Hydrodynamic Radii of Intrinsically Disordered Proteins Determined from Experimental Polyproline II Propensities

The properties of disordered proteins are thought to depend on intrinsic conformational propensities for polyproline II (PP _II) structure. While intrinsic PP _II propensities have been measured for the common biological amino acids in short peptides, the ability of these experimentally determined propensities to quantitatively reproduce structural behavior in intrinsically disordered proteins (IDPs) has not been established. Presented here are results from molecular simulations of disordered proteins showing that the hydrodynamic radius (R _h) can be predicted from experimental PP _II propensities with good agreement, even when charge-based considerations are omitted. The simulations demonstrate that R _h and chain propensity for PP _II structure are linked via a simple power-law scaling relationship, which was tested using the experimental R _h of 22 IDPs covering a wide range of peptide lengths, net charge, and sequence composition. Charge effects on R _h were found to be generally weak when compared to PP _II effects on R _h. Results from this study indicate that the hydrodynamic dimensions of IDPs are evidence of considerable sequence-dependent backbone propensities for PP _II structure that qualitatively, if not quantitatively, match conformational propensities measured in peptides.     

Temperature effects on the hydrodynamic radius of the intrinsically disordered N‐terminal region of the p53 protein

Intrinsically disordered proteins (IDPs) are often characterized in terms of the hydrodynamic radius, R_h. The R_h of IDPs are known to depend on fractional proline content and net charge, where increased numbers of proline residues and increased net charge cause larger R_h. Though sequence and charge effects on the R_h of IDPs have been studied, the temperature sensitivity has been noted only briefly. Reported here are R_h measurements in the temperature range of 5–75°C for the intrinsically disordered N‐terminal region of the p53 protein, p53(1–93). Of note, the R_h of this protein fragment was highly sensitive to temperature, decreasing from 35 Å at 5°C to 26 Å at 75°C. Computer generated simulations of conformationally dynamic and disordered polypeptide chains were performed to provide a hypothesis for the heat‐induced compaction of p53(1–93) structure, which was opposite to the heat‐induced increase in R_h observed for a model folded protein. The simulations demonstrated that heat caused R_h to trend toward statistical coil values for both proteins, indicating that the effects of heat on p53(1–93) structure could be interpreted as thermal denaturation. The simulation data also predicted that proline content contributed minimally to the native R_h of p53(1–93), which was confirmed by measuring R_h for a substitution variant that had all 22 proline residues changed for glycine. Proteins 2014; 82:668–678. © 2013 Wiley Periodicals, Inc.     

固有无序蛋白与蛋白质相互作用位点残基特征分析

本文对固有无序蛋白(IDPs)与其他蛋白质相互作用位点残基特征进行了研究.首先在数据库中选出满足条件的109条IDPs蛋白质链及与其他配体蛋白形成的299个IDPs-蛋白质复合物,然后提取复合物中作为相互作用位点的IDPs-蛋白质残基.这109条IDPs链中共含有50 031个氨基酸残基,其中处于作用位点的残基有4 822个.通过分析发现,20种氨基酸在形成IDPs-蛋白质相互作用位点残基时具有不同的倾向性,根据形成作用位点残基的倾向性,20种氨基酸可分成三大类:倾向型氨基酸(ILE、LEU、ARG、PHE、TYR、MET、TRP)、中间型氨基酸(GLN、GLU、THR、LYS、VAL、ASP、HIS)、非倾向型氨基酸(PRO、SER、GLY、ALA、ASN、CYS).研究结果还进一步表明,不同氨基酸在有序区域与无序区域形成IDPs-蛋白质作用位点残基的倾向性不同.其中,氨基酸TRP、LEU、ILE、CYS在有序和无序区域形成作用位点残基的差异性尤为明显,而氨基酸GLU、PHE、HIS、ALA则基本没有多大差别.对IDPs-蛋白质相互作用位点残基理化特征进行分析发现:疏水性强、侧链净电荷量较少、极性较小、溶剂可及性表面积较大、侧链体积较大、极化率较大的氨基酸比较倾向于形成作用位点残基.主成分分析结果显示,残基的极化率、侧链体积和溶剂可及表面积对作用位点残基影响最大.     

