Accelerating structural life science by paramagnetic lanthanide probe methods Saio, Tomohide; Ishimori, Koichiro
Biochimica et biophysica acta. G, General subjects/Biochimica et biophysica acta. General subjects (Online),
February 2020, 2020-Feb, 2020-02-00, 20200201, Letnik:
1864, Številka:
2
Journal Article
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We describe the recent progress of structural analysis methods exploiting paramagnetic lanthanide ions. In NMR spectroscopy, the paramagnetic effects induced by the trivalent lanthanide ions provide ...long-range (~40 Å) distance and angular information that can be exploited in protein structure determination, ligand screening, structure-based resonance assignment, and in-cell observation. The paramagnetic lanthanide ions can also be utilized in EPR spectroscopy, providing nanometer-scale distance measurement. These applications of the paramagnetic lanthanide probe are becoming more widespread by the use of a variety of lanthanide binding tags. Here, we introduce the basics of paramagnetic effects, several examples of lanthanide tags, and recent applications of paramagnetic lanthanide ions in NMR and EPR spectroscopy. Collectively, we show how the paramagnetic lanthanide probe accelerates research in protein science and drug design, and consequently life science.
•The recent progress in protein NMR and EPR using paramagnetic lanthanide ions.•Information obtainable from the paramagnetic effects observed in NMR.•The tags for site-specific immobilization of the lanthanide ions.•The applications investigating protein structure, dynamics, and interaction.
Molecular chaperones alter the folding properties of cellular proteins via mechanisms that are not well understood. Here, we show that Trigger Factor (TF), an ATP-independent chaperone, exerts ...strikingly contrasting effects on the folding of non-native proteins as it transitions between a monomeric and a dimeric state. We used NMR spectroscopy to determine the atomic resolution structure of the 100 kDa dimeric TF. The structural data show that some of the substrate-binding sites are buried in the dimeric interface, explaining the lower affinity for protein substrates of the dimeric compared to the monomeric TF. Surprisingly, the dimeric TF associates faster with proteins and it exhibits stronger anti-aggregation and holdase activity than the monomeric TF. The structural data show that the dimer assembles in a way that substrate-binding sites in the two subunits form a large contiguous surface inside a cavity, thus accounting for the observed accelerated association with unfolded proteins. Our results demonstrate how the activity of a chaperone can be modulated to provide distinct functional outcomes in the cell.
The dye-decolorizing peroxidase (DyP) protein from Vibrio cholerae (VcDyP) was expressed in Escherichia coli, and its DyP activity was assayed by monitoring degradation of a typical anthraquinone ...dye, reactive blue 19 (RB19). Its kinetic activity was obtained by fitting the data to the Michaelis–Menten equation, giving k cat and K m values of 1.3 ± 0.3 s–1 and 50 ± 20 μM, respectively, which are comparable to those of other DyP enzymes. The enzymatic activity of VcDyP was highest at pH 4. A mutational study showed that two distal residues, Asp144 and Arg230, which are conserved in a DyP family, are essential for the DyP reaction. The crystal structure and resonance Raman spectra of VcDyP indicate the transfer of a radical from heme to the protein surface, which was supported by the formation of the intermolecular covalent bond in the reaction with H2O2. To identify the radical site, each of nine tyrosine or two tryptophan residues was substituted. It was clarified that Tyr129 and Tyr235 are in the active site of the dye degradation reaction at lower pH, while Tyr109 and Tyr133 are the sites of an intermolecular covalent bond at higher pH. VcDyP degrades RB19 at lower pH, while it loses activity under neutral or alkaline conditions because of a change in the radical transfer pathway. This finding suggests the presence of a pH-dependent switch of the radical transfer pathway, probably including His178. Although the physiological function of the DyP reaction is unclear, our findings suggest that VcDyP enhances the DyP activity to survive only when it is placed under a severe condition such as being in gastric acid.
Despite recent developments in protein structure prediction, the process of the structure formation, folding, remains poorly understood. Notably, folding of multidomain proteins, which involves ...multiple steps of segmental folding, is one of the biggest questions in protein science. Multidomain protein folding often requires the assistance of molecular chaperones. Molecular chaperones promote or delay the folding of the client protein, but the detailed mechanisms are still unclear. This review summarizes the findings of biophysical and structural studies on the mechanism of multidomain protein folding mediated by molecular chaperones and explains how molecular chaperones recognize the client proteins and alter their folding properties. Furthermore, we introduce several recent studies that describe the concept of kinetics-activity relationships to explain the mechanism of functional diversity of molecular chaperones.
