Mzm1 Influences a Labile Pool of Mitochondrial Zinc Important for Respiratory Function

Zinc is essential for function of mitochondria as a cofactor for several matrix zinc metalloproteins. We demonstrate that a labile cationic zinc component of low molecular mass exists in the yeast mitochondrial matrix. This zinc pool is homeostatically regulated in response to the cellular zinc status. This pool of zinc is functionally important because matrix targeting of a cytosolic zinc-binding protein reduces the level of labile zinc and interferes with mitochondrial respiratory function. We identified a series of proteins that modulate the matrix zinc pool, one of which is a novel conserved mitochondrial protein designated Mzm1. Mutant mzm1Δ cells have reduced total and labile mitochondrial zinc, and these cells are hypersensitive to perturbations of the labile pool. In addition, mzm1Δ cells have a destabilized cytochrome c reductase (Complex III) without any effects on Complexes IV or V. Thus, we have established that a link exists between Complex III integrity and the labile mitochondrial zinc pool.     

Activation of the SARS-CoV-2 NSP14 3′–5′ exoribonuclease by NSP10 and response to antiviral inhibitors

Understanding the core replication complex of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to the development of novel coronavirus-specific antiviral therapeutics. Among the proteins required for faithful replication of the SARS-CoV-2 genome are nonstructural protein 14 (NSP14), a bifunctional enzyme with an N-terminal 3′-to-5′ exoribonuclease (ExoN) and a C-terminal N7-methyltransferase, and its accessory protein, NSP10. The difficulty in producing pure and high quantities of the NSP10/14 complex has hampered the biochemical and structural study of these important proteins. We developed a straightforward protocol for the expression and purification of both NSP10 and NSP14 from Escherichia coli and for the in vitro assembly and purification of a stoichiometric NSP10/14 complex with high yields. Using these methods, we observe that NSP10 provides a 260-fold increase in k_cat/K_m in the exoribonucleolytic activity of NSP14 and enhances protein stability. We also probed the effect of two small molecules on NSP10/14 activity, remdesivir monophosphate and the methyltransferase inhibitor S-adenosylhomocysteine. Our analysis highlights two important factors for drug development: first, unlike other exonucleases, the monophosphate nucleoside analog intermediate of remdesivir does not inhibit NSP14 activity; and second, S-adenosylhomocysteine modestly activates NSP14 exonuclease activity. In total, our analysis provides insights for future structure–function studies of SARS-CoV-2 replication fidelity for the treatment of coronavirus disease 2019.     

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III ε subunit (featuring 3’-5’ exonuclease activity) is highly improved when the same protein is co-expressed with the α and θ subunits (featuring DNA polymerase activity and stabilizing ε, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the α subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the ε subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type ε subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-β-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.     

Protein yoga: Conformational versatility of the Hemolysin II C‐terminal domain detailed by NMR structures for multiple states

The C‐terminal domain of Bacillus cereus hemolysin II (HlyIIC), stabilizes the trans‐membrane‐pore formed by the HlyII toxin and may aid in target cell recognition. Initial efforts to determine the NMR structure of HlyIIC were hampered by cis/trans isomerization about the single proline at position 405 that leads to doubling of NMR resonances. We used the mutant P405M‐HlyIIC that eliminates the cis proline to determine the NMR structure of the domain, which revealed a novel fold. Here, we extend earlier studies to the NMR structure determination of the cis and trans states of WT‐HlyIIC that exist simultaneously in solution. The primary structural differences between the cis and trans states are in the loop that contains P405, and structurally adjacent loops. Thermodynamic linkage analysis shows that at 25 C the cis proline, which already has a large fraction of 20% in the unfolded protein, increases to 50% in the folded state due to coupling with the global stability of the domain. The P405M or P405A substitutions eliminate heterogeneity due to proline isomerization but lead to the formation of a new dimeric species. The NMR structure of the dimer shows that it is formed through domain‐swapping of strand β5, the last segment of secondary structure following P405. The presence of P405 in WT‐HlyIIC strongly disfavors the dimer compared to the P405M‐HlyIIC or P405A‐HlyIIC mutants. The WT proline may thus act as a “gatekeeper,” warding off aggregative misfolding.     

