How Robust Is the Mechanism of Folding-Upon-Binding for an Intrinsically Disordered Protein?

The mechanism of interaction of an intrinsically disordered protein (IDP) with its physiological partner is characterized by a disorder-to-order transition in which a recognition and a binding step take place. Even if the mechanism is quite complex, IDPs tend to bind their partner in a cooperative manner such that it is generally possible to detect experimentally only the disordered unbound state and the structured complex. The interaction between the disordered C-terminal domain of the measles virus nucleoprotein (N_TAIL) and the X domain (XD) of the viral phosphoprotein allows us to detect and quantify the two distinct steps of the overall reaction. Here, we analyze the robustness of the folding of N_TAIL upon binding to XD by measuring the effect on both the folding and binding steps of N_TAIL when the structure of XD is modified. Because it has been shown that wild-type XD is structurally heterogeneous, populating an on-pathway intermediate under native conditions, we investigated the binding to 11 different site-directed variants of N_TAIL of one particular variant of XD (I504A XD) that populates only the native state. Data reveal that the recognition and the folding steps are both affected by the structure of XD, indicating a highly malleable pathway. The experimental results are briefly discussed in the light of previous experiments on other IDPs.     

Conformational Analysis of the Partially Disordered Measles Virus NTAIL-XD Complex by SDSL EPR Spectroscopy

To characterize the structure of dynamic protein systems, such as partly disordered protein complexes, we propose a novel approach that relies on a combination of site-directed spin-labeled electron paramagnetic resonance spectroscopy and modeling of local rotation conformational spaces. We applied this approach to the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (N_TAIL) both free and in complex with the X domain (XD, aa 459-507) of the viral phosphoprotein. By comparing measured and modeled temperature-dependent restrictions of the side-chain conformational spaces of 12 SL cysteine-substituted N_TAIL variants, we showed that the 490-500 region of N_TAIL is prestructured in the absence of the partner, and were able to quantitatively estimate, for the first time to our knowledge, the extent of the α-helical sampling of the free form. In addition, we showed that the 505-525 region of N_TAIL conserves a significant degree of freedom even in the bound form. The latter two findings provide a mechanistic explanation for the reported rather high affinity of the N_TAIL-XD binding reaction. Due to the nanosecond timescale of X-band EPR spectroscopy, we were also able to monitor the disordering in the 488-525 region of N_TAIL, in particular the unfolding of the α-helical region when the temperature was increased from 281 K to 310 K.     

Interaction between the C‐terminal domains of measles virus nucleoprotein and phosphoprotein: A tight complex implying one binding site

The intrinsically disordered C‐terminal domain (N_TAIL) of the measles virus (MeV) nucleoprotein undergoes α‐helical folding upon binding to the C‐terminal X domain (XD) of the phosphoprotein. The N_TAIL region involved in binding coupled to folding has been mapped to a conserved region (Box2) encompassing residues 489–506. In the previous studies published in this journal, we obtained experimental evidence supporting a K_D for the N_TAIL–XD binding reaction in the nM range and also showed that an additional N_TAIL region (Box3, aa 517–525) plays a role in binding to XD. In striking contrast with these data, studies published in this journal by Kingston and coworkers pointed out a much less stable complex (K_D in the μM range) and supported lack of involvement of Box3 in complex formation. The objective of this study was to critically re‐evaluate the role of Box3 in N_TAIL–XD binding. Since our previous studies relied on N_TAIL‐truncated forms possessing an irrelevant Flag sequence appended at their C‐terminus, we, herein, generated an N_TAIL devoid of Box3 and any additional C‐terminal residues, as well as a form encompassing only residues 482–525. We then used isothermal titration calorimetry to characterize the binding reactions between XD and these N_TAIL forms. Results effectively argue for the presence of a single XD‐binding site located within Box2, in agreement with the results by Kingston et al., while providing clear experimental support for a high‐affinity complex. Altogether, the present data provide mechanistic insights into the replicative machinery of MeV and clarify a hitherto highly debated point.     

