Sequence-specific dynamic DNA bending explains mitochondrial TFAM's dual role in DNA packaging and transcription initiation.

Mitochondrial transcription factor A (TFAM) employs DNA bending to package mitochondrial DNA (mtDNA) into nucleoids and recruit mitochondrial RNA polymerase (POLRMT) at specific promoter sites, light strand promoter (LSP) and heavy strand promoter (HSP). Herein, we characterize the conformational dynamics of TFAM on promoter and non-promoter sequences using single-molecule fluorescence resonance energy transfer (smFRET) and single-molecule protein-induced fluorescence enhancement (smPIFE) methods. The DNA-TFAM complexes dynamically transition between partially and fully bent DNA conformational states. The bending/unbending transition rates and bending stability are DNA sequence-dependent-LSP forms the most stable fully bent complex and the non-specific sequence the least, which correlates with the lifetimes and affinities of TFAM with these DNA sequences. By quantifying the dynamic nature of the DNA-TFAM complexes, our study provides insights into how TFAM acts as a multifunctional protein through the DNA bending states to achieve sequence specificity and fidelity in mitochondrial transcription while performing mtDNA packaging.

The mutation R107Q alters mtSSB ssDNA compaction ability and binding dynamics.

Mitochondrial single-stranded DNA-binding protein (mtSSB) is essential for mitochondrial DNA (mtDNA) replication. Recently, several mtSSB variants have been associated with autosomal dominant mitochondrial optic atrophy and retinal dystrophy. Here, we have studied at the molecular level the functional consequences of one of the most severe mtSSB variants, R107Q. We first studied the oligomeric state of this variant and observed that the mtSSBR107Q mutant forms stable tetramers in vitro. On the other hand, we showed, using complementary single-molecule approaches, that mtSSBR107Q displays a lower intramolecular ssDNA compaction ability and a higher ssDNA dissociation rate than the WT protein. Real-time competition experiments for ssDNA-binding showed a marked advantage of mtSSBWT over mtSSBR107Q. Combined, these results show that the R107Q mutation significantly impaired the ssDNA-binding and compacting ability of mtSSB, likely by weakening mtSSB ssDNA wrapping efficiency. These features are in line with our molecular modeling of ssDNA on mtSSB showing that the R107Q mutation may destabilize local interactions and results in an electronegative spot that interrupts an ssDNA-interacting-electropositive patch, thus reducing the potential mtSSB-ssDNA interaction sites.

Structural analysis of the SAM domain of the Arabidopsis mitochondrial tRNA import receptor.

Mitochondria are membrane-bound organelles of endosymbiotic origin with limited protein-coding capacity. The import of nuclear-encoded proteins and nucleic acids is required and essential for maintaining organelle mass, number, and activity. As plant mitochondria do not encode all the necessary tRNA types required, the import of cytosolic tRNA is vital for organelle maintenance. Recently, two mitochondrial outer membrane proteins, named Tric1 and Tric2, for tRNA import component, were shown to be involved in the import of cytosolic tRNA. Tric1/2 binds tRNAalavia conserved residues in the C-terminal Sterile Alpha Motif (SAM) domain. Here we report the X-ray crystal structure of the Tric1 SAM domain. We identified the ability of the SAM domain to form a helical superstructure with six monomers per helical turn and key amino acid residues responsible for its formation. We determined that the oligomerization of the Tric1 SAM domain may play a role in protein function whereby mutation of Gly241 introducing a larger side chain at this position disrupted the oligomer and resulted in the loss of RNA binding capability. Furthermore, complementation of Arabidopsis thaliana Tric1/2 knockout lines with a mutated Tric1 failed to restore the defective plant phenotype. AlphaFold2 structure prediction of both the SAM domain and Tric1 support a cyclic pentameric or hexameric structure. In the case of a hexameric structure, a pore of sufficient dimensions to transfer tRNA across the mitochondrial membrane is observed. Our results highlight the importance of oligomerization of Tric1 for protein function.

Order wrapped in chaos: On the roles of intrinsically disordered proteins and RNAs in the arrangement of the mitochondrial enzymatic machines.