Turning Saccharomyces cerevisiae into a Frataxin-Independent Organism

Frataxin (Yfh1 in yeast) is a conserved protein and deficiency leads to the neurodegenerative disease Friedreich’s ataxia. Frataxin is a critical protein for Fe-S cluster assembly in mitochondria, interacting with other components of the Fe-S cluster machinery, including cysteine desulfurase Nfs1, Isd11 and the Isu1 scaffold protein. Yeast Isu1 with the methionine to isoleucine substitution (M141I), in which the E. coli amino acid is inserted at this position, corrected most of the phenotypes that result from lack of Yfh1 in yeast. This suppressor Isu1 behaved as a genetic dominant. Furthermore frataxin-bypass activity required a completely functional Nfs1 and correlated with the presence of efficient scaffold function. A screen of random Isu1 mutations for frataxin-bypass activity identified only M141 substitutions, including Ile, Cys, Leu, or Val. In each case, mitochondrial Nfs1 persulfide formation was enhanced, and mitochondrial Fe-S cluster assembly was improved in the absence of frataxin. Direct targeting of the entire E. coli IscU to ∆yfh1 mitochondria also ameliorated the mutant phenotypes. In contrast, expression of IscU with the reverse substitution i.e. IscU with Ile to Met change led to worsening of the ∆yfh1 phenotypes, including severely compromised growth, increased sensitivity to oxygen, deficiency in Fe-S clusters and heme, and impaired iron homeostasis. A bioinformatic survey of eukaryotic Isu1/prokaryotic IscU database entries sorted on the amino acid utilized at the M141 position identified unique groupings, with virtually all of the eukaryotic scaffolds using Met, and the preponderance of prokaryotic scaffolds using other amino acids. The frataxin-bypassing amino acids Cys, Ile, Leu, or Val, were found predominantly in prokaryotes. This amino acid position 141 is unique in Isu1, and the frataxin-bypass effect likely mimics a conserved and ancient feature of the prokaryotic Fe-S cluster assembly machinery.     

The Complex Energy Landscape of the Protein IscU

IscU, the scaffold protein for iron-sulfur (Fe-S) cluster biosynthesis in Escherichia coli, traverses a complex energy landscape during Fe-S cluster synthesis and transfer. Our previous studies showed that IscU populates two interconverting conformational states: one structured (S) and one largely disordered (D). Both states appear to be functionally important because proteins involved in the assembly or transfer of Fe-S clusters have been shown to interact preferentially with either the S or D state of IscU. To characterize the complex structure-energy landscape of IscU, we employed NMR spectroscopy, small-angle x-ray scattering (SAXS), and differential scanning calorimetry. Results obtained for IscU at pH 8.0 show that its S state is maximally populated at 25°C and that heating or cooling converts the protein toward the D state. Results from NMR and DSC indicate that both the heat- and cold-induced S→D transitions are cooperative and two-state. Low-resolution structural information from NMR and SAXS suggests that the structures of the cold-induced and heat-induced D states are similar. Both states exhibit similar ¹H-¹⁵N HSQC spectra and the same pattern of peptidyl-prolyl peptide bond configurations by NMR, and both appear to be similarly expanded compared with the S state based on analysis of SAXS data. Whereas in other proteins the cold-denatured states have been found to be slightly more compact than the heat-denatured states, these two states occupy similar volumes in IscU.     

The Zinc Center Influences the Redox and Thermodynamic Properties of Escherichia coli Thioredoxin 2

Thioredoxins are small, ubiquitous redox enzymes that reduce protein disulfide bonds by using a pair of cysteine residues present in a strictly conserved WCGPC catalytic motif. The Escherichia coli cytoplasm contains two thioredoxins, Trx1 and Trx2. Trx2 is special because it is induced under oxidative stress conditions and it has an additional N-terminal zinc-binding domain. We have determined the redox potential of Trx2, the pK_a of the active site nucleophilic cysteine, as well as the stability of the oxidized and reduced form of the protein. Trx2 is more oxidizing than Trx1 (-221 mV versus -284 mV, respectively), which is in good agreement with the decreased value of the pK_a of the nucleophilic cysteine (5.1 versus 7.1, respectively). The difference in stability between the oxidized and reduced forms of an oxidoreductase is the driving force to reduce substrate proteins. This difference is smaller for Trx2 (ΔΔG°_H2O = 9 kJ/mol and ΔT_m = 7. 4 °C) than for Trx1 (ΔΔG°_H2O = 15 kJ/mol and ΔT_m = 13 °C). Altogether, our data indicate that Trx2 is a significantly less reducing enzyme than Trx1, which suggests that Trx2 has a distinctive function. We disrupted the zinc center by mutating the four Zn²⁺-binding cysteines to serine. This mutant has a more reducing redox potential (-254 mV) and the pK_a of its nucleophilic cysteine shifts from 5.1 to 7.1. The removal of Zn²⁺ also decreases the overall stability of the reduced and oxidized forms by 3.2 kJ/mol and 5.8 kJ/mol, respectively. In conclusion, our data show that the Zn²⁺-center of Trx2 fine-tunes the properties of this unique thioredoxin.     

