The Saccharomyces cerevisiae nucleoporin Nup2p is a natively unfolded protein

Little is known about the structure of the individual nucleoporins that form eukaryotic nuclear pore complexes (NPCs). We report here in vitro physical and structural characterizations of a full-length nucleoporin, the Saccharomyces cerevisiae protein Nup2p. Analyses of the Nup2p structure by far-UV circular dichroism (CD) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, protease sensitivity, gel filtration, and sedimentation velocity experiments indicate that Nup2p is a "natively unfolded protein," belonging to a class of proteins that exhibit little secondary structure, high flexibility, and low compactness. Nup2p possesses a very large Stokes radius (79 A) in gel filtration columns, sediments slowly in sucrose gradients as a 2.9 S particle, and is highly sensitive to proteolytic digestion by proteinase K; these characteristics suggest a structure of low compactness and high flexibility. Spectral analyses (CD and FTIR spectroscopy) provide additional evidence that Nup2p contains extensive regions of structural disorder with comparatively small contributions of ordered secondary structure. We address the possible significance of natively unfolded nucleoporins in the mechanics of nucleocytoplasmic trafficking across NPCs.     

The N-terminal domain of p53 is natively unfolded

p53 is one of the key molecules regulating cell proliferation, apoptosis and tumor suppression by integrating a wide variety of signals. The structural basis for this function is still poorly understood. p53 appears to exercise its function as a modular protein in which different functions are associated with distinct domains. Presumably, p53 contains both folded and partially structured parts. Here, we have investigated the structure of the isolated N-terminal part of p53 (amino acid residues 1-93) using biophysical techniques. We demonstrate that this domain is devoid of tertiary structure and largely missing secondary structure elements. It exhibits a large hydrodynamic radius, typical for unfolded proteins. These findings suggest strongly that the entire N-terminal part of p53 is natively unfolded under physiological conditions. Furthermore, the binding affinity to its functional antagonist Mdm2 was investigated. A comparison of the binding of human Mdm2 to the N-terminal part of p53 and full-length p53 suggests that unfolded and folded parts of p53 function synergistically.     

Dematin exhibits a natively unfolded core domain and an independently folded headpiece domain

Dematin is an actin‐binding protein originally identified in the junctional complex of the erythrocyte plasma membrane, and is present in many nonerythroid cells. Dematin headpiece knockout mice display a spherical red cell phenotype and develop a compensated anemia. Dematin has two domains: a 315‐residue, proline‐rich “core” domain and a 68‐residue carboxyl‐terminal villin‐type “headpiece” domain. Expression of full‐length dematin in E. coli as a GST recombinant protein results in truncation within a proline, glutamic acid, serine, threonine rich region (PEST). Therefore, we designed a mutant construct that replaces the PEST sequence. The modified dematin has high actin binding activity as determined by actin sedimentation assays. Negative stain electron microscopy demonstrates that the modified dematin also exhibits actin bundling activity like that of native dematin. Circular dichroism (CD) and NMR spectral analysis, however, show little secondary structure in the modified dematin. The lack of secondary structure is also observed in native dematin purified from human red blood cells. ¹⁵N‐HSQC NMR spectra of modified dematin indicate that the headpiece domain is fully folded whereas the core region is primarily unfolded. Our finding suggests that the core is natively unfolded and may serve as a scaffold to organize the components of the junctional complex.     

Evidences of a natively unfolded state for the human topoisomerase IB N-terminal domain

The N-terminal domain of human topoisomerase IB has been expressed, purified and characterized by spectroscopic techniques. CD spectra as a function of concentration and pH indicate that the domain does not possess any defined secondary structure. The protein is probably in a natively unfolded state since its denaturation curve is indicative of a non-cooperative transition. Evidence of a partially folded structure comes from the fluorescence spectrum of ANS, whose intensity increases in presence of the domain. Indication of a partial structural arrangement of the domain comes also from the endogenous fluorescence of tryptophans that is centred at 350 nm in the native and shifts to 354 nm in the fully denaturated protein. Interestingly despite the poor structural degree, as also confirmed by a predictive approach, the domain efficiently binds DNA, suggesting that the absence of a defined 3D structure has a functional meaning that permits the domain to be available for the interaction with different molecular partners.     

