Target-binding behavior of IDPs via pre-structured motifs.

Pre-Structured Motifs (PreSMos) are transient secondary structures observed in many intrinsically disordered proteins (IDPs) and serve as protein target-binding hot spots. The prefix "pre" highlights that PreSMos exist a priori in the target-unbound state of IDPs as the active pockets of globular proteins pre-exist before target binding. Therefore, a PreSMo is an "active site" of an IDP; it is not a spatial pocket, but rather a secondary structural motif. The classical and perhaps the most effective approach to understand the function of a protein has been to determine and investigate its structure. Ironically or by definition IDPs do not possess structure (here structure refers to tertiary structure only). Are IDPs then entirely structureless? The PreSMos provide us with an atomic-resolution answer to this question. For target binding, IDPs do not rely on the spatial pockets afforded by tertiary or higher structures. Instead, they utilize the PreSMos possessing particular conformations that highly presage the target-bound conformations. PreSMos are recognized or captured by targets via conformational selection (CS) before their conformations eventually become stabilized via structural induction into more ordered bound structures. Using PreSMos, a number of, if not all, IDPs can bind targets following a sequential pathway of CS followed by an induced fit (IF). This chapter presents several important PreSMos implicated in cancers, neurodegenerative diseases, and other diseases along with discussions on their conformational details that mediate target binding, a structural rationale for unstructured proteins.

Cellular Chaperone Function of Intrinsically Disordered Dehydrin ERD14.

Disordered plant chaperones play key roles in helping plants survive in harsh conditions, and they are indispensable for seeds to remain viable. Aside from well-known and thoroughly characterized globular chaperone proteins, there are a number of intrinsically disordered proteins (IDPs) that can also serve as highly effective protecting agents in the cells. One of the largest groups of disordered chaperones is the group of dehydrins, proteins that are expressed at high levels under different abiotic stress conditions, such as drought, high temperature, or osmotic stress. Dehydrins are characterized by the presence of different conserved sequence motifs that also serve as the basis for their categorization. Despite their accepted importance, the exact role and relevance of the conserved regions have not yet been formally addressed. Here, we explored the involvement of each conserved segment in the protective function of the intrinsically disordered stress protein (IDSP) A. thaliana's Early Response to Dehydration (ERD14). We show that segments that are directly involved in partner binding, and others that are not, are equally necessary for proper function and that cellular protection emerges from the balanced interplay of different regions of ERD14.

Interplay of Structural Disorder and Short Binding Elements in the Cellular Chaperone Function of Plant Dehydrin ERD14.

Details of the functional mechanisms of intrinsically disordered proteins (IDPs) in living cells is an area not frequently investigated. Here, we dissect the molecular mechanism of action of an IDP in cells by detailed structural analyses based on an in-cell nuclear magnetic resonance experiment. We show that the ID stress protein (IDSP) A. thaliana Early Response to Dehydration (ERD14) is capable of protecting E. coli cells under heat stress. The overexpression of ERD14 increases the viability of E. coli cells from 38.9% to 73.9% following heat stress (50 °C × 15 min). We also provide evidence that the protection is mainly achieved by protecting the proteome of the cells. In-cell NMR experiments performed in E. coli cells show that the protective activity is associated with a largely disordered structural state with conserved, short sequence motifs (K- and H-segments), which transiently sample helical conformations in vitro and engage in partner binding in vivo. Other regions of the protein, such as its S segment and its regions linking and flanking the binding motifs, remain unbound and disordered in the cell. Our data suggest that the cellular function of ERD14 is compatible with its residual structural disorder in vivo.

WT and A53T α-Synuclein Systems: Melting Diagram and Its New Interpretation.

The potential barriers governing the motions of α-synuclein (αS) variants' hydration water, especially energetics of them, is in the focus of the work. The thermodynamical approach yielded essential information about distributions and heights of the potential barriers. The proteins' structural disorder was measured by ratios of heterogeneous water-binding interfaces. They showed the αS monomers, oligomers and amyloids to possess secondary structural elements, although monomers are intrinsically disordered. Despite their disordered nature, monomers have 33% secondary structure, and therefore they are more compact than a random coil. At the lowest potential barriers with mobile hydration water, monomers are already functional, a monolayer of mobile hydration water is surrounding them. Monomers realize all possible hydrogen bonds with the solvent water. αS oligomers and amyloids have half of the mobile hydration water amount than monomers because aggregation involves less mobile hydration. The solvent-accessible surface of the oligomers is ordered or homogenous in its interactions with water to 66%. As a contrast, αS amyloids are disordered or heterogeneous to 75% of their solvent accessible surface and both wild type and A53T amyloids show identical, low-level hydration. Mobile water molecules in the first hydration shell of amyloids are the weakest bound compared to other forms.

