Structural and Kinetic Views of Molecular Chaperones in Multidomain Protein Folding

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

Dehydration of main-chain amides in the final folding step of single-chain monellin revealed by time-resolved infrared spectroscopy

Kinetic IR spectroscopy was used to reveal β-sheet formation and water expulsion in the folding of single-chain monellin (SMN) composed of a five-stranded β-sheet and an α-helix. The time-resolved IR spectra between 100 μs and 10 s were analyzed based on two consecutive intermediates, I1 and I2, appearing within 100 μs and with a time constant of ≈100 ms, respectively. The initial unfolded state showed broad amide I′ corresponded to a fluctuating conformation. In contrast, I1 possessed a feature at 1,636 cm−1 for solvated helix and weak features assignable to turns, demonstrating the rapid formation of helix and turns. I2 possessed a line for solvated helix at 1,637 cm−1 and major and minor lines for β-sheet at 1,625 and 1,680 cm−1, respectively. The splitting of the major and minor lines is smaller than that of the native state, implying an incomplete formation of the β-sheet. Furthermore, both major and minor lines demonstrated a low-frequency shift compared to those of the native state, which was interpreted to be caused by hydration of the C=O group in the β-sheet. Together with the identification of solvated helix, the core domain of I2 was interpreted as being hydrated. Finally, slow conversion of the water-penetrated core of I2 to the dehydrated core of the native state was observed. We propose that both the expulsion of water, hydrogen-bonded to main-chain amides, and the completion of the secondary structure formation contribute to the energetic barrier of the rate-limiting step in SMN folding

Zinc-Dependent Oligomerization of Thermus thermophilus Trigger Factor Chaperone

Metal ions often play important roles in biological processes. Thermus thermophilus trigger factor (TtTF) is a zinc-dependent molecular chaperone where Zn2+ has been shown to enhance its folding-arrest activity. However, the mechanisms of how Zn2+ binds to TtTF and how Zn2+ affects the activity of TtTF are yet to be elucidated. As a first step in understanding the mechanism, we performed in vitro biophysical experiments on TtTF to investigate the zinc-binding site on TtTF and unveil how Zn2+ alters the physical properties of TtTF, including secondary structure, thermal stability, and oligomeric state. Our results showed that TtTF binds Zn2+ in a 1:1 ratio, and all three domains of TtTF are involved in zinc-binding. We found that Zn2+ does not affect the thermal stability of TtTF, whereas it does induce partial structural change and promote the oligomerization of TtTF. Given that the folding-arrest activity of Escherichia coli TF (EcTF) is regulated by its oligomerization, our results imply that TtTF exploits Zn2+ to modulate its oligomeric state to regulate the activity.Thermus thermophilus trigger factor (TtTF) is a zinc-dependent molecular chaperone whose folding-arrest activity is regulated by Zn2+. However, little is known about the mechanism of zinc-dependent regulation of the TtTF activity. Here we exploit in vitro biophysical experiments to investigate zinc-binding, the oligomeric state, the secondary structure, and the thermal stability of TtTF in the absence and presence of Zn2+. The data show that full-length TtTF binds Zn2+, but the isolated domains and tandem domains of TtTF do not bind to Zn2+. Furthermore, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectra suggested that Zn2+-binding induces the partial structural changes of TtTF, and size exclusion chromatography-multi-angle light scattering (SEC-MALS) showed that Zn2+ promotes TtTF oligomerization. Given the previous work showing that the activity regulation of E. coli trigger factor is accompanied by oligomerization, the Data suggest that TtTF exploits zinc ions to induce the structural change coupled with the oligomerization to assemble the client-binding site, thereby effectively preventing proteins from misfolding in the thermal environment

Functional cooperativity between the trigger factor chaperone and the ClpXP proteolytic complex

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

C9orf72-derived arginine-rich poly-dipeptides impede phase modifiers

Nuclear import receptors (NIRs) not only transport RNA-binding proteins (RBPs) but also modify phase transitions of RBPs by recognizing nuclear localization signals (NLSs). Toxic arginine-rich poly-dipeptides from C9orf72 interact with NIRs and cause nucleocytoplasmic transport deficit. However, the molecular basis for the toxicity of arginine-rich poly-dipeptides toward NIRs function as phase modifiers of RBPs remains unidentified. Here we show that arginine-rich poly-dipeptides impede the ability of NIRs to modify phase transitions of RBPs. Isothermal titration calorimetry and size-exclusion chromatography revealed that proline:arginine (PR) poly-dipeptides tightly bind karyopherin-β2 (Kapβ2) at 1:1 ratio. The nuclear magnetic resonances of Kapβ2 perturbed by PR poly-dipeptides partially overlapped with those perturbed by the designed NLS peptide, suggesting that PR poly-dipeptides target the NLS binding site of Kapβ2. The findings offer mechanistic insights into how phase transitions of RBPs are disabled in C9orf72-related neurodegeneration

酸素結合ヘム蛋白質における構造と機能の分子制御に関する研究

京都大学0048新制・課程博士工学博士甲第4280号工博第1074号新制||工||761(附属図書館)UT51-89-G123京都大学大学院工学研究科分子工学専攻(主査)教授 本多 健一, 教授 山邊 時雄, 教授 清水 剛夫学位規則第5条第1項該当Kyoto UniversityDFA

Converting cytochrome c into a DyP-like metalloenzyme

Dye-decolorizing peroxidase (DyP), which can degrade anthraquinone dyes using H2O2, is an attractive prospect for potential biotechnological applications for environmental purification. We previously designed an artificial DyP with an optimal pH for reactive blue 19 (RB19) degradation shifting from pH 4.5 to 6.5. We then attempted to degrade RB19 using Escherichia coli expressing this mutant, but RB19 was degraded equally compared with bacteria expressing wild-type (WT) DyP because most DyP was expressed in a heme-free form. In this study, we attempted to design an artificial peroxidase based on cytochrome c (cyt c), whose heme is covalently bound to the protein. We found that cyt c can degrade RB19, but its ability at pH 7.0 was ~60% of that of DyP from Vibrio cholerae at pH 4.5. To enhance this activity we constructed several mutants using three approaches. Initially, to improve reactivity with H2O2, Met80 was replaced with a noncoordinating residue, Ala or Val, but catalytic efficiency (kcat/Km) was increased by only ~1.5-fold. To enhance the substrate binding affinity we introduced an additional Trp by replacing Pro76 (P76W). The catalytic efficiency of this mutant was ~3-fold greater than that of WT cyt c. Finally, to form a hydrogen bond to axial histidine Gly29 was replaced with Asp (G29D). This mutant exhibited an ~80-fold greater dye-decolorizing activity. Escherichia coli expressing the G29D mutant was unable to degrade RB19 in solution due to degradation of heme itself, but this study provides new insights into the design of artificial DyPs

Dual role of the active-center cysteine in human peroxiredoxin 1 : Peroxidase activity and heme binding

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 mu M. 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 (similar to 20 mu M), PRX1 thus functions as a scavenger of cytosolic hemin (<1 mu M). Collectively, our results indicate that PRX1 has a dual role; Cys-dependent peroxidase activity and cytosolic heme scavenger. (C) 2017 Elsevier Inc. All rights reserved