A natively unfolded toxin domain uses its receptor as a folding template

Natively unfolded proteins range from molten globules to disordered coils. They are abundant in eukaryotic genomes and commonly involved in molecular interactions. The essential N-terminal translocation domains of colicin toxins from Escherichia coli are disordered bacterial proteins that bind at least one protein of the Tol or Ton family. The colicin N translocation domain (ColN-(1-90)), which binds to the C-terminal domain of TolA (TolA-(296-421)), shows a disordered far-UV CD spectrum, no near-UV CD signal, and non-cooperative thermal unfolding. As expected, TolA-(296-421) displays both secondary structure in far-UV CD and tertiary structure in near-UV CD. Furthermore it shows a cooperative unfolding transition at 65 degrees C. CD spectra of the 1:1 complex show both increased secondary structure and colicin N-specific near-UV CD signals. A new cooperative thermal transition at 35 degrees C is followed by the unchanged unfolding behavior of TolA-(296-421). Fluorescence and surface plasmon resonance confirm that the new unfolding transition accompanies dissociation of ColN-(1-90). Hence upon binding the disordered structure of ColN-(1-90) converts to a cooperatively folded domain without altering the TolA-(296-421) structure.     

The N-terminal half of the peroxisomal cycling receptor Pex5p is a natively unfolded domain

Targeting of most newly synthesised peroxisomal matrix proteins to the organelle requires Pex5p, the so-called PTS1 receptor. According to current models of peroxisomal biogenesis, Pex5p interacts with these proteins in the cytosol, transports them to the peroxisomal membrane and catalyses their translocation across the membrane. Presently, our knowledge on the structural details behind the interaction of Pex5p with the cargo proteins is reasonably complete. In contrast, information regarding the structure of the Pex5p N-terminal half (a region containing its peroxisomal targeting domain) is still limited. We have recently observed that the Stokes radius of this Pex5p domain is anomalously large, suggesting that this portion of the protein is either a structured elongated domain or that it adopts a low compactness conformation. Here, we address this issue using a combination of biophysical and biochemical approaches. Our results indicate that the N-terminal half of Pex5p is best described as a natively unfolded pre-molten globule-like domain. The implications of these findings on the mechanism of protein import into the peroxisome are discussed.     

Rapid evolution exposes the boundaries of domain structure and function in natively unfolded FG nucleoporins

Nucleoporins with phenylalanine-glycine repeats (FG Nups) function at the nuclear pore complex (NPC) to facilitate nucleocytoplasmic transport. In Saccharomyces cerevisiae, each FG Nup contains a large natively unfolded domain that is punctuated by FG repeats. These FG repeats are surrounded by hydrophilic amino acids (AAs) common to disordered protein domains. Here we show that the FG domain of Nups from human, fly, worm, and other yeast species is also enriched in these disorder-associated AAs, indicating that structural disorder is a conserved feature of FG Nups and likely serves an important role in NPC function. Despite the conservation of AA composition, FG Nup sequences from different species show extensive divergence. A comparison of the AA substitution rates of proteins with syntenic orthologs in four Saccharomyces species revealed that FG Nups have evolved at twice the rate of average yeast proteins with most substitutions occurring in sequences between FG repeats. The rapid evolution of FG Nups is poorly explained by parameters known to influence AA substitution rate, such as protein expression level, interactivity, and essentiality; instead their rapid evolution may reflect an intrinsic permissiveness of natively unfolded structures to AA substitutions. The overall lack of AA sequence conservation in FG Nups is sharply contrasted by discrete stretches of conserved sequences. These conserved sequences highlight known karyopherin and nucleoporin binding sites as well as other uncharacterized sites that may have important structural and functional properties.     