Co-fibrillogenesis of Wild-type and D76N β2-Microglobulin: THE CRUCIAL ROLE OF FIBRILLAR SEEDS*

The amyloidogenic variant of β₂-microglobulin, D76N, can readily convert into genuine fibrils under physiological conditions and primes in vitro the fibrillogenesis of the wild-type β₂-microglobulin. By Fourier transformed infrared spectroscopy, we have demonstrated that the amyloid transformation of wild-type β₂-microglobulin can be induced by the variant only after its complete fibrillar conversion. Our current findings are consistent with preliminary data in which we have shown a seeding effect of fibrils formed from D76N or the natural truncated form of β₂-microglobulin lacking the first six N-terminal residues. Interestingly, the hybrid wild-type/variant fibrillar material acquired a thermodynamic stability similar to that of homogenous D76N β₂-microglobulin fibrils and significantly higher than the wild-type homogeneous fibrils prepared at neutral pH in the presence of 20% trifluoroethanol. These results suggest that the surface of D76N β₂-microglobulin fibrils can favor the transition of the wild-type protein into an amyloid conformation leading to a rapid integration into fibrils. The chaperone crystallin, which is a mild modulator of the lag phase of the variant fibrillogenesis, potently inhibits fibril elongation of the wild-type even once it is absorbed on D76N β₂-microglobulin fibrils.     

Beta turn propensity and a model polymer scaling exponent identify intrinsically disordered phase-separating proteins

The complex cellular milieu can spontaneously demix, or phase separate, in a process controlled in part by intrinsically disordered (ID) proteins. A protein''s propensity to phase separate is thought to be driven by a preference for protein–protein over protein–solvent interactions. The hydrodynamic size of monomeric proteins, as quantified by the polymer scaling exponent (v), is driven by a similar balance. We hypothesized that mean v, as predicted by protein sequence, would be smaller for proteins with a strong propensity to phase separate. To test this hypothesis, we analyzed protein databases containing subsets of proteins that are folded, disordered, or disordered and known to spontaneously phase separate. We find that the phase-separating disordered proteins, on average, had lower calculated values of v compared with their non-phase-separating counterparts. Moreover, these proteins had a higher sequence-predicted propensity for β-turns. Using a simple, surface area-based model, we propose a physical mechanism for this difference: transient β-turn structures reduce the desolvation penalty of forming a protein-rich phase and increase exposure of atoms involved in π/sp² valence electron interactions. By this mechanism, β-turns could act as energetically favored nucleation points, which may explain the increased propensity for turns in ID regions (IDRs) utilized biologically for phase separation. Phase-separating IDRs, non-phase-separating IDRs, and folded regions could be distinguished by combining v and β-turn propensity. Finally, we propose a new algorithm, ParSe (partition sequence), for predicting phase-separating protein regions, and which is able to accurately identify folded, disordered, and phase-separating protein regions based on the primary sequence.     

The A-kinase Anchoring Protein GSKIP Regulates GSK3β Activity and Controls Palatal Shelf Fusion in Mice

A-kinase anchoring proteins (AKAPs) represent a family of structurally diverse proteins, all of which bind PKA. A member of this family is glycogen synthase kinase 3β (GSK3β) interaction protein (GSKIP). GSKIP interacts with PKA and also directly interacts with GSK3β. The physiological function of the GSKIP protein in vivo is unknown. We developed and characterized a conditional knock-out mouse model and found that GSKIP deficiency caused lethality at birth. Embryos obtained through Caesarean section at embryonic day 18.5 were cyanotic, suffered from respiratory distress, and failed to initiate breathing properly. Additionally, all GSKIP-deficient embryos showed an incomplete closure of the palatal shelves accompanied by a delay in ossification along the fusion area of secondary palatal bones. On the molecular level, GSKIP deficiency resulted in decreased phosphorylation of GSK3β at Ser-9 starting early in development (embryonic day 10.5), leading to enhanced GSK3β activity. At embryonic day 18.5, GSK3β activity decreased to levels close to that of wild type. Our findings reveal a novel, crucial role for GSKIP in the coordination of GSK3β signaling in palatal shelf fusion.     

