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- PDB-7bes: CryoEM structure of Mycobacterium tuberculosis UMP Kinase (UMPK) ... -

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Basic information

Entry
Database: PDB / ID: 7bes
TitleCryoEM structure of Mycobacterium tuberculosis UMP Kinase (UMPK) in complex with UDP and UTP
ComponentsUridylate kinase
KeywordsTRANSFERASE / nucleotide metabolism / UMP kinase / allosteric regulation / antibacterial target
Function / homology
Function and homology information


UMP kinase / UMP kinase activity / 'de novo' CTP biosynthetic process / ATP binding / cytoplasm
Similarity search - Function
Uridylate kinase, bacteria / Uridylate kinase / Aspartate/glutamate/uridylate kinase / Amino acid kinase family / Acetylglutamate kinase-like superfamily
Similarity search - Domain/homology
URIDINE-5'-DIPHOSPHATE / URIDINE 5'-TRIPHOSPHATE / Uridylate kinase
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.85 Å
AuthorsBous, J. / Trapani, S. / Walter, P. / Bron, P. / Munier-Lehmann, H.
Funding supportEuropean Union, France, 2items
OrganizationGrant numberCountry
Marie Sklodowska-Curie Actions, FragNET ITN675555European Union
Agence Nationale de la Recherche (ANR)ANR-10-INBS-05 France
CitationJournal: FEBS J / Year: 2022
Title: Structural basis for the allosteric inhibition of UMP kinase from Gram-positive bacteria, a promising antibacterial target.
Authors: Patrick Walter / Ariel Mechaly / Julien Bous / Ahmed Haouz / Patrick England / Joséphine Lai-Kee-Him / Aurélie Ancelin / Sylviane Hoos / Bruno Baron / Stefano Trapani / Patrick Bron / ...Authors: Patrick Walter / Ariel Mechaly / Julien Bous / Ahmed Haouz / Patrick England / Joséphine Lai-Kee-Him / Aurélie Ancelin / Sylviane Hoos / Bruno Baron / Stefano Trapani / Patrick Bron / Gilles Labesse / Hélène Munier-Lehmann /
Abstract: Tuberculosis claims significantly more than one million lives each year. A feasible way to face the issue of drug resistance is the development of new antibiotics. Bacterial uridine 5'-monophosphate ...Tuberculosis claims significantly more than one million lives each year. A feasible way to face the issue of drug resistance is the development of new antibiotics. Bacterial uridine 5'-monophosphate (UMP) kinase is a promising target for novel antibiotic discovery as it is essential for bacterial survival and has no counterpart in human cells. The UMP kinase from M. tuberculosis is also a model of particular interest for allosteric regulation with two effectors, GTP (positive) and UTP (negative). In this study, using X-ray crystallography and cryo-electron microscopy, we report for the first time a detailed description of the negative effector UTP-binding site of a typical Gram-positive behaving UMP kinase. Comparison between this snapshot of low affinity for Mg-ATP with our previous 3D-structure of the GTP-bound complex of high affinity for Mg-ATP led to a better understanding of the cooperative mechanism and the allosteric regulation of UMP kinase. Thermal shift assay and circular dichroism experiments corroborate our model of an inhibition by UTP linked to higher flexibility of the Mg-ATP-binding domain. These new structural insights provide valuable knowledge for future drug discovery strategies targeting bacterial UMP kinases.
History
DepositionDec 24, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 12, 2022Provider: repository / Type: Initial release
Revision 1.1Mar 16, 2022Group: Database references / Derived calculations
Category: citation / citation_author / pdbx_struct_oper_list
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation
Revision 1.2Aug 24, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
A: Uridylate kinase
B: Uridylate kinase
C: Uridylate kinase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)91,0588
Polymers88,8783
Non-polymers2,1815
Water0
1
A: Uridylate kinase
B: Uridylate kinase
C: Uridylate kinase
hetero molecules

A: Uridylate kinase
B: Uridylate kinase
C: Uridylate kinase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)182,11716
Polymers177,7556
Non-polymers4,36210
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation1
MethodThe assembly is composed by 6 chains of UMPK.Those are assembled in 2 trimers symetric with each other. Each trimer bind to 2UTP and 3 UDP ligands.

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Components

#1: Protein Uridylate kinase / UK / Uridine monophosphate kinase / UMPK


Mass: 29625.893 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria)
Gene: pyrH, DSI38_19350, E5M52_15045, ERS007661_01512, ERS007665_01956, ERS007670_03545, ERS007679_03743, ERS007681_04226, ERS007703_02851, ERS007741_01760, ERS013471_00404, ERS023446_00770, ...Gene: pyrH, DSI38_19350, E5M52_15045, ERS007661_01512, ERS007665_01956, ERS007670_03545, ERS007679_03743, ERS007681_04226, ERS007703_02851, ERS007741_01760, ERS013471_00404, ERS023446_00770, ERS024276_02256, ERS027646_01450, ERS027661_02116, ERS075361_01995, ERS094182_02076, F6W99_01494, FRD82_11895, SAMEA2683035_02116
Production host: Escherichia coli (E. coli) / Strain (production host): BL21/pDIA17 / References: UniProt: A0A045IH65, UMP kinase
#2: Chemical ChemComp-UDP / URIDINE-5'-DIPHOSPHATE / Uridine diphosphate


Type: RNA linking / Mass: 404.161 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C9H14N2O12P2 / Comment: UDP*YM
#3: Chemical ChemComp-UTP / URIDINE 5'-TRIPHOSPHATE / Uridine triphosphate


Mass: 484.141 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C9H15N2O15P3 / Comment: UTP*YM
Has ligand of interestN

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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: UMPK-UDP-UTP / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.177558 MDa / Experimental value: NO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21/pDIA17
Buffer solutionpH: 8
Buffer component
IDConc.FormulaBuffer-ID
120 mMNa2HPO41
2100 mMNaClSodium chloride1
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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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 FIELDBright-field microscopy
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 53.6 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameCategory
1Gautomatchparticle selection
2DigitalMicrographimage acquisition
4GctfCTF correction
10RELIONfinal Euler assignment
11RELIONclassification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 793915
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.85 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 88841 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 87.61 / Space: REAL

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