The analysis of cryo-electron tomography images of human and rat mitochondria revealed that the mitochondrial matrix is at least as crowded as the cytosol. To mitigate the crowding effects, metabolite transport in the mitochondria primarily occurs through the intermembrane space, which is significantly less crowded. The scientific literature largely ignores how enzyme systems and metabolite transport are organized in the crowded environment of the mitochondrial matrix. Under crowded conditions, multivalent interactions carried out by disordered protein regions (IDRs), may become extremely important. We analyzed the human mitochondrial proteome to determine the presence and physiological significance of IDRs. Despite mitochondrial proteins being generally more ordered than cytosolic or overall proteome proteins, disordered regions plays a significant role in certain mitochondrial compartments and processes. Even in highly ordered enzyme systems, there are proteins with long IDRs. Some IDRs act as binding elements between highly ordered subunits, while the roles of others are not yet established. Mitochondrial systems, like their bacterial ancestors, rely less on IDRs and more on RNA for LLPS compartmentalization. More evolutionarily advanced subsystems that enable mitochondria-cell interactions contain more IDRs. The study highlights the crucial and often overlooked role played by IDRs and non-coding RNAs in mitochondrial organization.

Regulations of mitoNEET by the key redox homeostasis molecule glutathione.

Human mitoNEET (mNT) and CISD2 are two NEET proteins characterized by an atypical [2Fe-2S] cluster coordination involving three cysteines and one histidine. They act as redox switches with an active state linked to the oxidation of their cluster. In the present study, we show that reduced glutathione but also free thiol-containing molecules such as ß-mercaptoethanol can induce a loss of the mNT cluster under aerobic conditions, while CISD2 cluster appears more resistant. This disassembly occurs through a radical-based mechanism as previously observed with the bacterial SoxR. Interestingly, adding cysteine prevents glutathione-induced cluster loss. At low pH, glutathione can bind mNT in the vicinity of the cluster. These results suggest a potential new regulation mechanism of mNT activity by glutathione, an essential actor of the intracellular redox state.

CHCHD4 binding affects the active site of apoptosis inducing factor (AIF): Structural determinants for allosteric regulation.

Apoptosis-inducing factor (AIF), which is confined to mitochondria of normal healthy cells, is the first identified caspase-independent cell death effector. Moreover, AIF is required for the optimal functioning of the respiratory chain machinery. Recent findings have revealed that AIF fulfills its pro-survival function by interacting with CHCHD4, a soluble mitochondrial protein which promotes the entrance and the oxidative folding of different proteins in the inner membrane space. Here, we report the crystal structure of the ternary complex involving the N-terminal 27-mer peptide of CHCHD4, NAD+, and AIF harboring its FAD (flavin adenine dinucleotide) prosthetic group in oxidized form. Combining this information with biophysical and biochemical data on the CHCHD4/AIF complex, we provide a detailed structural description of the interaction between the two proteins, validated by both chemical cross-linking mass spectrometry analysis and site-directed mutagenesis.

Cryo-EM structure and molecular dynamic simulations explain the enhanced stability and ATP activity of the pathological chaperonin mutant.

Chaperonins Hsp60s are required for cellular vitality by assisting protein folding in an ATP-dependent mechanism. Although conserved, the human mitochondrial mHsp60 exhibits molecular characteristics distinct from the E. coli GroEL, with different conformational assembly and higher subunit association dynamics, suggesting a different mechanism. We previously found that the pathological mutant mHsp60V72I exhibits enhanced subunit association stability and ATPase activity. To provide structural explanations for the V72I mutational effects, here we determined a cryo-EM structure of mHsp60V72I. Our structural analysis combined with molecular dynamic simulations showed mHsp60V72I with increased inter-subunit interface, binding free energy, and dissociation force, all contributing to its enhanced subunit association stability. The gate to the nucleotide-binding (NB) site in mHsp60V72I mimicked the open conformation in the nucleotide-bound state with an additional open channel leading to the NB site, both promoting the mutant's ATPase activity. Our studies highlight the importance of mHsp60's characteristics in its biological function.

Biochemical and cellular characterization of the CISD3 protein: Molecular bases of cluster release and destabilizing effects of nitric oxide.