Prediction of natively unfolded regions in protein chain

We have shown that the ability of a protein to be in globular or in natively unfolded state (under native conditions) may be determined (besides low overall hydrophobicity and a large net charge) by such a property as the average environment density, the average number of residues enclosed at the given distance. A statistical scale of the average number of residues enclosed at the given distance for 20 types of amino acid residues in globular state has been created on the basis of 6626 protein structures. Using this scale for separation of 80 globular and 90 natively unfolded proteins we fail only in 11% of proteins (compared with 17% of errors which are observed if to use hydrophobicity scale). The present scale may be used both for prediction of form (folded or unfolded) of the native state of protein and for prediction of natively unfolded regions in protein chains. The results of comparison of our method of predicting natively unfolded regions with the other known methods show that our method has the highest fraction of correctly predicted natively unfolded regions (that is 87% and 77% if to make averaging over residues and over proteins correspondingly).     

Prediction of natively unfolded regions in protein chains

Analysis showed that the globular or natively unfolded state of a protein can be inferred not only from a lower hydrophobicity or a higher charge, but also from the average environment density (average number of close residues located within a certain distance of a given one) of its residues. A database of 6626 protein structures was used to construct a statistical scale of the average number of close residues in globular structures for the 20 amino acids. The portion of false predictions in distinguishing between 80 globular and 90 natively unfolded proteins was 11% with the new scale and 17% with a hydrophobicity scale. The new scale proved suitable for predicting the folded or unfolded state for native proteins or the natively unfolded regions for protein chains. In comparisons with the available algorithms, the new method yielded the highest portion of true predictions (87 and 77% with averaging over residues and over proteins, respectively).     

Studying the natively unfolded neuronal Tau protein by solution NMR spectroscopy

The neuronal Tau protein, whose physiological role is to stabilize the microtubules, is found under the form of aggregated filaments and tangles in Alzheimer's diseased neurons. Until recently detailed structural analysis of the natively unfolded Tau protein has been hindered due to its shear size and unfavourable amino acid composition. We review here the recent progress in the assignments of the full-length polypeptide using novel methods of product planes and peptide NMR mapping, and indicate the structural insights that can be obtained from this assignment. Preliminary NMR data on the fibers show that the assignment enables a precise mapping of the rigid core. Future NMR experiments should allow one to gain more insight into the conformational aspects of this intriguing protein.     

The intracellular domain of the Drosophila cholinesterase-like neural adhesion protein, gliotactin, is natively unfolded

Drosophila gliotactin (Gli) is a 109-kDa transmembrane, cholinesterase-like adhesion molecule (CLAM), expressed in peripheral glia, that is crucial for formation of the blood-nerve barrier. The intracellular portion (Gli-cyt) was cloned and expressed in the cytosolic fraction of Escherichia coli BLR(DE3) at 45 mg/L and purified by Ni-NTA (nitrilotriacetic acid) chromatography. Although migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), under denaturing conditions, was unusually slow, molecular weight determination by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) confirmed that the product was consistent with its theoretical size. Gel filtration chromatography yielded an anomalously large Stokes radius, suggesting a fully unfolded conformation. Circular dichroism (CD) spectroscopy demonstrated that Gli-cyt was >50% unfolded, further suggesting a nonglobular conformation. Finally, 1D-(1)H NMR conclusively demonstrated that Gli-cyt possesses an extended unfolded structure. In addition, Gli-cyt was shown to possess charge and hydrophobic properties characteristic of natively unfolded proteins (i.e., proteins that, when purified, are intrinsically disordered under physiologic conditions in vitro).     

