Gir2 is an intrinsically unstructured protein that is present in Saccharomyces cerevisiae as a group of heterogeneously electrophoretic migrating forms

Gir2 is a highly acidic cytoplasmic protein of Saccharomyces cerevisiae of unknown function that shows an anomalous migration on SDS-PAGE. Based on its large Stokes radius and thermostability, we have previously suggested that Gir2 lacks extensive secondary structure. Here we report that Gir2 is extremely sensitive to proteolysis when compared to glutathione-S-transferase, a highly structured protein, further indicating its unfolded nature. Prediction based on the FoldIndex program also indicates that Gir2 is a disordered protein. Using truncated forms of Gir2 we show that the N-terminal half of this protein, with its high content of acidic amino acid residues, is responsible for the anomalous electrophoretic behavior of Gir2. Because all these features are hallmarks of intrinsically unstructured proteins (IUP), we propose that Gir2 is another representative of the IUP group of proteins. Additionally, we describe that the endogenous yeast Gir2 shows heterogeneous electrophoretic mobility, which is not due to proteolytic cleavage.     

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.     

Impaired transport of intrinsically disordered proteins through the Sec61 and SecY translocon; implications for prion diseases

The prion protein (PrP) is composed of two major domains of similar size. The structured C-terminal domain contains three alpha-helical regions and a short two-stranded beta-sheet, while the N-terminal domain is intrinsically disordered. The analysis of PrP mutants with deletions in the C-terminal globular domain provided the first hint that intrinsically disordered domains are inefficiently transported into the endoplasmic reticulum through the Sec61 translocon. Interestingly, C-terminally truncated PrP mutants have been linked to inherited prion disease in humans and are characterized by inefficient ER import and the formation of neurotoxic PrP conformers. In a recent study we found that the Sec61 translocon in eukaryotic cells as well as the SecY translocon in bacteria is inherently deficient in translocating intrinsically disordered proteins. Moreover, our results suggest that translocon-associated components in eukaryotic cells enable the Sec61 complex to transport secretory proteins with extended unstructured domains such as PrP and shadoo.     

Intrinsically disordered C-terminal segments of voltage-activated potassium channels: a possible fishing rod-like mechanism for channel binding to scaffold proteins

Membrane-embedded voltage-activated potassium channels (Kv) bind intracellular scaffold proteins, such as the Post Synaptic Density 95 (PSD-95) protein, using a conserved PDZ-binding motif located at the channels' C-terminal tip. This interaction underlies Kv-channel clustering, and is important for the proper assembly and functioning of the synapse. Here we demonstrate that the C-terminal segments of Kv channels adjacent to the PDZ-binding motif are intrinsically disordered. Phylogenetic analysis of the Kv channel family reveals a cluster of channel sequences belonging to three out of the four main channel families, for which an association is demonstrated between the presence of the consensus terminal PDZ-binding motif and the intrinsically disordered nature of the immediately adjacent C-terminal segment. Our observations, combined with a structural analogy to the N-terminal intra-molecular ball-and-chain mechanism for Kv channel inactivation, suggest that the C-terminal disordered segments of these channel families encode an inter-molecular fishing rod-like mechanism for K(+) channel binding to scaffold proteins.     

In vitro acetylation of HMGB-1 and -2 proteins by CBP: the role of the acidic tail

Histone acetyltransferases CBP, PCAF, and Tip60 have been tested for their ability to in vitro acetylate HMGB-1 and -2 proteins and their truncated forms lacking the C-terminal tail. It was found that these proteins were substrates for CBP only. Analyses of modified proteins by electrophoresis, amino acid sequencing, and mass spectrometry showed that full-length HMGB-1 and -2 were monoacetylated at Lys2. Removal of the C terminus resulted in (i) an increased incorporation of radiolabeled acetate within the proteins to a level close to that observed with histones H3/H4 and (ii) creation of a novel target site at Lys81. Acetylated and nonmodified HMGB-1 and -2 protein lacking the acidic tail were compared relative to their binding affinity to distorted DNA and the ability to bend linear DNA. Both proteins showed similar affinities to cisplatin-damaged DNA; the acetylated protein, however, was 3-fold more effective in inducing ligase-mediated circularization of a 111-bp DNA fragment. The alterations in the acetylation pattern of HMGB-1 and -2 upon removal of the C-terminal tail are regarded as a means by which the acidic domain modulates some properties of these proteins.     