Modulation of p53 N-terminal transactivation domain 2 conformation ensemble and kinetics by phosphorylation

Abstract

Phosphorylation of protein is critical for various cell processes, which preferentially happens in intrinsically disordered proteins (IDPs). How phosphorylation modulates structural ensemble of disordered peptide remains largely unexplored. Here, using replica exchange molecular dynamics (REMD) and Markov state model (MSM), the conformational distribution and kinetics of p53 N-terminal transactivation domain (TAD) 2 as well as its dual-site phosphorylated form (pSer46, pThr55) were simulated. It reveals that the dual phosphorylation does not change overall size and secondary structure element fraction, while a change in the distribution of hydrogen bonds induces slightly more pre-existing bound helical conformations. MSM analysis indicates that the dual phosphorylation accelerates conformation exchange between disordered and order-like states in target-binding region. It suggests that p53 TAD2 after phosphorylation would be more apt to bind to both the human p62 pleckstrin homology (PH) domain and the yeast tfb1?PH domain through different binding mechanism, where experimentally it exhibits an extended and α-helix conformation, respectively, with increased binding strength in both complexes. Our study implies except binding interface, both conformation ensemble and kinetics should be considered for the effects of phosphorylation on IDPs. Abbreviations IDPs intrinsically disordered proteins

REMD replica exchange molecular dynamics

MSM Markov state model

TAD transactivation domain

PH pleckstrin homology

PRR proline-rich region

DBD DNA-binding domain

TET Tetramerization domain

REG regulatory domain

MD molecular dynamics

PME particle-mesh Ewald

TICA time-lagged independent component analysis

CK Chapman–Kolmogorov

GMRQ generalized matrix Rayleigh quotient

SARW self-avoiding random walk

KID kinase-inducible domain

MFPT mean first passage time

DSSP definition of secondary structure of proteins

RMSD root mean square deviation

R_g radius of gyration

R_ee end to end distance

Communicated by Ramaswamy H. Sarma     

Targeting intrinsically disordered proteins in neurodegenerative and protein dysfunction diseases: another illustration of the D2 concept

Many biologically active proteins, which are usually called intrinsically disordered or natively unfolded proteins, lack stable tertiary and/or secondary structure under physiological conditions in vitro. Their functions complement the functional repertoire of ordered proteins, with intrinsically disordered proteins (IDPs) often being involved in regulation, signaling and control. Their amino acid sequences and compositions are very different from those of ordered proteins, making reliable identification of IDPs possible at the proteome level. IDPs are highly abundant in various human diseases, including neurodegeneration and other protein dysfunction maladies and, therefore, represent attractive novel drug targets. Some of the aspects of IDPs, as well as their roles in neurodegeneration and protein dysfunction diseases, are discussed in this article, together with the peculiarities of IDPs as potential drug targets.     

Characterization of the disordered-to-α-helical transition of IA₃ by SDSL-EPR spectroscopy

Electron paramagnetic resonance (EPR) spectroscopy coupled with site-directed spin labeling (SDSL) is a valuable tool for characterizing the mobility and conformational changes of proteins but has seldom been applied to intrinsically disordered proteins (IDPs). Here, IA₃ is used as a model system demonstrating SDSL-EPR characterization of conformational changes in small IDP systems. IA₃ has 68 amino acids, is unstructured in solution, and becomes α-helical upon addition of the secondary structural stabilizer 2,2,2-trifluoroethanol (TFE). Two single cysteine substitutions, one in the N-terminus (S14C) and one in the C-terminus (N58C), were generated and labeled with three different nitroxide spin labels. The resultant EPR line shapes of each of the labels were compared and each reported changes in mobility upon addition of TFE. Specifically, the spectral line shape parameters h₍₊₁₎/h₍₀₎, the local tumbling volume (V_L), and the percent change of the h₍₋₁₎ intensity were utilized to quantitatively monitor TFE-induced conformational changes. The values of h_(+1)/h₍₀₎ as a function of TFE titration varied in a sigmoidal manner and were fit to a two-state Boltzmann model that provided values for the midpoint of the transition, thus, reporting on the global conformational change of IA₃. The other parameters provide site-specific information and show that S14C-SL undergoes a conformational change resulting in more restricted motion than N58C-SL, which is consistent with previously published results obtained by studies using NMR and circular dichroism spectroscopy indicating a higher degree of α-helical propensity of the N-terminal segment of IA₃. Overall, the results provide a framework for data analyzes that can be used to study induced unstructured-to-helical conformations in IDPs by SDSL.     

