Conformational Distribution of a Multidomain Protein Measured by Single-Pair Small-Angle X-ray Scattering.

The difficulty in evaluating the conformational distribution of proteins in solution often hinders mechanistic insights. One possible strategy for visualizing conformational distribution is distance distribution measurement by single-pair small-angle X-ray scattering (SAXS), in which the scattering interference from only a specific pair of atoms in the target molecule is extracted. Despite this promising concept, with few applications in synthetic small molecules and DNA, technical difficulties have prevented its application in protein conformational studies. This study used a synthetic tag to fix the lanthanide ion at desired sites on the protein and used single-pair SAXS with contrast matching to evaluate the conformational distribution of the multidomain protein enzyme MurD. These data highlighted the broad conformational and ligand-driven distribution shifts of MurD in solution. This study proposes an important strategy in solution structural biology that targets dynamic proteins, including multidomain and intrinsically disordered proteins.

Regulation of electron transfer in the terminal step of the respiratory chain.

In mitochondria, electrons are transferred along a series of enzymes and electron carriers that are referred to as the respiratory chain, leading to the synthesis of cellular ATP. The series of the interprotein electron transfer (ET) reactions is terminated by the reduction in molecular oxygen at Complex IV, cytochrome c oxidase (CcO) that is coupled with the proton pumping from the matrix to the inner membrane space. Unlike the ET reactions from Complex I to Complex III, the ET reaction to CcO, mediated by cytochrome c (Cyt c), is quite specific in that it is irreversible with suppressed electron leakage, which characterizes the ET reactions in the respiratory chain and is thought to play a key role in the regulation of mitochondrial respiration. In this review, we summarize the recent findings regarding the molecular mechanism of the ET reaction from Cyt c to CcO in terms of specific interaction between two proteins, a molecular breakwater, and the effects of the conformational fluctuation on the ET reaction, conformational gating. Both of these are essential factors, not only in the ET reaction from Cyt c to CcO, but also in the interprotein ET reactions in general. We also discuss the significance of a supercomplex in the terminal ET reaction, which provides information on the regulatory factors of the ET reactions that are specific to the mitochondrial respiratory chain.

Unveiling the impact of oxidation-driven endogenous protein interactions on the dynamics of amyloid-ß aggregation and toxicity.

Cytochrome c (Cyt c), a multifunctional protein with a crucial role in controlling cell fate, has been implicated in the amyloid pathology associated with Alzheimer's disease (AD); however, the interaction between Cyt c and amyloid-ß (Aß) with the consequent impact on the aggregation and toxicity of Aß is not known. Here we report that Cyt c can directly bind to Aß and alter the aggregation and toxicity profiles of Aß in a manner that is dependent on the presence of a peroxide. When combined with hydrogen peroxide (H2O2), Cyt c redirects Aß peptides into less toxic, off-pathway amorphous aggregates, whereas without H2O2, it promotes Aß fibrillization. The mechanisms behind these effects may involve a combination of the complexation between Cyt c and Aß, the oxidation of Aß by Cyt c and H2O2, and the modification of Cyt c by H2O2. Our findings demonstrate a new function of Cyt c as a modulator against Aß amyloidogenesis.

Heat-Induced Conformational Transition Mechanism of Heat Shock Factor 1 Investigated by Tryptophan Probe.

A transcriptional regulatory system called heat shock response (HSR) has been developed in eukaryotic cells to maintain proteome homeostasis under various stresses. Heat shock factor-1 (Hsf1) plays a central role in HSR, mainly by upregulating molecular chaperones as a transcription factor. Hsf1 forms a complex with chaperones and exists as a monomer in the resting state under normal conditions. However, upon heat shock, Hsf1 is activated by oligomerization. Thus, oligomerization of Hsf1 is considered an important step in HSR. However, the lack of information about Hsf1 monomer structure in the resting state, as well as the structural change via oligomerization at heat response, impeded the understanding of the thermosensing mechanism through oligomerization. In this study, we applied solution biophysical methods, including fluorescence spectroscopy, nuclear magnetic resonance, and circular dichroism spectroscopy, to investigate the heat-induced conformational transition mechanism of Hsf1 leading to oligomerization. Our study showed that Hsf1 forms an inactive closed conformation mediated by intramolecular contact between leucine zippers (LZs), in which the intermolecular contact between the LZs for oligomerization is prevented. As the temperature increases, Hsf1 changes to an open conformation, where the intramolecular LZ interaction is dissolved so that the LZs can form intermolecular contacts to form oligomers in the active form. Furthermore, since the interaction sites with molecular chaperones and nuclear transporters are also expected to be exposed in the open conformation, the conformational change to the open state can lead to understanding the regulation of Hsf1-mediated stress response through interaction with multiple cellular components.

