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- PDB-9b7y: Cryo-EM structure of TetR regulator Mce3R from Mycobacterium tube... -

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Basic information

Entry
Database: PDB / ID: 9b7y
TitleCryo-EM structure of TetR regulator Mce3R from Mycobacterium tuberculosis bound to a DNA oligonucleotide
Components
  • (DNA (29-MER)) x 2
  • Transcriptional repressor Mce3R
KeywordsDNA BINDING PROTEIN/DNA / TetR-like repressor stress resistance / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


regulation of lipid metabolic process / lipid catabolic process / transcription cis-regulatory region binding / DNA-binding transcription factor activity / negative regulation of DNA-templated transcription / regulation of DNA-templated transcription / DNA binding
Similarity search - Function
: / Bacterial regulatory proteins, tetR family / DNA-binding HTH domain, TetR-type / TetR-type HTH domain profile. / Homeobox-like domain superfamily
Similarity search - Domain/homology
: / DNA / DNA (> 10) / DNA (> 100) / Transcriptional repressor Mce3R
Similarity search - Component
Biological speciesMycobacterium tuberculosis H37Rv (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.51 Å
AuthorsPanagoda, N. / Sampson, N.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI134054 United States
CitationJournal: ACS Chem Biol / Year: 2024
Title: Mce3R TetR-like Repressor Forms an Asymmetric Four-Helix Bundle and Binds a Nonpalindrome Sequence†.
Authors: Navanjalee T Panagoda / Gábor Balázsi / Nicole S Sampson /
Abstract: (), the causative agent of tuberculosis, is a major global health concern. TetR family repressors (TFRs) are important for 's adaptation to the human host environment. Our study focuses on one ... (), the causative agent of tuberculosis, is a major global health concern. TetR family repressors (TFRs) are important for 's adaptation to the human host environment. Our study focuses on one notable repressor, Mce3R, composed of an unusual double TFR motif. Mce3R-regulated genes encode enzymes implicated in cholesterol metabolism, resistance against reactive oxygen species, and lipid transport activities important for survival and persistence in the host and for the cellular activity of a 6-azasteroid derivative. Here, we present the structure of Mce3R bound to its DNA operator, unveiling a unique asymmetric assembly previously unreported. We obtained a candidate DNA-binding motif through MEME motif analysis, comparing intergenic regions of orthologues and identifying nonpalindromic regions conserved between orthologues. Using an electrophoretic mobility shift assay (EMSA), we confirmed that Mce3R binds to a 123-bp sequence that includes the predicted motif. Using scrambled DNA and DNA oligonucleotides of varying lengths with sequences from the upstream region of the () operon, we elucidated the operator region to be composed of two Mce3R binding sites, each a 25-bp asymmetric sequence separated by 53 bp. Mce3R binds with a higher affinity to the downstream site with a of 2.4 ± 0.7 nM. The cryo-EM structure of Mce3R bound to the 123-bp sequence was refined to a resolution of 2.51 Å. Each Mce3R monomer comprises 21 α-helices (α1-α21) folded into an asymmetric TFR-like structure with a core asymmetric four-helix bundle. This complex has two nonidentical HTH motifs and a single ligand-binding domain. The two nonidentical HTHs from each TFR bind within the high-affinity, nonpalindromic operator motif, with Arg53 and Lys262 inserted into the major groove. Site-directed mutagenesis of Arg53 to alanine abrogated DNA binding, validating the Mce3R/DNA structure obtained. Among 811,645 particles, 63% were Mce3R homodimer bound to two duplex oligonucleotides. Mce3R homodimerizes primarily through α15, and each monomer binds to an identical site in the DNA duplex oligonucleotide.
History
DepositionMar 28, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 13, 2024Provider: repository / Type: Initial release
Revision 1.0Nov 13, 2024Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
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Revision 1.0Nov 13, 2024Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Nov 13, 2024Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.1Nov 27, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Revision 1.2Jan 1, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Revision 1.3May 28, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.1May 28, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Transcriptional repressor Mce3R
D: Transcriptional repressor Mce3R
E: DNA (29-MER)
F: DNA (29-MER)
B: DNA (29-MER)
C: DNA (29-MER)


Theoretical massNumber of molelcules
Total (without water)240,6186
Polymers240,6186
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Transcriptional repressor Mce3R / TetR family transcriptional regulator


Mass: 44400.602 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Gene: mce3R, Rv1963c / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P95251
#2: DNA chain DNA (29-MER)


Mass: 37845.172 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Production host: Escherichia coli BL21(DE3) (bacteria) / References: GenBank: 444893469
#3: DNA chain DNA (29-MER)


Mass: 38063.328 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Production host: Escherichia coli BL21(DE3) (bacteria) / References: GenBank: 444893469
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: TetR regulator Mce3R from Mycobacterium tuberculosis bound to a DNA oligonucleotide
Type: COMPLEX
Details: Protein was prepared from a maltose-binding protein fusion construct and proteolytic cleavage with Tev protease.
Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: NITROGEN

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 55 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.51 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 135814 / Symmetry type: POINT

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