How multisite phosphorylation impacts the conformations of intrinsically disordered proteins

Phosphorylation of intrinsically disordered proteins (IDPs) can produce changes in structural and dynamical properties and thereby mediate critical biological functions. How phosphorylation effects intrinsically disordered proteins has been studied for an increasing number of IDPs, but a systematic understanding is still lacking. Here, we compare the collapse propensity of four disordered proteins, Ash1, the C-terminal domain of RNA polymerase (CTD2’), the cytosolic domain of E-Cadherin, and a fragment of the p130Cas, in unphosphorylated and phosphorylated forms using extensive all-atom molecular dynamics (MD) simulations. We find all proteins to show V-shape changes in their collapse propensity upon multi-site phosphorylation according to their initial net charge: phosphorylation expands neutral or overall negatively charged IDPs and shrinks positively charged IDPs. However, force fields including those tailored towards and commonly used for IDPs overestimate these changes. We find quantitative agreement of MD results with SAXS and NMR data for Ash1 and CTD2’ only when attenuating protein electrostatic interactions by using a higher salt concentration (e.g. 350 mM), highlighting the overstabilization of salt bridges in current force fields. We show that phosphorylation of IDPs also has a strong impact on the solvation of the protein, a factor that in addition to the actual collapse or expansion of the IDP should be considered when analyzing SAXS data. Compared to the overall mild change in global IDP dimension, the exposure of active sites can change significantly upon phosphorylation, underlining the large susceptibility of IDP ensembles to regulation through post-translational modifications.     

<Superscript>15</Superscript>N transverse relaxation measurements for the characterization of µs–ms dynamics are deteriorated by the deuterium isotope effect on <Superscript>15</Superscript>N resulting from solvent exchange

¹⁵N R₂ relaxation measurements are key for the elucidation of the dynamics of both folded and intrinsically disordered proteins (IDPs). Here we show, on the example of the intrinsically disordered protein α-synuclein and the folded domain PDZ2, that at physiological pH and near physiological temperatures amide—water exchange can severely skew Hahn-echo based ¹⁵N R₂ relaxation measurements as well as low frequency data points in CPMG relaxation dispersion experiments. The nature thereof is the solvent exchange with deuterium in the sample buffer, which modulates the ¹⁵N chemical shift tensor via the deuterium isotope effect, adding to the apparent relaxation decay which leads to systematic errors in the relaxation data. This results in an artificial increase of the measured apparent ¹⁵N R₂ rate constants—which should not be mistaken with protein inherent chemical exchange contributions, R_ex, to ¹⁵N R₂. For measurements of ¹⁵N R₂ rate constants of IDPs and folded proteins at physiological temperatures and pH, we recommend therefore the use of a very low D₂O molar fraction in the sample buffer, as low as 1%, or the use of an external D₂O reference along with a modified ¹⁵N R₂ Hahn-echo based experiment. This combination allows for the measurement of R_ex contributions to ¹⁵N R₂ originating from conformational exchange in a time window from µs to ms.     

Intramolecular binding of a proximal PPII helix to an SH3 domain in the fusion protein SH3Hck: PPIIhGAP

SH3 domains are a conserved feature of many nonreceptor protein tyrosine kinases, such as Hck, and often function in substrate recruitment and regulation of kinase activity. SH3 domains modulate kinase activity by binding to polyproline helices (PP_II helix) either intramolecularly or in target proteins. The preponderance of bimolecular and distal interactions between SH3 domains and PP_II helices led us to investigate whether proximal placement of a PP_II helix relative to an SH3 domain would result in tight, intramolecular binding. We have fused the PP_II helix region of human GAP to the C-terminus of Hck SH3 and expressed the recombinant fusion protein in Eschericheria coli. The fusion protein, SH3_Hck: PPII_hGAP, folded spontaneously into a structure in which the PP_II helix was bound intramolecularly to the hydrophobic crevice of the SH3 domain. The SH3_Hck: PPII_hGAP fusion protein is useful for investigating SH3: PP_II helix interactions, for studying concepts in protein folding and design, and may represent a protein structural motif that is widely distributed in nature.     