Heme binding to cold shock protein D, CspD, from Vibrio cholerae.

Cold shock protein D (CspD) is one of the homologous proteins of cold shock protein A (CspA), inhibiting DNA replication by binding to single-stranded DNA. We found that CspD from Vibrio cholerae (VcCspD) possesses one heme regulatory motif (HRM) sequence and specifically binds heme with a stoichiometry of 1:1. The binding of a synthetic single-stranded DNA oligomer (ssDNA) was followed by fluorescence quenching of Trp. The fluorescence quenching associated with the addition of ssDNA was suppressed in the presence of heme, indicating that heme binding to VcCspD inhibited the formation of the VcCspD-ssDNA complex. Such heme-induced inhibition was not observed for the VcCspD mutant with replacement of Cys22 in the HRM with alanine (C22A). Heme binding at Cys22 is, therefore, essential for the inhibition of ssDNA binding for VcCspD. The growth of Escherichia coli at 37 °C was slowed when VcCspD was overexpressed, indicating that VcCspD hampers the growth of E. coli. When the production of heme in cells was promoted by the addition of a heme precursor, δ-aminolevulinic acid, the growth of E. coli expressing VcCspD was decelerated, but the growth of E. coli expressing the C22A mutant was not decelerated. These observations allow us to conclude that heme specifically binds to the HRM region in VcCspD and inhibits the binding of target ssDNA, which suggests that heme functions as a regulatory molecule for DNA replication.

Metal Sensing by a Glycine-Histidine Repeat Sequence Regulates the Heme Degradation Activity of PM0042 from Pasteurella multocida.

PM0042 protein from the Gram-negative bacterial pathogen Pasteurella multocida is homologous to the heme-degrading enzyme HutZ belonging to the pyridoxine-5-phosphate oxidase-like family. A characteristic feature of PM0042 is possession of a glycine-histidine (GH) repeat sequence at the C-terminal region. In this study, we examined the heme degradation ability of PM0042, with a particular focus on the role of the GH repeat sequence. PM0042 was expressed in Escherichia coli and successfully purified using a nickel (Ni2+)-affinity column without a histidine tag, suggesting that its GH motif facilitates binding to Ni2+. Reaction with ascorbic acid induced a significant decrease in the Soret band, suggesting the breakage of heme. While a Fe2+-ferrozine complex was not formed upon addition of ferrozine to the solution after the reaction, prior addition of metal ions to fill the metal binding site in the GH repeat sequence led to increased complex formation. In the presence of Fe2+, the heme degradation rate was accelerated ∼threefold, supporting the theory that Fe2+ binds the PM0042 protein (possibly at the GH repeat sequence) and enhances its heme degradation activity. In contrast to HutZ from Vibrio cholerae in which enzymatic activity is regulated by the protonation status of the heme proximal ligand, heme reduction is not the rate-determining step for PM0042. Rather, proton transfer to reduced oxyheme is affected, as established with the H2O/D2O isotope experiment. Based on the collective findings, the GH repeat sequence of PM0042 is proposed to function as a metal sensor that modulates iron uptake via the heme-degrading process in P. multocida.

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.

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.

Regulation of the expression of the nickel uptake system in Vibrio cholerae by iron and heme via ferric uptake regulator (Fur).