Salient Features of Monomeric Alpha-Synuclein Revealed by NMR Spectroscopy.

Elucidating the structural details of proteins is highly valuable and important for the proper understanding of protein function. In the case of intrinsically disordered proteins (IDPs), however, obtaining the structural details is quite challenging, as the traditional structural biology tools have only limited use. Nuclear magnetic resonance (NMR) is a unique experimental tool that provides ensemble conformations of IDPs at atomic resolution, and when studying IDPs, a slightly different experimental strategy needs to be employed than the one used for globular proteins. We address this point by reviewing many NMR investigations carried out on the α-synuclein protein, the aggregation of which is strongly correlated with Parkinson's disease.

Intrinsically Disordered Linkers Impart Processivity on Enzymes by Spatial Confinement of Binding Domains.

(1) Background: Processivity is common among enzymes and mechanochemical motors that synthesize, degrade, modify or move along polymeric substrates, such as DNA, RNA, polysaccharides or proteins. Processive enzymes can make multiple rounds of modification without releasing the substrate/partner, making their operation extremely effective and economical. The molecular mechanism of processivity is rather well understood in cases when the enzyme structurally confines the substrate, such as the DNA replication factor PCNA, and also when ATP energy is used to confine the succession of molecular events, such as with mechanochemical motors. Processivity may also result from the kinetic bias of binding imposed by spatial confinement of two binding elements connected by an intrinsically disordered (ID) linker. (2) Method: By statistical physical modeling, we show that this arrangement results in processive systems, in which the linker ensures an optimized effective concentration around novel binding site(s), favoring rebinding over full release of the polymeric partner. (3) Results: By analyzing 12 such proteins, such as cellulase, and RNAse-H, we illustrate that in these proteins linker length and flexibility, and the kinetic parameters of binding elements, are fine-tuned for optimizing processivity. We also report a conservation of structural disorder, special amino acid composition of linkers, and the correlation of their length with step size. (4) Conclusion: These observations suggest a unique type of entropic chain function of ID proteins, that may impart functional advantages on diverse enzymes in a variety of biological contexts.

Transient Secondary Structures as General Target-Binding Motifs in Intrinsically Disordered Proteins.

Intrinsically disordered proteins (IDPs) are unorthodox proteins that do not form three-dimensional structures under non-denaturing conditions, but perform important biological functions. In addition, IDPs are associated with many critical diseases including cancers, neurodegenerative diseases, and viral diseases. Due to the generic name of "unstructured" proteins used for IDPs in the early days, the notion that IDPs would be completely unstructured down to the level of secondary structures has prevailed for a long time. During the last two decades, ample evidence has been accumulated showing that IDPs in their target-free state are pre-populated with transient secondary structures critical for target binding. Nevertheless, such a message did not seem to have reached with sufficient clarity to the IDP or protein science community largely because similar but different expressions were used to denote the fundamentally same phenomenon of presence of such transient secondary structures, which is not surprising for a quickly evolving field. Here, we summarize the critical roles that these transient secondary structures play for diverse functions of IDPs by describing how various expressions referring to transient secondary structures have been used in different contexts.

PreSMo Target-Binding Signatures in Intrinsically Disordered Proteins.

Intrinsically disordered proteins (IDPs) are highly unorthodox proteins that do not form three-dimensional structures under physiological conditions. The discovery of IDPs has destroyed the classical structure-function paradigm in protein science, 3-D structure = function, because IDPs even without well-folded 3-D structures are still capable of performing important biological functions and furthermore are associated with fatal diseases such as cancers, neurodegenerative diseases and viral pandemics. Pre-structured motifs (PreSMos) refer to transient local secondary structural elements present in the target-unbound state of IDPs. During the last two decades PreSMos have been steadily acknowledged as the critical determinants for target binding in dozens of IDPs. To date, the PreSMo concept provides the most convincing structural rationale explaining the IDP-target binding behavior at an atomic resolution. Here we present a brief developmental history of PreSMos and describe their common characteristics. We also provide a list of newly discovered PreSMos along with their functional relevance.