A comparative study of the relationship between protein structure and beta-aggregation in globular and intrinsically disordered proteins

A growing number of proteins are being identified that are biologically active though intrinsically disordered, in sharp contrast with the classic notion that proteins require a well-defined globular structure in order to be functional. At the same time recent work showed that aggregation and amyloidosis are initiated in amino acid sequences that have specific physico-chemical properties in terms of secondary structure propensities, hydrophobicity and charge. In intrinsically disordered proteins (IDPs) such sequences would be almost exclusively solvent-exposed and therefore cause serious solubility problems. Further, some IDPs such as the human prion protein, synuclein and Tau protein are related to major protein conformational diseases. However, this scenario contrasts with the large number of unstructured proteins identified, especially in higher eukaryotes, and the fact that the solubility of these proteins is often particularly good. We have used the algorithm TANGO to compare the beta aggregation tendency of a set of globular proteins derived from SCOP and a set of 296 experimentally verified, non-redundant IDPs but also with a set of IDPs predicted by the algorithms DisEMBL and GlobPlot. Our analysis shows that the beta-aggregation propensity of all-alpha, all-beta and mixed alpha/beta globular proteins as well as membrane-associated proteins is fairly similar. This illustrates firstly that globular structures possess an appreciable amount of structural frustration and secondly that beta-aggregation is not determined by hydrophobicity and beta-sheet propensity alone. We also show that globular proteins contain almost three times as much aggregation nucleating regions as IDPs and that the formation of highly structured globular proteins comes at the cost of a higher beta-aggregation propensity because both structure and aggregation obey very similar physico-chemical constraints. Finally, we discuss the fact that although IDPs have a much lower aggregation propensity than globular proteins, this does not necessarily mean that they have a lower potential for amyloidosis.     

Self-assembling systems comprising intrinsically disordered protein polymers like elastin-like recombinamers

Despite lacking cooperatively folded structures under native conditions, numerous intrinsically disordered proteins (IDPs) nevertheless have great functional importance. These IDPs are hybrids containing both ordered and intrinsically disordered protein regions (IDPRs), the structure of which is highly flexible in this unfolded state. The conformational flexibility of these disordered systems favors transitions between disordered and ordered states triggered by intrinsic and extrinsic factors, folding into different dynamic molecular assemblies to enable proper protein functions. Indeed, prokaryotic enzymes present less disorder than eukaryotic enzymes, thus showing that this disorder is related to functional and structural complexity. Protein-based polymers that mimic these IDPs include the so-called elastin-like polypeptides (ELPs), which are inspired by the composition of natural elastin. Elastin-like recombinamers (ELRs) are ELPs produced using recombinant techniques and which can therefore be tailored for a specific application. One of the most widely used and studied characteristic structures in this field is the pentapeptide (VPGXG)_n. The structural disorder in ELRs probably arises due to the high content of proline and glycine in the ELR backbone, because both these amino acids help to keep the polypeptide structure of elastomers disordered and hydrated. Moreover, the recombinant nature of these systems means that different sequences can be designed, including bioactive domains, to obtain specific structures for each application. Some of these structures, along with their applications as IDPs that self-assemble into functional vesicles or micelles from diblock copolymer ELRs, will be studied in the following sections. The incorporation of additional order- and disorder-promoting peptide/protein domains, such as α-helical coils or β-strands, in the ELR sequence, and their influence on self-assembly, will also be reviewed. In addition, chemically cross-linked systems with controllable order–disorder balance, and their role in biomineralization, will be discussed. Finally, we will review different multivalent IDPs-based coatings and films for different biomedical applications, such as spatially controlled cell adhesion, osseointegration, or biomaterial-associated infection (BAI).     