The NEET proteins, an important family of iron-sulfur (Fe-S) proteins, have generated a strong interest due to their involvement in diverse diseases such as cancer, diabetes, and neurodegenerative disorders. Among the human NEET proteins, CISD3 has been the least studied, and its functional role is still largely unknown. We have investigated the biochemical features of CISD3 at the atomic and in cellulo levels upon challenge with different stress conditions i.e., iron deficiency, exposure to hydrogen peroxide, and nitric oxide. The redox and cellular stability properties of the protein agree on a predominance of reduced form of CISD3 in the cells. Upon the addition of iron chelators, CISD3 loses its Fe-S clusters and becomes unstructured, and its cellular level drastically decreases. Chemical shift perturbation measurements suggest that, upon cluster oxidation, the protein undergoes a conformational change at the C-terminal CDGSH domain, which determines the instability of the oxidized state. This redox-associated conformational change may be the source of cooperative electron transfer via the two [Fe2S2] clusters in CISD3, which displays a single sharp voltammetric signal at -31 mV versus SHE. Oxidized CISD3 is particularly sensitive to the presence of hydrogen peroxide in vitro, whereas only the reduced form is able to bind nitric oxide. Paramagnetic NMR provides clear evidence that, upon NO binding, the cluster is disassembled but iron ions are still bound to the protein. Accordingly, in cellulo CISD3 is unaffected by oxidative stress induced by hydrogen peroxide but it becomes highly unstable in response to nitric oxide treatment.

Stress response silencing by an E3 ligase mutated in neurodegeneration.

Stress response pathways detect and alleviate adverse conditions to safeguard cell and tissue homeostasis, yet their prolonged activation induces apoptosis and disrupts organismal health1-3. How stress responses are turned off at the right time and place remains poorly understood. Here we report a ubiquitin-dependent mechanism that silences the cellular response to mitochondrial protein import stress. Crucial to this process is the silencing factor of the integrated stress response (SIFI), a large E3 ligase complex mutated in ataxia and in early-onset dementia that degrades both unimported mitochondrial precursors and stress response components. By recognizing bifunctional substrate motifs that equally encode protein localization and stability, the SIFI complex turns off a general stress response after a specific stress event has been resolved. Pharmacological stress response silencing sustains cell survival even if stress resolution failed, which underscores the importance of signal termination and provides a roadmap for treating neurodegenerative diseases caused by mitochondrial import defects.

Repurposing the inhibitors of MMP-9 and SGLT-2 against ubiquitin specific protease 30 in Parkinson's disease: computational modelling studies.

Ubiquitin specific protease 30 (USP30) has been attributed to mitochondrial dysfunction and impediment of mitophagy in Parkinson's disease (PD). This happens once ubiquitin that supposed to bind with deformed mitochondria at the insistence of Parkin, it's been recruited by USP30 via the distal ubiquitin binding domain. This is a challenge when PINK1 and Parkin loss their functions due to mutation. Although, there are reports on USP30s' inhibitors but no study on the repurposing of inhibitors approved against MMP-9 and SGLT-2 as potential inhibitors of USP30 in PD. Thus, the highlight therein, is to repurpose approved inhibitors of MMP-9 and SGLT-2 against USP30 in PD using extensive computational modelling framework. 3D structures of Ligands and USP30 were obtained from PubChem and protein database (PDB) servers respectively, and were subjected to molecular docking, ADMET evaluation, DFT calculation, molecular dynamics simulation (MDS) and free energy calculations. Out of the 18 drugs, 2 drugs showed good binding affinity to the distal ubiquitin binding domain, moderate pharmacokinetic properties and good stability. The findings showed canagliflozin and empagliflozin as potential inhibitors of USP30. Thus, we present these drugs as repurposing candidates for the treatment of PD. However, the findings in this current study needs to be validated experimentally.Communicated by Ramaswamy H. Sarma.

The role of lysine acetylation in the function of mitochondrial ribosomal protein L12.