A subset of functional adaptation mutations alter propensity for α-helical conformation in the intrinsically disordered glucocorticoid receptor tau1core activation domain.

BACKGROUND: Adaptive mutations that alter protein functionality are enriched within intrinsically disordered protein regions (IDRs), thus conformational flexibility correlates with evolvability. Pre-structured motifs (PreSMos) with transient propensity for secondary structure conformation are believed to be important for IDR function. The glucocorticoid receptor tau1core transcriptional activation domain (GR tau1core) domain contains three α-helical PreSMos in physiological buffer conditions. METHODS: Sixty change-of-function mutants affecting the intrinsically disordered 58-residue GR tau1core were studied using disorder prediction and molecular dynamics simulations. RESULTS: Change-of-function mutations were partitioned into seven clusters based on their effect on IDR predictions and gene activation activity. Some mutations selected from clusters characterized by mutations altering the IDR prediction score, altered the apparent stability of the α-helical form of one of the PreSMos in molecular dynamics simulations, suggesting PreSMo stabilization or destabilization as strategies for functional adaptation. Indeed all tested gain-of-function mutations affecting this PreSMo were associated with increased stability of the α-helical PreSMo conformation, suggesting that PreSMo stabilization may be the main mechanism by which adaptive mutations can increase the activity of this IDR type. Some mutations did not appear to affect PreSMo stability. CONCLUSIONS: Changes in PreSMo stability account for the effects of a subset of change-of-function mutants affecting the GR tau1core IDR. GENERAL SIGNIFICANCE: Long IDRs occur in about 50% of human proteins. They are poorly characterized despite much recent attention. Our results suggest the importance of a subtle balance between PreSMo stability and IDR activity, which may provide a novel target for future pharmaceutical intervention.

An NMR study on the intrinsically disordered core transactivation domain of human glucocorticoid receptor.

A large number of transcriptional activation domains (TADs) are intrinsically unstructured, meaning they are devoid of a three-dimensional structure. The fact that these TADs are transcriptionally active without forming a 3-D structure raises the question of what features in these domains enable them to function. One of two TADs in human glucocorticoid receptor (hGR) is located at its N-terminus and is responsible for ∼70% of the transcriptional activity of hGR. This 58-residue intrinsically-disordered TAD, named tau1c in an earlier study, was shown to form three helices under trifluoroethanol, which might be important for its activity. We carried out heteronuclear multi-dimensional NMR experiments on hGR tau1c in a more physiological aqueous buffer solution and found that it forms three helices that are ∼30% pre-populated. Since pre-populated helices in several TADs were shown to be key elements for transcriptional activity, the three pre-formed helices in hGR tau1c delineated in this study should be critical determinants of the transcriptional activity of hGR. The presence of prestructured helices in hGR tau1c strongly suggests that the existence of pre-structured motifs in target-unbound TADs is a very broad phenomenon. [BMB Reports 2017; 50(10): 522-527].

Rescuing p53 from mdm2 by a pre-structured motif in intrinsically unfolded SUMO specific protease 4.

Many intrinsically unstructured/unfolded proteins (IUPs) contain transient local secondary structures even though they are "unstructured" in a tertiary sense. These local secondary structures are named "pre-structured motifs (PreSMos)" and in fact are the specificity determinants for IUP-target binding, i.e., the active sites in IUPs. Using high-resolution NMR we have delineated a PreSMo active site in the intrinsically unfolded mid-domain (residues 201-300) of SUMO-specific protease 4 (SUSP4). This 29-residue motif which we termed a p53 rescue motif can protect p53 from mdm2 quenching by binding to the p53-helix binding pocket in mdm2(3-109). Our work demonstrates that the PreSMo approach is quite effective in providing a structural rationale for interactions of p53-mdm2- SUSP4 and opens a novel avenue for designing mdm2- inhibiting anticancer compounds. [BMB Reports 2017; 50(10): 485-486].