Structural characterization by NMR of the natively unfolded extracellular domain of beta-dystroglycan: toward the identification of the binding epitope for alpha-dystroglycan

Dystroglycan (DG) is an adhesion molecule playing a crucial role for tissue stability during both early embriogenesis and adulthood and is composed by two tightly interacting subunits: alpha-DG, membrane-associated and highly glycosylated, and the transmembrane beta-DG. Recently, by solid-phase binding assays and NMR experiments, we have shown that the C-terminal domain of alpha-DG interacts with a recombinant extracellular fragment of beta-DG (positions 654-750) independently from glycosylation and that the linear binding epitope is located between residues 550 and 565 of alpha-DG. In order to elucidate which moieties of beta-DG are specifically involved in the complex with alpha-DG, the ectodomain has been recombinantly expressed and purified in a labeled ((13)C,(15)N) form and studied by multidimensional NMR. Although it represents a natively unfolded protein domain, we obtained an almost complete backbone assignment. Chemical shift index, (1)H-(15)N heteronuclear single-quantum coherence and nuclear Overhauser effect (HSQC-NOESY) spectra and (3)J(HN,H)(alpha) coupling constant values confirm that this protein is highly disordered, but (1)H-(15)N steady-state NOE experiments indicate that the protein presents two regions of different mobility. The first one, between residues 659 and 722, is characterized by a limited degree of mobility, whereas the C-terminal portion, containing about 30 amino acids, is highly flexible. The binding of beta-DG(654-750) to the C-terminal region of the alpha subunit, alpha-DG(485-620), has been investigated, showing that the region of beta-DG(654-750) between residues 691 and 719 is involved in the interaction.     

Structural memory of natively unfolded tau protein detected by small-angle X-ray scattering

Small-angle X-ray scattering (SAXS) is a universal low-resolution method to study size and shape of globular proteins in solution but recent developments facilitate the quantitative characterization of the structure and structural transitions of metastable systems like partially or completely unfolded proteins. We present here a study of temperature induced transitions in tau, a natively unfolded protein involved in Alzheimer's disease. Previous studies on full length tau and several disease-related mutants provided information about the residual structure in different domains revealing a specific role and extended conformations of the so-called repeat domains, which are considered to be responsible for the formation of amyloid-like fibrils ("paired helical filaments"). Here, we employ SAXS to investigate the temperature dependent properties of tau. Slow heating/cooling of the full length protein from 10°C to 50°C did not lead to detectable changes in the overall size. Surprisingly, quick heating/cooling caused tau to adopt a significantly more compact conformation, which was stable over up to 3 h and represents a structural "memory" effect. This compaction is not observed for the shorter tau constructs containing largely the repeat domains. The structural and functional implications of the observed unusual behavior of tau under nonequilibrium conditions are discussed.     

Backbone dynamics of the natively unfolded pro-peptide of subtilisin by heteronuclear NMR relaxation studies

The dynamics of the natively unfolded form of the pro-peptide of subtilisin (PPS) have been characterized at two different pHs (6.0 and 3.0) by ¹⁵N relaxation experiments. ¹⁵N relaxation data is obtained at multiple field strengths and a detailed comparison of spectral density mapping, the model free approach and the recently proposed Cole–Cole model free (CC-MF) analysis is presented. The CC-MF analysis provides a better fit to the observed magnetic field dependence of ¹⁵N relaxation data of unfolded PPS than conventional model free approaches and shows that fluctuations in R₂ may be accounted for by a distribution of correlation times on the nanosecond timescale. A new parameter derives from the analysis and represents the width of the distribution function and the heterogeneity of the dynamics on the nanosecond timescale at a particular site. Particularly interesting is the observation that is sensitive to pH changes and that PPS samples a wider distribution of nanosecond time scale motions at less acidic pHs than at more acidic pHs. These results suggest that PPS experiences a higher degree of correlated motion at pH 6.0 and that electrostatic interactions may be important for inducing correlated motions on the nanosecond timescale in unfolded PPS.     

Critical scaffolding regions of the tumor suppressor Axin1 are natively unfolded

The Wnt pathway tumor-suppressor protein Axin coordinates the formation of a critical multiprotein destruction complex that serves to downregulate β-catenin protein levels, thereby preventing target gene activation. Given the lack of structural information on some of the major functional parts of Axin, it remains unresolved how the recruitment and positioning of Wnt pathway kinases, such as glycogen synthase kinase 3β, are coordinated to bring about β-catenin phosphorylation. Using various biochemical and biophysical methods, we demonstrate here that the central region of Axin that is implicated in binding glycogen synthase kinase 3β and β-catenin is natively unfolded. Our results support a model in which the unfolded nature of these critical scaffolding regions in Axin facilitates dynamic interactions with a kinase and its substrate, which in turn act upon each other.     