Mechanism of Assembly of the Non-Covalent Spectrin Tetramerization Domain from Intrinsically Disordered Partners

Interdomain interactions of spectrin are critical for maintenance of the erythrocyte cytoskeleton. In particular, “head-to-head” dimerization occurs when the intrinsically disordered C-terminal tail of β-spectrin binds the N-terminal tail of α-spectrin, folding to form the “spectrin tetramer domain”. This non-covalent three-helix bundle domain is homologous in structure and sequence to previously studied spectrin domains. We find that this tetramer domain is surprisingly kinetically stable. Using a protein engineering Φ-value analysis to probe the mechanism of formation of this tetramer domain, we infer that the domain folds by the docking of the intrinsically disordered β-spectrin tail onto the more structured α-spectrin tail.     

Intrinsic protein disorder, amino acid composition, and histone terminal domains

Core and linker histones are the most abundant protein components of chromatin. Even though they lack intrinsic structure, the N-terminal "tail" domains (NTDs) of the core histones and the C-terminal tail domain (CTD) of linker histones bind to many different macromolecular partners while functioning in chromatin. Here we discuss the underlying physicochemical basis for how the histone terminal domains can be disordered and yet specifically recognize and interact with different macromolecules. The relationship between intrinsic disorder and amino acid composition is emphasized. We also discuss the potential structural consequences of acetylation and methylation of lysine residues embedded in intrinsically disordered histone tail domains.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N assignments of the C-terminal intrinsically disordered cytosolic fragment of the receptor tyrosine kinase ErbB2

ErbB2 (or HER2) is a receptor tyrosine kinase that is involved in signaling pathways controlling cell division, motility and apoptosis. Though important in development and cell growth homeostasis, this protein, when overexpressed, participates in triggering aggressive HER2+ breast cancers. It is composed of an extracellular part and a transmembrane domain, both important for activation by dimerization, and a cytosolic tyrosine kinase, which activates its intrinsically disordered C-terminal end (CtErbB2). Little is known about this C-terminal part of 268 residues, despite its crucial role in interacting with adaptor proteins involved in signaling. Understanding its structural and dynamic characteristics could eventually lead to the design of new interaction inhibitors, and treatments complementary to those already targeting other parts of ErbB2. Here we report backbone and side-chain assignment of CtErbB2, which, together with structural predictions, confirms its intrinsically disordered nature.     

Rapid purification of truncated tau proteins: model approach to purification of functionally active fragments of disordered proteins, implication for neurodegenerative diseases

Truncated tau is of great interest because of its important role in neurofibrillary pathogenesis in Alzheimer's disease (AD). A major obstacle for characterization of detailed biochemical and biological properties of truncated tau species and their fragments has been the lack of reliable and quick purification methods. Uneven distribution of acidic and basic residues in tau determines that the N- and C-terminal tau fragments require entirely different purification conditions. Conventional methods take several days; they do not allow purification of the acidic N-terminal tau fragments and do not prevent aggregation during purification that makes purified truncated tau unusable in functional studies. To prevent these inherent problems, we have designed a two-step, highly efficient purification procedure yielding a fully functional, non-aggregated homogeneous population of truncated tau molecules. Various forms of tau produced in bacteria without the need for a heat pre-treatment step were subjected to anion- and cation-exchange chromatography. Conditions were developed that allowed effective separation and purification of acidic and/or basic tau species. Following the gel filtration step, up to 10mg of tau proteins with 96% purity was obtained within one working day. Purified truncated tau exhibited an unmodified immunoreactivity and allowed its functional activity analysis. Since many neurodegenerative diseases have implicated similar disordered proteins in their pathogenesis, our procedure will allow their detailed analysis and characterization.     