Differential dehydration effects on globular proteins and intrinsically disordered proteins during film formation

Globular proteins composed of different secondary structures and fold types were examined by synchrotron radiation circular dichroism spectroscopy to determine the effects of dehydration on their secondary structures. They exhibited only minor changes upon removal of bulk water during film formation, contrary to previously reported studies of proteins dehydrated by lyophilization (where substantial loss of helical structure and gain in sheet structure was detected). This near lack of conformational change observed for globular proteins contrasts with intrinsically disordered proteins (IDPs) dried in the same manner: the IDPs, which have almost completely unordered structures in solution, exhibited increased amounts of regular (mostly helical) secondary structures when dehydrated, suggesting formation of new intra‐protein hydrogen bonds replacing solvent‐protein hydrogen bonds, in a process which may mimic interactions that occur when IDPs bind to partner molecules. This study has thus shown that the secondary structures of globular and intrinsically disordered proteins behave very differently upon dehydration, and that films are a potentially useful format for examining dehydrated soluble proteins and assessing IDPs structures.     

固有无序蛋白质及逆境胁迫下的分子功能

固有无序蛋白质是一类在生理条件下缺乏稳定三维结构而具有正常功能,参与信号转导、转录调控、胁迫应答等多种生物学过程的蛋白质.植物中许多逆境响应蛋白是固有无序蛋白质,通过其结构无序或部分无序区域在蛋白质 蛋白质、蛋白质 膜脂、蛋白质 核酸的互作中发挥重要作用.本文主要对固有无序蛋白质的类别、氨基酸组成和结构特点以及在逆境胁迫下其稳定细胞膜、保护核酸和蛋白质、调控基因表达等分子功能进行综述,以拓展对逆境胁迫下蛋白质作用分子机制的认识.     

Small Molecule Sequestration of the Intrinsically Disordered Protein,p27Kip1, Within Soluble Oligomers

Proteins that exhibit intrinsically disordered regions (IDRs) are prevalent in the human proteome and perform diverse biological functions, including signaling and regulation. Due to these important roles, misregulation of intrinsically disordered proteins (IDPs) is associated with myriad human diseases, including neurodegeneration and cancer. The inherent flexibility of IDPs limits the applicability of the traditional structure-based drug design paradigm; therefore, IDPs have long been considered “undruggable”. Using NMR spectroscopy and other methods, we previously discovered small, drug-like molecules that bind specifically, albeit weakly, to dynamic clusters of aromatic residues within p27^Kip1 (p27), an archetypal disordered protein involved in cell cycle regulation. Here, using synthetic chemistry, NMR spectroscopy and other biophysical methods, we discovered elaborated analogs of our previously reported molecules with 30-fold increased affinity for p27 (apparent K_d = 57 ± 19 μM). Strikingly, using analytical ultracentrifugation methods, we showed that the highest affinity compounds caused p27 to form soluble, disordered oligomers. Based on these observations, we propose that sequestration within soluble oligomers may represent a general strategy for therapeutically targeting disease-associated IDPs in the future.     

Alzheimer's-disease-associated conformation of intrinsically disordered tau protein studied by intrinsically disordered protein liquid-phase competitive enzyme-linked immunosorbent assay

Tau protein, the major constituent of paired helical filaments in Alzheimer's disease, belongs to the intrinsically disordered proteins (IDPs). IDPs are an emerging group in the protein kingdom characterized by the absence of a rigid three-dimensional structure. Disordered proteins usually acquire a "functional fold" upon binding to their interaction partner(s). This property of IDPs implies the need for innovative approaches to measure their binding affinity. We have mapped and measured the Alzheimer's-disease-associated epitope on intrinsically disordered tau protein with a novel two-step sandwich competitive enzyme-linked immunosorbent assay (ELISA). This approach allowed us to determine the binding affinity of disordered tau protein in liquid phase without any disturbance to the competitive equilibrium and without any need for covalent or noncovalent modification of tau protein. Furthermore, the global fitting method, used for the reconstruction of tau binding curves, significantly improved the assay readout. The proposed novel competitive ELISA allowed us to determine the changes in the standard Gibbs energy of binding, thus enabling measurement of tau protein conformation in the core of paired helical filaments. IDP competitive ELISA results showed, for the first time, that the tau protein C terminus of the Alzheimer's-disease-derived paired helical filaments core subunit adopts beta-turn type I' fold and is accessible from solution.     