Mitochondria play a central role in energy production and cellular metabolism. Mitochondria contain their own small genome (mitochondrial DNA, mtDNA) that carries the genetic instructions for proteins required for ATP synthesis. The mitochondrial proteome, including the mitochondrial transcriptional machinery, is subject to post-translational modifications (PTMs), including acetylation and phosphorylation. We set out to determine whether PTMs of proteins associated with mtDNA may provide a potential mechanism for the regulation of mitochondrial gene expression. Here, we focus on mitochondrial ribosomal protein L12 (MRPL12), which is thought to stabilize mitochondrial RNA polymerase (POLRMT) and promote transcription. Numerous acetylation sites of MRPL12 were identified by mass spectrometry. We employed amino acid mimics of the acetylated (lysine to glutamine mutants) and deacetylated (lysine to arginine mutants) versions of MRPL12 to interrogate the role of lysine acetylation in transcription initiation in vitro and mitochondrial gene expression in HeLa cells. MRPL12 acetyl and deacetyl protein mimics were purified and assessed for their ability to impact mtDNA promoter binding of POLRMT. We analyzed mtDNA content and mitochondrial transcript levels in HeLa cells upon overexpression of acetyl and deacetyl mimics of MRPL12. Our results suggest that MRPL12 single-site acetyl mimics do not change the mtDNA promoter binding ability of POLRMT or mtDNA content in HeLa cells. Individual acetyl mimics may have modest effects on mitochondrial transcript levels. We found that the mitochondrial deacetylase, Sirtuin 3, is capable of deacetylating MRPL12 in vitro, suggesting a potential role for dynamic acetylation controlling MRPL12 function in a role outside of the regulation of gene expression.

ATP-Bound State of the Uncoupling Protein 1 (UCP1) from Molecular Simulations.

The uncoupling protein 1 (UCP1) dissipates the transmembrane (TM) proton gradient in the inner mitochondrial membrane (IMM) by leaking protons across the membrane and producing heat in the process. Such a nonshivering production of heat in the brown adipose tissue can combat obesity-related diseases. UCP1-associated proton leak is activated by free fatty acids and inhibited by purine nucleotides. The mechanism of proton leak and the binding sites of the activators (fatty acids) remain unknown, while the binding site of the inhibitors (nucleotides) was described recently. Using molecular dynamics simulations, we generated a conformational ensemble of UCP1. Using metadynamics-based free energy calculations, we obtained the most likely ATP-bound conformation of UCP1. Our conformational ensemble provides a molecular basis for a breadth of prior biochemical data available for UCP1. Based on the simulations, we make the following testable predictions about the mechanisms of activation of proton leak and proton leak inhibition by ATP: (1) R277 plays the dual role of stabilizing ATP at the binding site for inhibition and acting as a proton surrogate for D28 in the absence of a proton during proton transport, (2) the binding of ATP to UCP1 is mediated by residues R84, R92, R183, and S88, (3) R92 shuttles ATP from the E191-R92 gate in the intermembrane space to the nucleotide binding site and serves to increase ATP affinity, (4) ATP can inhibit proton leak by controlling the ionization states of matrix facing lysine residues such as K269 and K56, and (5) fatty acids can bind to UCP1 from the IMM either via the cavity between TM1 and TM2 or between TM5 and TM6. Our simulations set the platform for future investigations into the proton transport and inhibition mechanisms of UCP1.

NMR-Based Characterization of the Interaction between Yeast Oxa1-CTD and Ribosomes.

In mitochondria, the major subunits of oxidative phosphorylation complexes are translated by the mitochondrial ribosome (mito-ribosome). The correct insertion and assembly of these subunits into the inner mitochondrial membrane (IMM) are facilitated by mitochondrial oxidase assembly protein 1 (Oxa1) during the translation process. This co-translational insertion process involves an association between the mito-ribosome and the C-terminus of Oxa1 (Oxa1-CTD) Nuclear magnetic resonance (NMR) methods were mainly used to investigate the structural characterization of yeast Oxa1-CTD and its mode of interaction with the E. coli 70S ribosome. Oxa1-CTD forms a transient α-helical structure within the residues P342-Q385, which were reported to form an α-helix when combining with the ribosome. Two conserved contact sites that could interact with the ribosome were further identified. The first site was located on the very end of the N-terminus (V321-I327), and the second one encompassed a stretch of amino acid residues I348-Q370. Based on our discoveries and previous reports, a model has been proposed in which Oxa1-CTD interacts with ribosomes, accompanied by transient-to-stable transitions at the second contact site. These observations may enhance our understanding of the potential role of Oxa1-CTD in facilitating the assembly of oxidative phosphorylation complexes and provide insight into the structural characteristics of Oxa1-CTD.