The Mechanism of p53 Rescue by SUSP4.

p53 is an important tumor-suppressor protein deactivation of which by mdm2 results in cancers. A SUMO-specific protease 4 (SUSP4) was shown to rescue p53 from mdm2-mediated deactivation, but the mechanism is unknown. The discovery by NMR spectroscopy of a "p53 rescue motif" in SUSP4 that disrupts p53-mdm2 binding is presented. This 29-residue motif is pre-populated with two transient helices connected by a hydrophobic linker. The helix at the C-terminus binds to the well-known p53-binding pocket in mdm2 whereas the N-terminal helix serves as an affinity enhancer. The hydrophobic linker binds to a previously unidentified hydrophobic crevice in mdm2. Overall, SUSP4 appears to use two synergizing modules, the p53 rescue motif described here and a globular-structured SUMO-binding catalytic domain, to stabilize p53. A p53 rescue motif peptide exhibits an anti-tumor activity in cancer cell lines expressing wild-type p53. A pre-structures motif in the intrinsically disordered proteins is thus important for target recognition.

Structural investigation on the intrinsically disordered N-terminal region of HPV16 E7 protein.

Human papillomavirus (HPV) is the major cause of cervical cancer, a deadly threat to millions of females. The early oncogene product (E7) of the high-risk HPV16 is the primary agent associated with HPV-related cervical cancers. In order to understand how E7 contributes to the transforming activity, we investigated the structural features of the flexible N-terminal region (46 residues) of E7 by carrying out N-15 heteronuclear NMR experiments and replica exchange molecular dynamics simulations. Several NMR parameters as well as simulation ensemble structures indicate that this intrinsically disordered region of E7 contains two transient (10-20% populated) helical pre-structured motifs that overlap with important target binding moieties such as an E2F-mimic motif and a pRb-binding LXCXE segment. Presence of such target-binding motifs in HPV16 E7 provides a reasonable explanation for its promiscuous target-binding behavior associated with its transforming activity. [BMB Reports 2016; 49(8): 431-436].

Wide-line NMR and DSC studies on intrinsically disordered p53 transactivation domain and its helically pre-structured segment.

Wide-line 1H NMR intensity and differential scanning calorimetry measurements were carried out on the intrinsically disordered 73-residue full transactivation domain (TAD) of the p53 tumor suppressor protein and two peptides: one a wild type p53 TAD peptide with a helix pre-structuring property, and a mutant peptide with a disabled helix-forming propensity. Measurements were carried out in order to characterize their water and ion binding characteristics. By quantifying the number of hydrate water molecules, we provide a microscopic description for the interactions of water with a wild-type p53 TAD and two p53 TAD peptides. The results provide direct evidence that intrinsically disordered proteins (IDPs) and a less structured peptide not only have a higher hydration capacity than globular proteins, but are also able to bind a larger amount of charged solute ions. [BMB Reports 2016; 49(9): 497-501].

Biophysical characterization of the structural change of Nopp140, an intrinsically disordered protein, in the interaction with CK2α.

Nucleolar phosphoprotein 140 (Nopp140) is a nucleolar protein, more than 80% of which is disordered. Previous studies have shown that the C-terminal region of Nopp140 (residues 568-596) interacts with protein kinase CK2α, and inhibits the catalytic activity of CK2. Although the region of Nopp140 responsible for the interaction with CK2α was identified, the structural features and the effect of this interaction on the structure of Nopp140 have not been defined due to the difficulty of structural characterization of disordered protein. In this study, the disordered feature of Nopp140 and the effect of CK2α on the structure of Nopp140 were examined using single-molecule fluorescence resonance energy transfer (smFRET) and electron paramagnetic resonance (EPR). The interaction with CK2α was increased conformational rigidity of the CK2α-interacting region of Nopp140 (Nopp140C), suggesting that the disordered and flexible conformation of Nopp140C became more rigid conformation as it binds to CK2α. In addition, site specific spin labeling and EPR analysis confirmed that the residues 574-589 of Nopp140 are critical for binding to CK2α. Similar technical approaches can be applied to analyze the conformational changes in other IDPs during their interactions with binding partners.

A pre-structured helix in the intrinsically disordered 4EBP1.