Fur (ferric uptake regulator) is a transcription factor that regulates expression of downstream genes containing a specific Fe2+-binding sequence called the Fur box. In Vibrio cholerae, a Fur box is located upstream of the nik operon, which is responsible for nickel uptake, suggesting that its expression is regulated by Fur. However, there are no reports that Ni2+ induces expression of Fur box genes. Accordingly, we here investigated whether Ni2+ or Fe2+ binds to Fur to regulate expression of the nik operon. We found that Fur binds to the Fur box in the presence of Fe2+ with a dissociation constant (Kd) of 1.2 µM, whereas only non-specific binding was observed in the presence of Ni2+. Thus, Fur-mediated expression of the nik operon is dependent on Fe2+, but not Ni2+. Since most iron in cells exists as heme, we examined the effect of heme on the Fur box binding activity of V. cholerae Fur (VcFur). Addition of heme to the VcFur-Fur box complex induced dissociation of VcFur from the Fur box, indicating that expression of the V. cholerae nik operon is regulated by both iron and heme. Furthermore, VCA1098, a nik operon-encoded protein, bound heme with a Kd of 1.3 µM. Collectively, our results suggest that the V. cholerae nik operon is involved not only in nickel uptake but also in heme uptake, and depends on iron and heme concentrations within bacteria.

Zinc-Dependent Oligomerization of Thermus thermophilus Trigger Factor Chaperone.

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.

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-ß2 (Kapß2) at 1:1 ratio. The nuclear magnetic resonances of Kapß2 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 Kapß2. The findings offer mechanistic insights into how phase transitions of RBPs are disabled in C9orf72-related neurodegeneration.

Conformational ensemble of a multidomain protein explored by Gd3+ electron paramagnetic resonance.

Despite their importance in function, the conformational state of proteins and its changes are often poorly understood, mainly because of the lack of an efficient tool. MurD, a 47-kDa protein enzyme responsible for peptidoglycan biosynthesis, is one of those proteins whose conformational states and changes during their catalytic cycle are not well understood. Although it has been considered that MurD takes a single conformational state in solution as shown by a crystal structure, the solution nuclear magnetic resonance (NMR) study suggested the existence of multiple conformational state of apo MurD in solution. However, the conformational distribution has not been evaluated. In this work, we investigate the conformational states of MurD by the use of electron paramagnetic resonance (EPR), especially intergadolinium distance measurement using double electron-electron resonance (DEER) measurement. The gadolinium ions are fixed on specific positions on MurD via a rigid double-arm paramagnetic lanthanide tag that has been originally developed for paramagnetic NMR. The combined use of NMR and EPR enables accurate interpretation of the DEER distance information to the structural information of MurD. The DEER distance measurement for apo MurD shows a broad distance distribution, whereas the presence of the inhibitor narrows the distance distribution. The results suggest that MurD exists in a wide variety of conformational states in the absence of ligands, whereas binding of the inhibitor eliminates variation in conformational states. The multiple conformational states of MurD were previously implied by NMR experiments, but our DEER data provided structural characterization of the conformational variety of MurD.

Radical transfer but not heme distal residues is essential for pH dependence of dye-decolorizing activity of peroxidase from Vibrio cholerae.

Dye-decolorizing peroxidase (DyP) is a heme-containing enzyme that catalyzes the degradation of anthraquinone dyes. A main feature of DyP is the acidic optimal pH for dye-decolorizing activity. In this study, we constructed several mutant DyP enzymes from Vibrio cholerae (VcDyP), with a view to identifying the decisive factor of the low pH preference of DyP. Initially, distal Asp144, a conserved residue, was replaced with His, which led to significant loss of dye-decolorizing activity. Introduction of His into a position slightly distant from heme resulted in restoration of activity but no shift in optimal pH, indicating that distal residues do not contribute to the pH dependence of catalytic activity. His178, an essential residue for dye decolorization, is located near heme and forms hydrogen bonds with Asp138 and Thr278. While Trp and Tyr mutants of His178 were inactive, the Phe mutant displayed ~35% activity of wild-type VcDyP, indicating that this position is a potential radical transfer route from heme to the active site on the protein surface. The Thr278Val mutant displayed similar enzymatic properties as WT VcDyP, whereas the Asp138Val mutant displayed significantly increased activity at pH 6.5. On the basis of these findings, we propose that neither distal amino acid residues, including Asp144, nor hydrogen bonds between His178 and Thr278 are responsible while the hydrogen bond between His178 and Asp138 plays a key role in the pH dependence of activity.