Structure and dynamics of the mitochondrial DNA-compaction factor Abf2 from S. cerevisiae.

Mitochondria are essential organelles that produce most of the energy via the oxidative phosphorylation (OXPHOS) system in all eukaryotic cells. Several essential subunits of the OXPHOS system are encoded by the mitochondrial genome (mtDNA) despite its small size. Defects in mtDNA maintenance and expression can lead to severe OXPHOS deficiencies and are amongst the most common causes of mitochondrial disease. The mtDNA is packaged as nucleoprotein structures, referred to as nucleoids. The conserved mitochondrial proteins, ARS-binding factor 2 (Abf2) in the budding yeast Saccharomyces cerevisiae (S. cerevisiae) and mitochondrial transcription factor A (TFAM) in mammals, are nucleoid associated proteins (NAPs) acting as condensing factors needed for packaging and maintenance of the mtDNA. Interestingly, gene knockout of Abf2 leads, in yeast, to the loss of mtDNA and respiratory growth, providing evidence for its crucial role. On a structural level, the condensing factors usually contain two DNA binding domains called high-mobility group boxes (HMG boxes). The co-operating mechanical activities of these domains are crucial in establishing the nucleoid architecture by bending the DNA strands. Here we used advanced solution NMR spectroscopy methods to characterize the dynamical properties of Abf2 together with its interaction with DNA. We find that the two HMG-domains react notably different to external cues like temperature and salt, indicating distinct functional properties. Biophysical characterizations show the pronounced difference of these domains upon DNA-binding, suggesting a refined picture of the Abf2 functional cycle.

An integrative analysis of genotype-phenotype correlation in Charcot Marie Tooth type 2A disease with MFN2 variants: A case and systematic review.

Dominant genetic variants in the mitofusin 2 (MFN2) gene lead to Charcot-Marie-Tooth type 2A (CMT2A), a neurodegenerative disease caused by genetic defects that directly damage axons. In this study, we reported a proband with a pathogenic variant in the GTPase domain of MFN2, c.494A > G (p.His165Arg). To date, at least 184 distinct MFN2 variants identified in 944 independent probands have been reported in 131 references. However, the field of medical genetics has long been challenged by how genetic variation in the MFN2 gene is associated with disease phenotypes. Here, by collating the MFN2 variant data and patient clinical information from Leiden Open Variant Database 3.0, NCBI clinvar database, and available related references in PubMed, we determined the mutation frequency, age of onset, sex ratio, and geographical distribution. Furthermore, the results of an analysis examining the relationship between variants and phenotypes from multiple genetic perspectives indicated that insertion and deletions (indels), copy number variants (CNVs), duplication variants, and nonsense mutations in single nucleotide variants (SNVs) tend to be pathogenic, and the results emphasized the importance of the GTPase domain to the structure and function of MFN2. Overall, three reliable classification methods of MFN2 genotype-phenotype associations provide insights into the prediction of CMT2A disease severity. Of course, there are still many MFN2 variants that have not been given clear clinical significance, which requires clinicians to make more accurate clinical diagnoses.

Frontotemporal Dementia-Related V57E Mutation Impairs Mitochondrial Function and Alters the Structural Properties of CHCHD10.