The eIF4E-binding protein 1 (4EBP1) has long been known to be completely unstructured without any secondary structures, which contributed significantly to the proposal of the induced fit mechanism for target binding of intrinsically disordered proteins. We show here that 4EBP1 is not completely unstructured, but contains a pre-structured helix.

Prognostic significance of catalase expression and its regulatory effects on hepatitis B virus X protein (HBx) in HBV-related advanced hepatocellular carcinomas.

Hepatitis B virus X protein (HBx) plays a role in liver cancer development. We previously showed that ROS increased HBx levels and here, we investigated the role of antioxidants in the regulation of HBx expression and their clinical relevance. We found that overexpression of catalase induced a significant loss in HBx levels. The cysteine null mutant of HBx (Cys-) showed a dramatic reduction in its protein stability. In clonogenic proliferation assays, Huh7-X cells produced a significant number of colonies whereas Huh7-Cys- cells failed to generate them. The Cys at position 69 of HBx was crucial to maintain its protein stability and transactivation function in response to ROS. Among 50 HBV-related hepatocellular carcinoma (HCC) specimens, 72% of HCCs showed lower catalase levels than those of surrounding non-tumor tissues. In advanced stage IV, catalase levels in non-tumor tissues were increased whereas those in tumors were further reduced. Accordingly, patients with a high T/N ratio for catalase showed significantly longer survival than those with a low T/N ratio. Together, catalase expression in HCC patients can be clinically useful for prediction of patient survival, and restoration of catalase expression in HCCs could be an important strategy for intervention in HBV-induced liver diseases.

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.

Contribution of proline to the pre-structuring tendency of transient helical secondary structure elements in intrinsically disordered proteins.

BACKGROUND: IDPs function without relying on three-dimensional structures. No clear rationale for such a behavior is available yet. PreSMos are transient secondary structures observed in the target-free IDPs and serve as the target-binding "active" motifs in IDPs. Prolines are frequently found in the flanking regions of PreSMos. Contribution of prolines to the conformational stability of the helical PreSMos in IDPs is investigated. METHODS: MD simulations are performed for several IDP segments containing a helical PreSMo and the flanking prolines. To measure the influence of flanking-prolines on the structural content of a helical PreSMo calculations were done for wild type as well as for mutant segments with Pro→Asp, His, Lys, or Ala. The change in the helicity due to removal of a proline was measured both for the PreSMo region and for the flanking regions. RESULTS: The α-helical content in ~70% of the helical PreSMos at the early stage of simulation decreases due to replacement of an N-terminal flanking proline by other residues whereas the helix content in nearly all PreSMos increases when the same replacements occur at the C-terminal flanking region. The helix destabilizing/terminating role of the C-terminal flanking prolines is more pronounced than the helix promoting effect of the N-terminal flanking prolines. GENERAL SIGNIFICANCE: This work represents a novel example demonstrating that a proline is encoded in an IDP with a defined purpose. The helical PreSMos presage their target-bound conformations. As they most likely mediate IDP-target binding via conformational selection their helical content can be an important feature for IDP function.

Structural disorder and local order of hNopp140.

Human nucleolar phosphoprotein p140 (hNopp 140) is a highly phosphorylated protein inhibitor of casein kinase 2 (CK2). As in the case of many kinase-inhibitor systems, the inhibitor has been described to belong to the family of intrinsically disordered proteins (IDPs), which often utilize transient structural elements to bind their cognate enzyme. Here we investigated the structural status of this protein both to provide distinct lines of evidence for its disorder and to point out its transient structure potentially involved in interactions and also its tendency to aggregate. Structural disorder of hNopp140 is apparent by its anomalous electrophoretic mobility, protease sensitivity, heat stability, hydrodynamic behavior on size-exclusion chromatography, (1)H NMR spectrum and differential scanning calorimetry scan. hNopp140 has a significant tendency to aggregate and the change of its circular dichroism spectrum in the presence of 0-80% TFE suggests a tendency to form local helical structures. Wide-line NMR measurements suggest the overall disordered character of the protein. In all, our data suggest that this protein falls into the pre-molten globule state of IDPs, with a significant tendency to become ordered in the presence of its partner as demonstrated in the presence of transcription factor IIB (TFIIB).