Cooperative Functions of ZnT1, Metallothionein and ZnT4 in the Cytoplasm Are Required for Full Activation of TNAP in the Early Secretory Pathway

The activation process of secretory or membrane-bound zinc enzymes is thought to be a highly coordinated process involving zinc transport, trafficking, transfer and coordination. We have previously shown that secretory and membrane-bound zinc enzymes are activated in the early secretory pathway (ESP) via zinc-loading by the zinc transporter 5 (ZnT5)-ZnT6 hetero-complex and ZnT7 homo-complex (zinc transport complexes). However, how other proteins conducting zinc metabolism affect the activation of these enzymes remains unknown. Here, we investigated this issue by disruption and re-expression of genes known to be involved in cytoplasmic zinc metabolism, using a zinc enzyme, tissue non-specific alkaline phosphatase (TNAP), as a reporter. We found that TNAP activity was significantly reduced in cells deficient in ZnT1, Metallothionein (MT) and ZnT4 genes (ZnT1 ^−/− MT ^−/− ZnT4 ^−/− cells), in spite of increased cytosolic zinc levels. The reduced TNAP activity in ZnT1 ^−/− MT ^−/− ZnT4 ^−/− cells was not restored when cytosolic zinc levels were normalized to levels comparable with those of wild-type cells, but was reversely restored by extreme zinc supplementation via zinc-loading by the zinc transport complexes. Moreover, the reduced TNAP activity was adequately restored by re-expression of mammalian counterparts of ZnT1, MT and ZnT4, but not by zinc transport-incompetent mutants of ZnT1 and ZnT4. In ZnT1 ^−/− MT ^−/− ZnT4 ^−/− cells, the secretory pathway normally operates. These findings suggest that cooperative zinc handling of ZnT1, MT and ZnT4 in the cytoplasm is required for full activation of TNAP in the ESP, and present clear evidence that the activation process of zinc enzymes is elaborately controlled.     

Functional characterization of Fdx1: evidence for an evolutionary relationship between P450-type and ISC-type ferredoxins

Ferredoxins are ubiquitous proteins with electron transfer activity involved in a variety of biological processes. In this work, we investigated the characteristics and function of Fdx1 from Sorangium cellulosum So ce56 by using a combination of bioinformatics and of biochemical/biophysical approaches. We were able to experimentally confirm a role of Fdx1 in the iron-sulfur cluster biosynthesis by in vitro reduction studies with cluster-loaded So ce56 IscU and by transfer studies of the cluster from the latter protein to apo-aconitase A. Moreover, we found that Fdx1 can replace mammalian adrenodoxin in supporting the activity of bovine CYP11A1. This makes S. cellulosum Fdx1 the first prokaryotic ferredoxin reported to functionally interact with this mammalian enzyme. Although the interaction with CYP11A1 is non-physiological, this is—to the best of our knowledge—the first study to experimentally prove the activity of a postulated ISC-type ferredoxin in both the ISC assembly and a cytochrome P450 system. This proves that a single ferredoxin can be structurally able to provide electrons to both cytochromes P450 and IscU and thus support different biochemical processes. Combining this finding with phylogenetic and evolutionary trace analyses led us to propose the evolution of eukaryotic mitochondrial P450-type ferredoxins and ISC-type ferredoxins from a common prokaryotic ISC-type ancestor.     