Abstract
To quantitatively categorize protein structures, we developed a quantitative coarse-grained model of protein structures with a novel amino acid network, the interaction selective network ...(ISN), characterized by the links based on interactions in
both
the main and side chains. We found that the ISN is a novel robust network model to show the higher classification probability in the plots of average vertex degree (
k
) versus average clustering coefficient (
C
), both of which are typical network parameters for protein structures, and successfully distinguished between “all-α” and “all-β” proteins. On the other hand, one of the typical conventional networks, the α-carbon network (CAN), was found to be less robust than the ISN, and another typical network, atomic distance network (ADN), failed to distinguish between these two protein structures. Considering that the links in the CAN and ADN are defined by the interactions only between the main chain atoms and by the distance of the closest atom pair between the two amino acid residues, respectively, we can conclude that reflecting structural information from both secondary and tertiary structures in the network parameters improves the quantitative evaluation and robustness in network models, resulting in a quantitative and more robust description of three-dimensional protein structures in the ISN.
Progesterone-receptor membrane component 1 (PGRMC1/Sigma-2 receptor) is a haem-containing protein that interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 to regulate cancer ...proliferation and chemoresistance; its structural basis remains unknown. Here crystallographic analyses of the PGRMC1 cytosolic domain at 1.95 Å resolution reveal that it forms a stable dimer through stacking interactions of two protruding haem molecules. The haem iron is five-coordinated by Tyr113, and the open surface of the haem mediates dimerization. Carbon monoxide (CO) interferes with PGRMC1 dimerization by binding to the sixth coordination site of the haem. Haem-mediated PGRMC1 dimerization is required for interactions with EGFR and cytochromes P450, cancer proliferation and chemoresistance against anti-cancer drugs; these events are attenuated by either CO or haem deprivation in cancer cells. This study demonstrates protein dimerization via haem-haem stacking, which has not been seen in eukaryotes, and provides insights into its functional significance in cancer.
HBP23, a 23-kDa heme-binding protein identified in rats, is a member of the peroxiredoxin (Prx) family, the primary peroxidases involved in hydrogen peroxide catabolism. Although HBP23 has a ...characteristic Cys-Pro heme-binding motif, the significance of heme binding to Prx family proteins remains to be elucidated. Here, we examined the effect of heme binding to human peroxiredoxin-1 (PRX1), which has 97% amino acid identity to HBP23. PRX1 was expressed in Escherichia coli and purified to homogeneity. Spectroscopic titration demonstrated that PRX1 binds heme with a 1:1 stoichiometry and a dissociation constant of 0.17 μM. UV–vis spectra of heme-PRX1 suggested that Cys52 is the axial ligand of ferric heme. PRX1 peroxidase activity was lost upon heme binding, reflecting the fact that Cys52 is not only the heme-binding site but also the active center of peroxidase activity. Interestingly, heme binding to PRX1 caused a decrease in the toxicity and degradation of heme, significantly suppressing H2O2-dependent heme peroxidase activity and degradation of PRX1-bound heme compared with that of free hemin. By virtue of its cytosolic abundance (∼20 μM), PRX1 thus functions as a scavenger of cytosolic hemin (<1 μM). Collectively, our results indicate that PRX1 has a dual role; Cys-dependent peroxidase activity and cytosolic heme scavenger.
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•Human peroxiredoxin-1 (PRX1) is a heme-binding protein.•Cys52 in PRX1 is the heme-binding site as well as the active center.•Heme-bound PRX1 loses the peroxidase activity.•Heme that is bound to PRX1 is protected from the reaction with H2O2.
A functional association is uncovered between the ribosome-associated trigger factor (TF) chaperone and the ClpXP degradation complex. Bioinformatic analyses demonstrate conservation of the close ...proximity of tig, the gene coding for TF, and genes coding for ClpXP, suggesting a functional interaction. The effect of TF on ClpXP-dependent degradation varies based on the nature of substrate. While degradation of some substrates are slowed down or are unaffected by TF, surprisingly, TF increases the degradation rate of a third class of substrates. These include λ phage replication protein λO, master regulator of stationary phase RpoS, and SsrA-tagged proteins. Globally, TF acts to enhance the degradation of about 2% of newly synthesized proteins. TF is found to interact through multiple sites with ClpX in a highly dynamic fashion to promote protein degradation. This chaperone-protease cooperation constitutes a unique and likely ancestral aspect of cellular protein homeostasis in which TF acts as an adaptor for ClpXP.
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