Transition from natively unfolded to folded state induced by desiccation in an anhydrobiotic nematode protein

Late embryogenesis abundant (LEA) proteins are associated with desiccation tolerance in resurrection plants and in plant seeds, and the recent discovery of a dehydration-induced Group 3 LEA-like gene in the nematode Aphelenchus avenae suggests a similar association in anhydrobiotic animals. Despite their importance, little is known about the structure of Group 3 LEA proteins, although computer modeling and secondary structure algorithms predict a largely alpha-helical monomer that forms coiled coil oligomers. We have therefore investigated the structure of the nematode protein, AavLEA1, in the first such analysis of a well characterized Group 3 LEA-like protein. Immunoblotting and subunit cross-linking experiments demonstrate limited oligomerization of AavLEA1, but analytical ultracentrifugation and gel filtration show that the vast majority of the protein is monomeric. Moreover, CD, fluorescence emission, and Fourier transform-infrared spectroscopy indicate an unstructured conformation for the nematode protein. Therefore, in solution, no evidence was found to support structure predictions; instead, AavLEA1 seems to be natively unfolded with a high degree of hydration and low compactness. Such proteins can, however, be induced to fold into more rigid structures by partner molecules or by altered physiological conditions. Because AavLEA1 is associated with desiccation stress, its Fourier transform-infrared spectrum in the dehydrated state was examined. A dramatic but reversible increase in alpha-helix and, possibly, coiled coil formation was observed on drying, indicating that computer predictions of secondary structure may be correct for the solid state. This unusual finding offers the possibility that structural shifts in Group 3 LEA proteins occur on dehydration, perhaps consistent with their role in anhydrobiosis.     

Membrane fusion mediated by coiled coils: a hypothesis

A molecular model of the low-pH-induced membrane fusion by influenza hemagglutinin (HA) is proposed based upon the hypothesis that the conformational change to the extended coiled coil creates a high-energy hydrophobic membrane defect in the viral envelope or HA expressing cell. It is known that 1) an aggregate of at least eight HAs is required at the fusion site, yet only two or three of these HAs need to undergo the "essential" conformational change for the first fusion pore to form (Bentz, J. 2000. Biophys. J. 78:000-000); 2) the formation of the first fusion pore signifies a stage of restricted lipid flow into the nascent fusion site; and 3) some HAs can partially insert their fusion peptides into their own viral envelopes at low pH. This suggests that the committed step for HA-mediated fusion begins with a tightly packed aggregate of HAs whose fusion peptides are inserted into their own viral envelope, which causes restricted lateral lipid flow within the HA aggregate. The transition of two or three HAs in the center of the aggregate to the extended coiled coil extracts the fusion peptide and creates a hydrophobic defect in the outer monolayer of the virion, which is stabilized by the closely packed HAs. These HAs are inhibited from diffusing away from the site to admit lateral lipid flow, in part because that would initially increase the surface area of hydrophobic exposure. The other obvious pathway to heal this hydrophobic defect, or some descendent, is recruitment of lipids from the outer monolayer of the apposed target membrane, i.e., fusion. Other viral fusion proteins and the SNARE fusion protein complex appear to fit within this hypothesis.     

The regions of securin and cyclin B proteins recognized by the ubiquitination machinery are natively unfolded

The proteins securin and cyclin B are destroyed in mitosis by the ubiquitin/proteasome system. This destruction is important to mitotic progression. The N-terminal regions of these proteins contain the sequence features recognized by the ubiquitination system. We have demonstrated using circular dichroism and 1-D and 2-D nuclear magnetic resonance that these rather substantial regions are natively unfolded. Based on these findings, we propose a model that helps to explain previously enigmatic observations.