The V57E pathological variant of the mitochondrial coiled-coil-helix-coiled-coil-helix domain-containing protein 10 (CHCHD10) plays a role in frontotemporal dementia. The wild-type and V57E mutant CHCHD10 proteins contain intrinsically disordered regions, and therefore, these regions hampered structural characterization of these proteins using conventional experimental tools. For the first time in the literature, we represent that the V57E mutation is pathogenic to mitochondria as it increases mitochondrial superoxide and impairs mitochondrial respiration. In addition, we represent here the structural ensemble properties of the V57E mutant CHCHD10 and describe the impacts of V57E mutation on the structural ensembles of wild-type CHCHD10 in aqueous solution. We conducted experimental and computational studies for this research. Namely, MitoSOX Red staining and Seahorse Mito Stress experiments, atomic force microscopy measurements, bioinformatics, homology modeling, and multiple-run molecular dynamics simulation computational studies were conducted. Our experiments show that the V57E mutation results in mitochondrial dysfunction, and our computational studies present that the structural ensemble properties of wild-type CHCHD10 are impacted by the frontotemporal dementia-associated V57E genetic mutation.

Protein transport along the presequence pathway.

Most mitochondrial proteins are nuclear-encoded and imported by the protein import machinery based on specific targeting signals. The proteins that carry an amino-terminal targeting signal (presequence) are imported via the presequence import pathway that involves the translocases of the outer and inner membranes - TOM and TIM23 complexes. In this article, we discuss how mitochondrial matrix and inner membrane precursor proteins are imported along the presequence pathway in Saccharomyces cerevisiae with a focus on the dynamics of the TIM23 complex, and further update with some of the key findings that advanced the field in the last few years.

Chemical inhibition of mitochondrial fission via targeting the DRP1-receptor interaction.

Mitochondrial fission is critical for mitochondrial dynamics and homeostasis. The dynamin superfamily GTPase DRP1 is recruited by three functionally redundant receptors, MFF, MiD49, and MiD51, to mitochondria to drive fission. Here, we exploit high-content live-cell imaging to screen for mitochondrial fission inhibitors and have developed a covalent compound, mitochondrial division inhibitor (MIDI). MIDI treatment potently blocks mitochondrial fragmentation induced by mitochondrial toxins and restores mitochondrial morphology in fusion-defective cells carrying pathogenic mitofusin and OPA1 mutations. Mechanistically, MIDI does not affect DRP1 tetramerization nor DRP1 GTPase activity but does block DRP1 recruitment to mitochondria. Subsequent biochemical and cellular characterizations reveal an unexpected mechanism that MIDI targets DRP1 interaction with multiple receptors via covalent interaction with DRP1-C367. Taken together, beyond developing a potent mitochondrial fission inhibitor that profoundly impacts mitochondrial morphogenesis, our study establishes proof of concept for developing protein-protein interaction inhibitors targeting DRP1.

Structures of BIRC6-client complexes provide a mechanism of SMAC-mediated release of caspases.

Tight regulation of apoptosis is essential for metazoan development and prevents diseases such as cancer and neurodegeneration. Caspase activation is central to apoptosis, and inhibitor of apoptosis proteins (IAPs) are the principal actors that restrain caspase activity and are therefore attractive therapeutic targets. IAPs, in turn, are regulated by mitochondria-derived proapoptotic factors such as SMAC and HTRA2. Through a series of cryo-electron microscopy structures of full-length human baculoviral IAP repeat-containing protein 6 (BIRC6) bound to SMAC, caspases, and HTRA2, we provide a molecular understanding for BIRC6-mediated caspase inhibition and its release by SMAC. The architecture of BIRC6, together with near-irreversible binding of SMAC, elucidates how the IAP inhibitor SMAC can effectively control a processive ubiquitin ligase to respond to apoptotic stimuli.

Structural basis for regulation of apoptosis and autophagy by the BIRC6/SMAC complex.

Inhibitor of apoptosis proteins (IAPs) bind to pro-apoptotic proteases, keeping them inactive and preventing cell death. The atypical ubiquitin ligase BIRC6 is the only essential IAP, additionally functioning as a suppressor of autophagy. We performed a structure-function analysis of BIRC6 in complex with caspase-9, HTRA2, SMAC, and LC3B, which are critical apoptosis and autophagy proteins. Cryo-electron microscopy structures showed that BIRC6 forms a megadalton crescent shape that arcs around a spacious cavity containing receptor sites for client proteins. Multivalent binding of SMAC obstructs client binding, impeding ubiquitination of both autophagy and apoptotic substrates. On the basis of these data, we discuss how the BIRC6/SMAC complex can act as a stress-induced hub to regulate apoptosis and autophagy drivers.