Mycobacterium tuberculosis ferritin: a suitable workhorse protein for cryo-EM development.
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ABSTRACT:
SUBMITTER: Gijsbers A
PROVIDER: S-EPMC8329864 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cryo-electron microscopy has become an essential tool to understand structure and function of biological samples. Especially for pathogens, such as disease-causing bacteria and viruses, insights gained by cryo-EM can aid in developing cures. However, due to the biosafety restrictions of pathogens, samples are often treated by chemical fixation to render the pathogen inert, affecting the ultrastructure of the sample. Alternatively, researchers use in vitro or ex vivo models, which are non-pathogenic but lack the complexity of the pathogen of interest. Here we show that ultraviolet-C (UVC) radiation applied at cryogenic temperatures can be used to eliminate or dramatically reduce the infectivity of Vibrio cholerae and the bacterial virus, the ICP1 bacteriophage. We show no discernable structural impact of this treatment of either sample using two cryo-EM methods: cryo-electron tomography followed by sub-tomogram averaging, and single particle analysis (SPA). Additionally, we applied the UVC irradiation to the protein apoferritin (ApoF), which is a widely used test sample for high-resolution SPA studies. The UVC-treated ApoF sample resulted in a 2.1 Å structure indistinguishable from an untreated published map. This research demonstrates that UVC treatment is an effective and inexpensive addition to the cryo-EM sample preparation toolbox.
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Project description:Human transferrin receptor 1 (CD71) guarantees iron supply by endocytosis upon binding of iron-loaded transferrin and ferritin. Arenaviruses and the malaria parasite exploit CD71 for cell invasion and epitopes on CD71 for interaction with transferrin and pathogenic hosts were identified. Here, we provide the molecular basis of the CD71 ectodomain-human ferritin interaction by determining the 3.9 Å resolution single-particle cryo-electron microscopy structure of their complex and by validating our structural findings in a cellular context. The contact surfaces between the heavy-chain ferritin and CD71 largely overlap with arenaviruses and Plasmodium vivax binding regions in the apical part of the receptor ectodomain. Our data account for transferrin-independent binding of ferritin to CD71 and suggest that select pathogens may have adapted to enter cells by mimicking the ferritin access gate.
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Project description:Resolution advances in cryoelectron microscopy (cryo-EM) now offer the possibility to visualize structural effects of naturally occurring resistance mutations in proteins and also of understanding the binding mechanisms of small drug molecules. In Mycobacterium tuberculosis the multifunctional heme enzyme KatG is indispensable for activation of isoniazid (INH), a first-line pro-drug for treatment of tuberculosis. We present a cryo-EM methodology for structural and functional characterization of KatG and INH resistance variants. The cryo-EM structure of the 161 kDa KatG dimer in the presence of INH is reported to 2.7 Å resolution allowing the observation of potential INH binding sites. In addition, cryo-EM structures of two INH resistance variants, identified from clinical isolates, W107R and T275P, are reported. In combination with electronic absorbance spectroscopy our cryo-EM approach reveals how these resistance variants cause disorder in the heme environment preventing heme uptake and retention, providing insight into INH resistance.
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Project description:The 300-kDa ClpP1P2 protease from Mycobacterium tuberculosis collaborates with the AAA+ (ATPases associated with a variety of cellular activities) unfoldases, ClpC1 and ClpX, to degrade substrate proteins. Unlike in other bacteria, all of the components of the Clp system are essential for growth and virulence of mycobacteria, and their inhibitors show promise as antibiotics. MtClpP1P2 is unique in that it contains a pair of distinct ClpP1 and ClpP2 rings and also requires the presence of activator peptides, such as benzoyl-leucyl-leucine (Bz-LL), for function. Understanding the structural basis for this requirement has been elusive but is critical for the rational design and improvement of antituberculosis (anti-TB) therapeutics that target the Clp system. Here, we present a combined biophysical and biochemical study to explore the structure-dynamics-function relationship in MtClpP1P2. Electron cryomicroscopy (cryo-EM) structures of apo and acyldepsipeptide-bound MtClpP1P2 explain their lack of activity by showing loss of a key ?-sheet in a sequence known as the handle region that is critical for the proper formation of the catalytic triad. Methyl transverse relaxation-optimized spectroscopy (TROSY)-based NMR, cryo-EM, and biochemical assays show that, on binding Bz-LL or covalent inhibitors, MtClpP1P2 undergoes a conformational change from an inactive compact state to an active extended structure that can be explained by a modified Monod-Wyman-Changeux model. Our study establishes a critical role for the handle region as an on/off switch for function and shows extensive allosteric interactions involving both intra- and interring communication that regulate MtClpP1P2 activity and that can potentially be exploited by small molecules to target M. tuberculosis.
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Project description:The role of abnormal brain iron metabolism in neurodegenerative diseases is still insufficiently understood. Here, we investigate the molecular basis of the neurodegenerative disease hereditary ferritinopathy (HF), in which dysregulation of brain iron homeostasis is the primary cause of neurodegeneration. We mutagenized ferritin's three-fold pores (3FPs), i.e. the main entry route for iron, to investigate ferritin's iron management when iron must traverse the protein shell through the disrupted four-fold pores (4FPs) generated by mutations in the ferritin light chain (FtL) gene in HF. We assessed the structure and properties of ferritins using cryo-electron microscopy and a range of functional analyses in vitro. Loss of 3FP function did not alter ferritin structure but led to a decrease in protein solubility and iron storage. Abnormal 4FPs acted as alternate routes for iron entry and exit in the absence of functional 3FPs, further reducing ferritin iron-storage capacity. Importantly, even a small number of MtFtL subunits significantly compromises ferritin solubility and function, providing a rationale for the presence of ferritin aggregates in cell types expressing different levels of FtLs in patients with HF. These findings led us to discuss whether modifying pores could be used as a pharmacological target in HF.