Probing Structural Transitions in the Intrinsically Disordered C-Terminal Domain of the Measles Virus Nucleoprotein by Vibrational Spectroscopy of Cyanylated Cysteines

Four single-cysteine variants of the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (N_TAIL) were cyanylated at cysteine and their infrared spectra in the C≡N stretching region were recorded both in the absence and in the presence of one of the physiological partners of N_TAIL, namely the C-terminal X domain (XD) of the viral phosphoprotein. Consistent with previous studies showing that XD triggers a disorder-to-order transition within N_TAIL, the C≡N stretching bands of the infrared probe were found to be significantly affected by XD, with this effect being position-dependent. When the cyanylated cysteine side chain is solvent-exposed throughout the structural transition, its changing linewidth reflects a local gain of structure. When the probe becomes partially buried due to binding, its frequency reports on the mean hydrophobicity of the microenvironment surrounding the labeled side chain of the bound form. The probe moiety is small compared to other common covalently attached spectroscopic probes, thereby minimizing possible steric hindrance/perturbation at the binding interface. These results show for the first time to our knowledge the suitability of site-specific cysteine mutagenesis followed by cyanylation and infrared spectroscopy to document structural transitions occurring within intrinsically disordered regions, with regions involved in binding and folding being identifiable at the residue level.     

Interaction between the C-terminal domains of N and P proteins of measles virus investigated by NMR

In this paper we investigate the interaction between the C-terminal domains of the measles virus phosphoprotein (XD) and nucleoprotein (N_TAIL) by using nuclear magnetic resonance chemical shift perturbation experiments. Using both N_TAIL constructs and peptides, we show that contrary to the conserved Box2 region (N^489-506), the C-terminal region of N_TAIL (N^513-525) does not directly interact with XD, and yet affects binding to XD. We tentatively propose a model where the C-terminus of N_TAIL would stabilize the N_TAIL-XD complex either via a functional coupling with N^489-506 or by reducing the entropic penalty associated to the binding-coupled-to-folding process.

Structured summary

MINT-7009780, MINT-7009793, MINT-7009808: N-tail (uniprotkb:Q89933) and P (uniprotkb:P03422) bind (MI:0407) by nuclear magnetic resonance (MI:0077)     

Compaction and binding properties of the intrinsically disordered C-terminal domain of Henipavirus nucleoprotein as unveiled by deletion studies

Henipaviruses are recently emerged severe human pathogens within the Paramyxoviridae family. Their genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that recruits the polymerase complex via the phosphoprotein (P). We have previously shown that in Henipaviruses the N protein possesses an intrinsically disordered C-terminal domain, N(TAIL), which undergoes α-helical induced folding in the presence of the C-terminal domain (P(XD)) of the P protein. Using computational approaches, we previously identified within N(TAIL) four putative molecular recognition elements (MoREs) with different structural propensities, and proposed a structural model for the N(TAIL)-P(XD) complex where the MoRE encompassing residues 473-493 adopt an α-helical conformation at the P(XD) surface. In this work, for each N(TAIL) protein, we designed four deletion constructs bearing different combinations of the predicted MoREs. Following purification of the N(TAIL) truncated proteins from the soluble fraction of E. coli, we characterized them in terms of their conformational, spectroscopic and binding properties. These studies provided direct experimental evidence for the structural state of the four predicted MoREs, and showed that two of them have clear α-helical propensities, with the one spanning residues 473-493 being strictly required for binding to P(XD). We also showed that Henipavirus N(TAIL) and P(XD) form heterologous complexes, indicating that the P(XD) binding regions are functionally interchangeable between the two viruses. By combining spectroscopic and conformational analyses, we showed that the content in regular secondary structure is not a major determinant of protein compaction.     

hsp47 and hsp70 gene expression is differentially regulated in a stress- and tissue-specific manner in zebrafish embryos

We have examined differences in the spatial and temporal regulation of stress-induced hsp47 and hsp70 gene expression following exposure of zebrafish embryos to heat shock or ethanol. Using Northern blot analysis, we found that levels of hsp47 and hsp70 mRNA were dramatically elevated during heat shock in 2-day-old embryos. In contrast, ethanol exposure resulted in strong upregulation of the hsp47 gene whereas hsp70 mRNA levels increased only slightly following the same treatment. Whole-mount in situ hybridization analysis revealed that hsp47 mRNA was expressed predominantly in precartilagenous cells, as well as several other connective tissue cell populations within the embryo following exposure to either stress. hsp70 mRNA displayed a very different cell-specific distribution. For example, neither stress induced hsp70 mRNA accumulation in precartilagenous cells. However, high levels of hsp70 mRNA were detectable in epithelial cells of the developing epidermis following exposure to heat shock, but not to ethanol. These cells did not express the hsp47 gene following exposure to either of these stresses. The results suggest the presence of different inducible regulatory mechanisms for these genes which operate in a cell- and stress-specific manner in zebrafish embryos. Dev. Genet. 21:123–133, 1997. © 1997 Wiley-Liss, Inc.     