Identification of intrinsic order and disorder in the DNA repair protein XPA

The DNA-repair protein XPA is required to recognize a wide variety of bulky lesions during nucleotide excision repair. Independent NMR solution structures of a human XPA fragment comprising approximately 40% of the full-length protein, the minimal DNA-binding domain, revealed that one-third of this molecule was disordered. To better characterize structural features of full-length XPA, we performed time-resolved trypsin proteolysis on active recombinant Xenopus XPA (xXPA). The resulting proteolytic fragments were analyzed by electrospray ionization interface coupled to a Fourier transform ion cyclotron resonance mass spectrometry and SDS-PAGE. The molecular weight of the full-length xXPA determined by mass spectrometry (30922.02 daltons) was consistent with that calculated from the sequence (30922.45 daltons). Moreover, the mass spectrometric data allowed the assignment of multiple xXPA fragments not resolvable by SDS-PAGE. The neural network program Predictor of Natural Disordered Regions (PONDR) applied to xXPA predicted extended disordered N- and C-terminal regions with an ordered internal core. This prediction agreed with our partial proteolysis results, thereby indicating that disorder in XPA shares sequence features with other well-characterized intrinsically unstructured proteins. Trypsin cleavages at 30 of the possible 48 sites were detected and no cleavage was observed in an internal region (Q85-I179) despite 14 possible cut sites. For the full-length xXPA, there was strong agreement among PONDR, partial proteolysis data, and the NMR structure for the corresponding XPA fragment.     

Structural insights into the dynamics and function of the C-terminus of the E. coli RNA chaperone Hfq

The hexameric Escherichia coli RNA chaperone Hfq (Hfq(Ec)) is involved in riboregulation of target mRNAs by small trans-encoded RNAs. Hfq proteins of different bacteria comprise an evolutionarily conserved core, whereas the C-terminus is variable in length. Although the structure of the conserved core has been elucidated for several Hfq proteins, no structural information has yet been obtained for the C-terminus. Using bioinformatics, nuclear magnetic resonance spectroscopy, synchrotron radiation circular dichroism (SRCD) spectroscopy and small angle X-ray scattering we provide for the first time insights into the conformation and dynamic properties of the C-terminal extension of Hfq(Ec). These studies indicate that the C-termini are flexible and extend laterally away from the hexameric core, displaying in this way features typical of intrinsically disordered proteins that facilitate intermolecular interactions. We identified a minimal, intrinsically disordered region of the C-terminus supporting the interactions with longer RNA fragments. This minimal region together with rest of the C-terminal extension provides a flexible moiety capable of tethering long and structurally diverse RNA molecules. Furthermore, SRCD spectroscopy supported the hypothesis that RNA fragments exceeding a certain length interact with the C-termini of Hfq(Ec).     

Modular organization of SARS coronavirus nucleocapsid protein

The SARS-CoV nucleocapsid (N) protein is a major antigen in severe acute respiratory syndrome. It binds to the viral RNA genome and forms the ribonucleoprotein core. The SARS-CoV N protein has also been suggested to be involved in other important functions in the viral life cycle. Here we show that the N protein consists of two non-interacting structural domains, the N-terminal RNA-binding domain (RBD) (residues 45–181) and the C-terminal dimerization domain (residues 248–365) (DD), surrounded by flexible linkers. The C-terminal domain exists exclusively as a dimer in solution. The flexible linkers are intrinsically disordered and represent potential interaction sites with other protein and protein-RNA partners. Bioinformatics reveal that other coronavirus N proteins could share the same modular organization. This study provides information on the domain structure partition of SARS-CoV N protein and insights into the differing roles of structured and disordered regions in coronavirus nucleocapsid proteins. CK Chang and SC Sue contributed equally to this project.     

