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- PDB-7bix: Crystal structure of UMPK from M. tuberculosis in complex with UD... -

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Basic information

Entry
Database: PDB / ID: 7bix
TitleCrystal structure of UMPK from M. tuberculosis in complex with UDP and UTP (C2 form)
ComponentsUridylate kinase
KeywordsTRANSFERASE / UMP kinase
Function / homology
Function and homology information


UMP kinase / UMP kinase activity / 'de novo' CTP biosynthetic process / UDP biosynthetic process / phosphorylation / magnesium ion binding / ATP binding / plasma membrane / 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 H37Rv (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.12 Å
AuthorsLabesse, G. / Walter, P. / Haouz, A. / Mechaly, A.E. / Munier-Lehmann, H.
Funding support France, 2items
OrganizationGrant numberCountry
H2020 Marie Curie Actions of the European Commission675555 France
French Infrastructure for Integrated Structural Biology (FRISBI)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
DepositionJan 13, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 2, 2022Provider: repository / Type: Initial release
Revision 1.1Sep 14, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jan 31, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Uridylate kinase
B: Uridylate kinase
C: Uridylate kinase
D: Uridylate kinase
E: Uridylate kinase
F: Uridylate kinase
G: Uridylate kinase
H: Uridylate kinase
I: Uridylate kinase
J: Uridylate kinase
K: Uridylate kinase
L: Uridylate kinase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)361,64926
Polymers355,51112
Non-polymers6,13814
Water99155
1
A: Uridylate kinase
B: Uridylate kinase
C: Uridylate kinase
D: Uridylate kinase
E: Uridylate kinase
F: Uridylate kinase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)180,82413
Polymers177,7556
Non-polymers3,0697
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area18070 Å2
ΔGint-123 kcal/mol
Surface area48180 Å2
MethodPISA
2
G: Uridylate kinase
H: Uridylate kinase
I: Uridylate kinase
J: Uridylate kinase
K: Uridylate kinase
L: Uridylate kinase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)180,82413
Polymers177,7556
Non-polymers3,0697
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area18090 Å2
ΔGint-129 kcal/mol
Surface area48280 Å2
MethodPISA
Unit cell
Length a, b, c (Å)197.470, 152.820, 125.110
Angle α, β, γ (deg.)90.000, 121.390, 90.000
Int Tables number5
Space group name H-MC121
Symmetry operation#1: x,y,z
#2: -x,y,-z
#3: x+1/2,y+1/2,z
#4: -x+1/2,y+1/2,-z

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Components

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


Mass: 29625.893 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Gene: pyrH, Rv2883c, MTCY274.14c / Production host: Escherichia coli (E. coli) / References: UniProt: P9WHK5, UMP kinase
#2: Chemical
ChemComp-UTP / URIDINE 5'-TRIPHOSPHATE / Uridine triphosphate


Mass: 484.141 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C9H15N2O15P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: UTP*YM
#3: Chemical
ChemComp-UDP / URIDINE-5'-DIPHOSPHATE / Uridine diphosphate


Type: RNA linking / Mass: 404.161 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Formula: C9H14N2O12P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: UDP*YM
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 55 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.27 Å3/Da / Density % sol: 45.73 %
Crystal growTemperature: 293 K / Method: vapor diffusion / pH: 8.7 / Details: 1 M LiCl, 20 % (w/v) PEG 8K and 0.1 M Tris pH 8.7

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SOLEIL / Beamline: PROXIMA 1 / Wavelength: 0.9801 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jul 13, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9801 Å / Relative weight: 1
ReflectionResolution: 3.12→27.98 Å / Num. obs: 56229 / % possible obs: 93.92 % / Redundancy: 7.1 % / Biso Wilson estimate: 59.27 Å2 / CC1/2: 0.992 / CC star: 0.998 / Rmerge(I) obs: 0.2052 / Rpim(I) all: 0.08247 / Rrim(I) all: 0.2214 / Net I/σ(I): 7.45
Reflection shellResolution: 3.12→3.231 Å / Rmerge(I) obs: 1.219 / Mean I/σ(I) obs: 1.23 / Num. unique obs: 3643 / CC1/2: 0.579 / CC star: 0.856 / Rpim(I) all: 0.4977 / Rrim(I) all: 1.319

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Processing

Software
NameVersionClassification
PHENIX1.18refinement
PHASERphasing
Aimlessdata scaling
XDSdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3NWY

3nwy


Resolution: 3.12→27.98 Å / Cross valid method: THROUGHOUT
RfactorNum. reflection% reflection
Rwork0.1906 --
obs-56229 93.92 %
Displacement parametersBiso max: 168.51 Å2 / Biso mean: 64.297 Å2 / Biso min: 2.32 Å2
Refinement stepCycle: LAST / Resolution: 3.12→27.98 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms19800 0 374 55 20229

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