Selective postmortem degradation of inducible heat shock protein 70 (hsp70) mRNAs in rat Brain

Summary 1. Altered mRNA levels in postmortem brain tissue from persons with Alzheimer's disease (AD) or other neurological diseases are usually presumed to be characteristic of the disease state, even though both agonal state (the physiological state immediately premortem) and postmortem interval (PMI) (the time between death and harvesting the tissue) have the potential to affect levels of mRNAs measured in postmortem tissue. Although the possible effect of postmortem interval on mRNA levels has been more carefully evaluated than that of agonal state, many studies assume that all mRNAs have similar rates of degradation postmortem.2. To determine the postmortem stability of inducible heat shock protein 70 (hsp70) mRNAs, themselves unstablein vivo at normal body temperature, rats were heat shocked in order to induce synthesis of the hsp70 mRNAs. hsp70 mRNA levels in cerebellum and cortex were then compared to those of their heat shock cognate 70 (hsc70) mRNAs, as well as to levels of 18S rRNAs, at 0 and at 24 hr postmortem.3. Quantiation of northern blots after hybridization with an hsp70 mRNA-specific oligo probe indicated a massive loss of hsp70 mRNA signal in RNAs isolated from 24-hr postmortem brains; quantitation by slot-blot hybridization was 5- to 15-fold more efficient. Even using the latter technique, hsp70 mRNA levels were reduced by 59% in 24-hr-postmortem cerebellum and by 78% in cortex compared to mRNA levels in the same region of 0-hr-postmortem brain. There was little reduction postmortem in levels of the hsp70 mRNAs or of 18S rRNAs in either brain region.4.In situ hybridization analysis indicated that hsp70 mRNAs were less abundant in all major classes of cerebellar cells after 24 hr postmortem and mRNAs had degraded severalfold more rapidly in neurons than in glia. There was no corresponding loss of intracellular 18S rRNA in any cell type.5. We conclude from these results that the effect of postmortem interval on mRNA degradation must be carefully evaluated when analyzing levels of inducible hsp70 mRNAs, and perhaps other short-lived mRNAs, in human brain.     

烯啶虫胺、 毒死蜱和高效氯氰菊酯对烟粉虱地中海隐种hsp70 mRNA的诱导表达

为深入理解杀虫剂胁迫对烟粉虱Bemisia tabaci适应逆境能力的影响, 本研究运用实时荧光定量PCR技术测定了LC₂₅, LC₅₀和LC₇₅ 3种浓度的烯啶虫胺、 毒死蜱和高效氯氰菊酯分别处理对烟粉虱地中海隐种B. tabaci Mediterranean成虫体内热激蛋白hsp70表达水平的影响。结果表明： 在低温（15℃）条件下, LC₅₀和LC₇₅的烯啶虫胺、 毒死蜱、 高效氯氰菊酯可显著诱导烟粉虱地中海隐种hsp70的表达量增加, 此后随时间延长hsp70表达量逐渐下降, 到72 h时恢复到对照水平, 但LC₂₅的3种杀虫剂对hsp70的表达无明显影响; 在常温（25℃）下, 较高浓度（LC₅₀和LC₇₅）的烯啶虫胺、 毒死蜱和高效氯氰菊酯处理24 h同样可促进hsp70表达量的增加, 然后hsp70表达量逐渐下降, 到72 h时恢复至正常水平, 但低浓度(LC₂₅)的3种药剂处理后hsp70表达量随时间延长而增加, 到72 h时达到最高; 在高温（32℃）条件下, LC₂₅和LC₅₀的烯啶虫胺、 毒死蜱和高效氯氰菊酯处理24 h可显著增强hsp70的表达水平, 此后随时间的延长hsp70表达恢复至正常水平。杀虫药剂诱导的hsp70表达量增加增强了烟粉虱地中海隐种对杀虫药剂和高温的耐受能力, 这可能是导致其在我国快速扩张的原因之一。     

Cloning and nucleotide sequencing of three heat shock protein genes (hsp90, hsc70, and hsp19.5) from the diamondback moth, Plutella xylostella (L.) and their expression in relation to developmental stage and temperature