Conformational selection and folding-upon-binding of intrinsically disordered protein CP12 regulate photosynthetic enzymes assembly

Carbon assimilation in plants is regulated by the reduction of specific protein disulfides by light and their re-oxidation in the dark. The redox switch CP12 is an intrinsically disordered protein that can form two disulfide bridges. In the dark oxidized CP12 forms an inactive supramolecular complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase, two enzymes of the carbon assimilation cycle. Here we show that binding of CP12 to GAPDH, the first step of ternary complex formation, follows an integrated mechanism that combines conformational selection with induced folding steps. Initially, a CP12 conformation characterized by a circular structural motif including the C-terminal disulfide is selected by GAPDH. Subsequently, the induced folding of the flexible C-terminal tail of CP12 in the active site of GAPDH stabilizes the binary complex. Formation of several hydrogen bonds compensates the entropic cost of CP12 fixation and terminates the interaction mechanism that contributes to carbon assimilation control.     

Defining Structural Domains of an Intrinsically Disordered Protein: Sic1, the Cyclin-Dependent Kinase Inhibitor of <Emphasis Type="Italic">Saccharomyces</Emphasis><Emphasis Type="Italic">cerevisiae</Emphasis>

The cyclin-dependent kinase inhibitor Sic1 is an intrinsically disordered protein (IDP) involved in cell–cycle regulation in the yeast Saccharomyces cerevisiae. Notwithstanding many studies on its biological function, structural characterization has been attempted only recently, fostering the development of production and purification protocols suitable to yield large amounts of this weakly expressed protein. In this study, we describe the identification of protein domains by the heterologous expression, purification, and characterization of Sic1-derived fragment. Four C-terminal fragments (Sic1^C-ter) were produced based on functional studies and limited-proteolysis results. The N-terminal fragment (Sic1^1–186) was complementary to the most stable C-terminal fragments (Sic1^Δ186). Both Sic1^1–186 and Sic1^C-ter fragments were, in general, less susceptible to spontaneous proteolysis than the full-length protein. The boundaries of the C-terminal fragments turned out to be crucial for integrity of the recombinant proteins and required two rounds of design and production. Sic1 fragments were purified by a simple procedure, based on their resistance to heat treatment, at the amount and purity required for structural characterization. Circular dichroism (CD) measurements and nuclear magnetic resonance (NMR) spectra of N- and C-terminal fragments confirm their disordered nature but reveal minor structural differences that may reflect their distinct functional roles.     

Hydrogen-exchange mass spectrometry for the study of intrinsic disorder in proteins

Amide hydrogen/deuterium exchange detected by mass spectrometry (HXMS) is seeing wider use for the identification of intrinsically disordered parts of proteins. In this review, we discuss examples of how discovery of intrinsically disordered regions and their removal can aid in structure determination, biopharmaceutical quality control, the characterization of how post-translational modifications affect weak structuring of disordered regions, the study of coupled folding and binding, and the characterization of amyloid formation. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.     

Conformational stability of six truncated cHMG1a proteins studied in their mixture by H/D exchange and electrospray ionization mass spectrometry

The high mobility group (HMG) proteins are abundant non-histone components of eukaryotic chromatin. The presence of C-terminal acidic tails is a common feature of the majority of HMG proteins. Although the biological significance of the acidic domains is not clear, they are conferring conformational and metabolic stability to the proteins in vitro. Moreover, the length and net charge of the acidic tails affect the strength of HMG protein interaction with DNA. Synthesis of an insect HMG protein by standard recombinant technology in bacteria leads to a mixture of the intact protein (cHMG1a-(1-113) (I)) and a series of its degradation products truncated at the C tail: cHMG1a-(1-111) (II); cHMG1a-(1-110) (III); cHuMGla-(1-109) (IV); cHMG1a-(1-108) (V); cHMG1a-(1-107) (VI); cHMG1a-(1-106) (VII). The proteins differ from each other only by the number of amino-acid residues at the C-terminal tail. We used H/D exchange mass spectrometry to characterize the stability of the proteins directly in their mixture. The results show that the proteins I-V and VII have very similar conformations. The protein VI is less compact and exchanges its protons faster than the others. It may be concluded that the C-terminal tail influences the conformation of the cHMG1a protein and that individual residues in this part of the protein play a key role in its compactness.