The disordered PCI‐binding human proteins CSNAP and DSS1 have diverged in structure and function

Intrinsically disordered proteins (IDPs) regularly constitute components of larger protein assemblies contributing to architectural stability. Two small, highly acidic IDPs have been linked to the so‐called PCI complexes carrying PCI‐domain subunits, including the proteasome lid and the COP9 signalosome. These two IDPs, DSS1 and CSNAP, have been proposed to have similar structural propensities and functions, but they display differences in their interactions and interactome sizes. Here we characterized the structural properties of human DSS1 and CSNAP at the residue level using NMR spectroscopy and probed their propensities to bind ubiquitin. We find that distinct structural features present in DSS1 are completely absent in CSNAP, and vice versa, with lack of relevant ubiquitin binding to CSNAP, suggesting the two proteins to have diverged in both structure and function. Our work additionally highlights that different local features of seemingly similar IDPs, even subtle sequence variance, may endow them with different functional traits. Such traits may underlie their potential to engage in multiple interactions thereby impacting their interactome sizes.     

Unusual biophysics of intrinsically disordered proteins

Research of a past decade and a half leaves no doubt that complete understanding of protein functionality requires close consideration of the fact that many functional proteins do not have well-folded structures. These intrinsically disordered proteins (IDPs) and proteins with intrinsically disordered protein regions (IDPRs) are highly abundant in nature and play a number of crucial roles in a living cell. Their functions, which are typically associated with a wide range of intermolecular interactions where IDPs possess remarkable binding promiscuity, complement functional repertoire of ordered proteins. All this requires a close attention to the peculiarities of biophysics of these proteins. In this review, some key biophysical features of IDPs are covered. In addition to the peculiar sequence characteristics of IDPs these biophysical features include sequential, structural, and spatiotemporal heterogeneity of IDPs; their rough and relatively flat energy landscapes; their ability to undergo both induced folding and induced unfolding; the ability to interact specifically with structurally unrelated partners; the ability to gain different structures at binding to different partners; and the ability to keep essential amount of disorder even in the bound form. IDPs are also characterized by the “turned-out” response to the changes in their environment, where they gain some structure under conditions resulting in denaturation or even unfolding of ordered proteins. It is proposed that the heterogeneous spatiotemporal structure of IDPs/IDPRs can be described as a set of foldons, inducible foldons, semi-foldons, non-foldons, and unfoldons. They may lose their function when folded, and activation of some IDPs is associated with the awaking of the dormant disorder. It is possible that IDPs represent the “edge of chaos” systems which operate in a region between order and complete randomness or chaos, where the complexity is maximal. This article is part of a Special Issue entitled: The emerging dynamic view of proteins: Protein plasticity in allostery, evolution and self-assembly.     

Discrete Molecular Dynamics Can Predict Helical Prestructured Motifs in Disordered Proteins

Intrinsically disordered proteins (IDPs) lack a stable tertiary structure, but their short binding regions termed Pre-Structured Motifs (PreSMo) can form transient secondary structure elements in solution. Although disordered proteins are crucial in many biological processes and designing strategies to modulate their function is highly important, both experimental and computational tools to describe their conformational ensembles and the initial steps of folding are sparse. Here we report that discrete molecular dynamics (DMD) simulations combined with replica exchange (RX) method efficiently samples the conformational space and detects regions populating α-helical conformational states in disordered protein regions. While the available computational methods predict secondary structural propensities in IDPs based on the observation of protein-protein interactions, our ab initio method rests on physical principles of protein folding and dynamics. We show that RX-DMD predicts α-PreSMos with high confidence confirmed by comparison to experimental NMR data. Moreover, the method also can dissect α-PreSMos in close vicinity to each other and indicate helix stability. Importantly, simulations with disordered regions forming helices in X-ray structures of complexes indicate that a preformed helix is frequently the binding element itself, while in other cases it may have a role in initiating the binding process. Our results indicate that RX-DMD provides a breakthrough in the structural and dynamical characterization of disordered proteins by generating the structural ensembles of IDPs even when experimental data are not available.