Heat shock protein genes, hsp90, hsc70, and hsp19.5, were cloned and sequenced from the diamondback moth, Plutella xylostella (L.) by RT-PCR and RACE method. The cDNA sequence analysis of hsp90 and hsp19.5 revealed open reading frames (ORFs) of 2,151 and 522 bp in length, which encode proteins with calculated molecular weights of 82.4 and 19.5 kDa, respectively. Analysis of cDNA from hsc70 revealed an ORF of 1,878 bp coding a protein with a calculated molecular weight of 69.3 kDa. Furthermore, the analysis of genomic DNA from hsc70 confirmed the presence of introns while no introns were apparent in hsp90 and hsp19.5. Southern blot analysis suggested the presence of multiple copies of each gene family in the DBM genome. Detectable expression of hsp19.5 was observed at the pupal stage while expression of hsp90 and hsc70 was detected at both pupal and adult stages. At adult stage, females showed a higher expression of hsp90 and hsc70 than males. An increased expression was observed in all three genes after exposure to a high temperature in both sexes. These results suggest that in addition to a heat shock response, these HSP genes might be involved in other functions during the course of development in DBM.     

Response to natural and laboratory selection at the Drosophila hsp70 genes

Abstract.— To determine whether and how laboratory and natural selection act on the hsp70 (70-Kd heat-shock protein) genes of Drosophila melanogaster , we examined hsp70 allele frequencies in two sets of populations. First, five populations reared at different temperatures for more than 20 years differentially fixed both a large insertion/deletion (indel) polymorphism at the 87A7 hsp70 cluster ("56H8"/"122") and a single nucleotide polymorphism at the 87C1 hsp70 cluster. In both cases, the 18°C and 25°C populations fixed one allele and the 28°C populations the other, consistent with previously described evolved differences among these populations in Hsp70 expression and thermo-tolerance. Second, we examined 56H8 and 122 frequencies in a set of 11 populations founded from flies collected along a latitudinal transect of eastern Australia. The 56H8 allele frequencies are positively associated with latitude, consistent with maintenance of the 56H8/122 polymorphism by natural selection. Thermal extremes and average values are negatively correlated with latitude. These results suggest that natural selection imposed by temperature and thermal variability may affect hsp70 allele frequencies.     

Modulation of the Stress-Induced Synthesis of hsp27 and αB-Crystallin by Cyclic AMP in C6 Rat Glioma Cells

Abstract: The possible participation of cyclic AMP in the stress-induced synthesis of two small stress proteins, hsp27 and αB-crystallin, in C6 rat glioma cells was examined by specific immunoassays, western blot analysis, and northern blot analysis. When C6 cells were exposed to arsenite (50–100 µM for 1 h) or heat (42°C for 30 min), expression of hsp27 and αB-crystallin was stimulated, with levels of the two proteins reaching a maximum after 10–16 h of culture. Induction of hsp27 was markedly enhanced when cells were exposed to arsenite in the presence of isoproterenol (20 µM) or epinephrine (20 µM) but not in the presence of phenylephrine. The stimulatory effects of isoproterenol and epinephrine were blocked completely by propranolol, an antagonist of β-adrenergic receptors. Cholera toxin (2 µg/ml), forskolin (20 µM), and dibutyryl cyclic AMP (2.5 mM), all of which are known to increase intracellular levels of cyclic AMP, also stimulated the arsenite- or heat-induced accumulation of hsp27. Treatment of cells with each of these modulators alone did not result in the induction of hsp27. The level of hsp70 in C6 cells, as estimated by western blot analysis, was also enhanced by arsenite or heat stress. However, induction of hsp70 by stress was barely stimulated by isoproterenol. By contrast, induction of αB-crystallin by heat or arsenite stress was suppressed when isoproterenol, cholera toxin, forskolin, or dibutyryl cyclic AMP was present during the stress period. Northern blot analysis of the expression of mRNAs for hsp70, hsp27, and αB-crystallin showed that the modulation of the stress-induced accumulation of the three hsps by the various agents was regulated at the level of the corresponding mRNA. These results indicate that stress responses of hsp70, hsp27, and αB-crystallin in C6 rat glioma cells are regulated differently and, moreover, that when the level of cyclic AMP increases in cells, the response to stress of hsp27 is stimulated but that of αB-crystallin is suppressed.