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Project description:Encapsulins containing dye-decolorizing peroxidase (DyP)-type peroxidases are ubiquitous among prokaryotes, protecting cells against oxidative stress. However, little is known about how they interact and function. Here, we have isolated a native cargo-packaging encapsulin from Mycobacterium smegmatis and determined its complete high-resolution structure by cryogenic electron microscopy (cryo-EM). This encapsulin comprises an icosahedral shell and a dodecameric DyP cargo. The dodecameric DyP consists of two hexamers with a twofold axis of symmetry and stretches across the interior of the encapsulin. Our results reveal that the encapsulin shell plays a role in stabilizing the dodecameric DyP. Furthermore, we have proposed a potential mechanism for removing the hydrogen peroxide based on the structural features. Our study also suggests that the DyP is the primary cargo protein of mycobacterial encapsulins and is a potential target for antituberculosis drug discovery.
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Project description:Iron plays a critical role in the pathogenesis of many organisms including Mtb. It is the preferred redox cofactor in many basic cellular processes but due to its insolubility and potential toxicity under physiological conditions is a limiting nutrient in the host environment. Previously, we demonstrated that Mtb requires the iron storage protein ferritin (BfrB), for adaptation to both low and sufficient concentrations of environmental iron. We also showed that absence of bfrB compromises the ability of Mtb to overcome iron deficiency and prevent excess iron toxicity. In this study, we tested whether vaccination with ?bfrB could elicit host protective immune response against virulent Mtb infection. The results show that immunization of mice with the ?bfrB stimulates protective immunity associated with reduced immunopathology and better containment of the infection compared to vaccination with BCG. Genome-wide transcriptome analysis showed a distinct expression pattern of significantly differentially expressed genes (SDEG) between the ?bfrB and BCG-vaccinated, Mtb-infected mice lungs. Our network/pathway analysis of SDEG revealed significant inhibition of inflammatory response genes and activation of fibrosis genes in the ?bfrB, compared to BCG vaccinated, Mtb-infected mice lungs. The results provide a frame work for the study of mechanisms of protection relevant for the design of new and improved preventive strategies for TB. Female C57BL/6 mice were vaccinated subcutaneously with ?bfrB or BCG . At 8 weeks post-vaccination, mice were aerosol infected with Mtb H37Rv. At 4 weeks post infection, mice were sacrificed and lungs were removed. Lung total RNA from vaccinated and Mtb-infected mice was extracted, purified and were processed separately for microarray analysis.
2014-09-01 | E-GEOD-58810 | biostudies-arrayexpress
Project description:Iron plays a critical role in the pathogenesis of many organisms including Mtb. It is the preferred redox cofactor in many basic cellular processes but due to its insolubility and potential toxicity under physiological conditions is a limiting nutrient in the host environment. Previously, we demonstrated that Mtb requires the iron storage protein ferritin (BfrB), for adaptation to both low and sufficient concentrations of environmental iron. We also showed that absence of bfrB compromises the ability of Mtb to overcome iron deficiency and prevent excess iron toxicity. In this study, we tested whether vaccination with ∆bfrB could elicit host protective immune response against virulent Mtb infection. The results show that immunization of mice with the ∆bfrB stimulates protective immunity associated with reduced immunopathology and better containment of the infection compared to vaccination with BCG. Genome-wide transcriptome analysis showed a distinct expression pattern of significantly differentially expressed genes (SDEG) between the ∆bfrB and BCG-vaccinated, Mtb-infected mice lungs. Our network/pathway analysis of SDEG revealed significant inhibition of inflammatory response genes and activation of fibrosis genes in the ∆bfrB, compared to BCG vaccinated, Mtb-infected mice lungs. The results provide a frame work for the study of mechanisms of protection relevant for the design of new and improved preventive strategies for TB.
2014-09-01 | GSE58810 | GEO
Project description:Tuberculosis (TB) is the leading infectious disease caused by Mycobacterium tuberculosis (Mtb). Clarithromycin (CTY), an analog of erythromycin (ERY), is more potent against multidrug-resistance (MDR) TB. ERY and CTY were previously reported to bind to the nascent polypeptide exit tunnel (NPET) near peptidyl transferase center (PTC), but the only available CTY structure in complex with D. radiodurans (Dra) ribosome could be misinterpreted due to resolution limitation. To date, the mechanism of specificity and efficacy of CTY for Mtb remains elusive since the Mtb ribosome-CTY complex structure is still unknown. Here, we employed new sample preparation methods and solved the Mtb ribosome-CTY complex structure at 3.3Å with cryo-EM technique, where the crucial gate site A2062 (E. coli numbering) is located at the CTY binding site within NPET. Two alternative conformations of A2062, a novel syn-conformation as well as a swayed conformation bound with water molecule at interface, may play a role in coordinating the binding of specific drug molecules. The previously overlooked C-H hydrogen bond (H-bond) and π interaction may collectively contribute to the enhanced binding affinity. Together, our structure data provide a structural basis for the dynamic binding as well as the specificity of CTY and explain of how a single methyl group in CTY improves its potency, which provides new evidence to reveal previously unclear mechanism of translational modulation for future drug design and anti-TB therapy. Furthermore, our sample preparation method may facilitate drug discovery based on the complexes with low water solubility drugs by cryo-EM technique.
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