Turning Escherichia coli into a Frataxin-Dependent Organism

Fe-S bound proteins are ubiquitous and contribute to most basic cellular processes. A defect in the ISC components catalyzing Fe-S cluster biogenesis leads to drastic phenotypes in both eukaryotes and prokaryotes. In this context, the Frataxin protein (FXN) stands out as an exception. In eukaryotes, a defect in FXN results in severe defects in Fe-S cluster biogenesis, and in humans, this is associated with Friedreich’s ataxia, a neurodegenerative disease. In contrast, prokaryotes deficient in the FXN homolog CyaY are fully viable, despite the clear involvement of CyaY in ISC-catalyzed Fe-S cluster formation. The molecular basis of the differing importance in the contribution of FXN remains enigmatic. Here, we have demonstrated that a single mutation in the scaffold protein IscU rendered E. coli viability strictly dependent upon a functional CyaY. Remarkably, this mutation changed an Ile residue, conserved in prokaryotes at position 108, into a Met residue, conserved in eukaryotes. We found that in the double mutant IscU_IM ΔcyaY, the ISC pathway was completely abolished, becoming equivalent to the ΔiscU deletion strain and recapitulating the drastic phenotype caused by FXN deletion in eukaryotes. Biochemical analyses of the “eukaryotic-like” IscU_IM scaffold revealed that it exhibited a reduced capacity to form Fe-S clusters. Finally, bioinformatic studies of prokaryotic IscU proteins allowed us to trace back the source of FXN-dependency as it occurs in present-day eukaryotes. We propose an evolutionary scenario in which the current mitochondrial Isu proteins originated from the IscU_IM version present in the ancestor of the Rickettsiae. Subsequent acquisition of SUF, the second Fe-S cluster biogenesis system, in bacteria, was accompanied by diminished contribution of CyaY in prokaryotic Fe-S cluster biogenesis, and increased tolerance to change in the amino acid present at the 108^th position of the scaffold.     

The asymmetric trimeric architecture of [2Fe-2S] IscU: implications for its scaffolding during iron-sulfur cluster biosynthesis

IscU is a key component of the ISC machinery and is involved in the biogenesis of iron-sulfur (Fe-S) proteins. IscU serves as a scaffold for assembly of a nascent Fe-S cluster prior to its delivery to an apo protein. Here, we report the first crystal structure of IscU with a bound [2Fe-2S] cluster from the hyperthermophilic bacterium Aquifex aeolicus, determined at a resolution of 2.3 Å, using multiwavelength anomalous diffraction of the cluster. The holo IscU formed a novel asymmetric trimer that harbored only one [2Fe-2S] cluster. One iron atom of the cluster was coordinated by the S^γ atom of Cys36 and the N^ε atom of His106, and the other was coordinated by the S^γ atoms of Cys63 and Cys107 on the surface of just one of the protomers. However, the cluster was buried inside the trimer between the neighboring protomers. The three protomers were conformationally distinct from one another and associated around a noncrystallographic pseudo-3-fold axis. The three flexible loop regions carrying the ligand-binding residues (Cys36, Cys63, His106 and Cys107) and the N-terminal α1 helices were positioned at the interfaces and underwent substantial conformational rearrangement, which stabilized the association of the asymmetric trimer. This unique trimeric A. aeolicus holo-IscU architecture was clearly distinct from other known monomeric apo-IscU/SufU structures, indicating that asymmetric trimer organization, as well as its association/dissociation, would be involved in the scaffolding function of IscU.     

Construction of a robust and sensitive arginine biosensor through ancestral protein reconstruction

Biosensors for signaling molecules allow the study of physiological processes by bringing together the fields of protein engineering, fluorescence imaging, and cell biology. Construction of genetically encoded biosensors generally relies on the availability of a binding “core” that is both specific and stable, which can then be combined with fluorescent molecules to create a sensor. However, binding proteins with the desired properties are often not available in nature and substantial improvement to sensors can be required, particularly with regard to their durability. Ancestral protein reconstruction is a powerful protein-engineering tool able to generate highly stable and functional proteins. In this work, we sought to establish the utility of ancestral protein reconstruction to biosensor development, beginning with the construction of an l-arginine biosensor. l-arginine, as the immediate precursor to nitric oxide, is an important molecule in many physiological contexts including brain function. Using a combination of ancestral reconstruction and circular permutation, we constructed a Förster resonance energy transfer (FRET) biosensor for l-arginine (cpFLIPR). cpFLIPR displays high sensitivity and specificity, with a K_d of ∼14 µM and a maximal dynamic range of 35%. Importantly, cpFLIPR was highly robust, enabling accurate l-arginine measurement at physiological temperatures. We established that cpFLIPR is compatible with two-photon excitation fluorescence microscopy and report l-arginine concentrations in brain tissue.