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.

Mechanistic insights into heme-mediated transcriptional regulation via a bacterial manganese-binding iron regulator, iron response regulator (Irr).

The transcription factor iron response regulator (Irr) is a key regulator of iron homeostasis in the nitrogen-fixating bacterium Bradyrhizobium japonicum Irr acts by binding to target genes, including the iron control element (ICE), and is degraded in response to heme binding. Here, we examined this binding activity using fluorescence anisotropy with a 6-carboxyfluorescein-labeled ICE-like oligomer (FAM-ICE). In the presence of Mn2+, Irr addition increased the fluorescence anisotropy, corresponding to formation of the Irr-ICE complex. The addition of EDTA to the Irr-ICE complex reduced fluorescence anisotropy, but fluorescence was recovered after Mn2+ addition, indicating that Mn2+ binding is a prerequisite for complex formation. Binding activity toward ICE was lost upon introduction of substitutions in a His-cluster region of Irr, revealing that Mn2+ binds to this region. We observed that the His-cluster region is also the heme binding site; results from fluorescence anisotropy and electrophoretic mobility shift analyses disclosed that the addition of a half-equivalent of heme dissociates Irr from ICE, likely because of Mn2+ release due to heme binding. We hypothesized that heme binding to another heme binding site, Cys-29, would also inhibit the formation of the Irr-ICE complex because it is proximal to the ICE binding site, which was supported by the loss of ICE binding activity in a Cys-29-mutated Irr. These results indicate that Irr requires Mn2+ binding to form the Irr-ICE complex and that the addition of heme dissociates Irr from ICE by replacing Mn2+ with heme or by heme binding to Cys-29.

Spectroscopic Characterization of Halorhodopsin Reconstituted into Nanodisks Using Native Lipids.

We successfully reconstituted single Natronomonas pharaonis halorhodopsin (NpHR) trimers into a nanodisk (ND) using the native archaeal lipid (NL) and an artificial lipid having a zwitterionic headgroup, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Incorporation of single trimeric NpHR into NDs was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, size-exclusion chromatography, and visible circular dichroism spectroscopy. The Cl- binding affinity of NpHR in NDs using NL (NL-ND NpHR) or POPC (POPC-ND NpHR) was examined by absorption spectroscopy, showing that the Cl--releasing affinities (Kd,N↔O) of these ND-reconstituted NpHRs are more than 10 times higher than that obtained from native NpHR membrane fragments (MFs) harvested from a NpHR-overexpressing archaeal strain (MF NpHR). The photoreaction kinetics of these ND-reconstituted NpHRs revealed that the Cl- uptake was faster than that of MF NpHR. These differences in the Cl--releasing and uptake properties of ND-reconstituted NpHRs and MF NpHR may arise from suppression of protein conformational changes associated with Cl- release from the trimeric NpHR caused by ND reconstitution, conformational perturbation in the trimeric state, and loss of the trimer-trimer interactions. On the other hand, POPC-ND NpHR demonstrated accelerated Cl- uptake compared to NL-ND NpHR, suggesting that the negative charge on the archaeal membrane surface regulates the photocycle of NpHR. Although NL-ND NpHR and MF NpHR are embedded in the same lipid, the lower Cl--binding affinity at the initial state (Kd,initial) and faster recovering from the NpHR' state to the original state of the photoreaction cycle were observed for NL-ND NpHR, probably because of insufficient interactions with a chromophore in the native membrane, bacterioruberin in reconstituted NDs. Our results indicate that specific interactions of NpHR with surrounding lipids and bacterioruberin, structural flexibility of the membrane, and interactions between trimeric NpHRs may be necessary for efficient Cl- pumping.