand, presence of the unstructured C‐terminal domain has a negative effect on the interaction strength. In protein interactions networks, the most highly connected proteins or “hubs” are significantly enriched in unstructured regions, and among them the transient hub proteins contain the largest and most highly flexible regions. The results presented here of our analysis of Knr4 protein suggest that these large disordered regions are not always involved in promoting the protein–protein interactions of hub proteins, but in some cases, might rather inhibit them. We propose that this type of regions could prevent unspecific protein interactions, or ensure the correct timing of occurrence of transient interactions, which may be of crucial importance for different signaling and regulation processes.