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- PDB-7b5t: S. agalactiae BusR transcription factor -

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Basic information

Entry
Database: PDB / ID: 7b5t
TitleS. agalactiae BusR transcription factor
ComponentsGntR family transcriptional regulator
KeywordsTRANSCRIPTION / Transcription factor / full length / repressor
Function / homology
Function and homology information


monoatomic cation transmembrane transporter activity / potassium ion transport / DNA-binding transcription factor activity
Similarity search - Function
GntR-type HTH domain profile. / helix_turn_helix gluconate operon transcriptional repressor / Transcription regulator HTH, GntR / Bacterial regulatory proteins, gntR family / Regulator of K+ conductance, C-terminal / Regulator of K+ conductance, C-terminal domain superfamily / TrkA-C domain / RCK C-terminal domain profile. / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
GntR family transcriptional regulator
Similarity search - Component
Biological speciesStreptococcus agalactiae (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 2.8 Å
AuthorsBandera, A.M. / Witte, G.
Funding support Germany, 2items
OrganizationGrant numberCountry
German Research Foundation (DFG)WI3717/3-1 Germany
German Research Foundation (DFG)GRK1721 Germany
CitationJournal: Nucleic Acids Res / Year: 2021
Title: BusR senses bipartite DNA binding motifs by a unique molecular ruler architecture.
Authors: Adrian M Bandera / Joseph Bartho / Katja Lammens / David Jan Drexler / Jasmin Kleinschwärzer / Karl-Peter Hopfner / Gregor Witte /
Abstract: The cyclic dinucleotide second messenger c-di-AMP is a major player in regulation of potassium homeostasis and osmolyte transport in a variety of bacteria. Along with various direct interactions with ...The cyclic dinucleotide second messenger c-di-AMP is a major player in regulation of potassium homeostasis and osmolyte transport in a variety of bacteria. Along with various direct interactions with proteins such as potassium channels, the second messenger also specifically binds to transcription factors, thereby altering the processes in the cell on the transcriptional level. We here describe the structural and biochemical characterization of BusR from the human pathogen Streptococcus agalactiae. BusR is a member of a yet structurally uncharacterized subfamily of the GntR family of transcription factors that downregulates transcription of the genes for the BusA (OpuA) glycine-betaine transporter upon c-di-AMP binding. We report crystal structures of full-length BusR, its apo and c-di-AMP bound effector domain, as well as cryo-EM structures of BusR bound to its operator DNA. Our structural data, supported by biochemical and biophysical data, reveal that BusR utilizes a unique domain assembly with a tetrameric coiled-coil in between the binding platforms, serving as a molecular ruler to specifically recognize a 22 bp separated bipartite binding motif. Binding of c-di-AMP to BusR induces a shift in equilibrium from an inactivated towards an activated state that allows BusR to bind the target DNA, leading to transcriptional repression.
History
DepositionDec 7, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 11, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 6, 2021Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / database_PDB_rev / database_PDB_rev_record / pdbx_database_proc
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Oct 13, 2021Group: Data collection / Category: pdbx_database_proc

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: GntR family transcriptional regulator
B: GntR family transcriptional regulator
D: GntR family transcriptional regulator
C: GntR family transcriptional regulator


Theoretical massNumber of molelcules
Total (without water)96,2714
Polymers96,2714
Non-polymers00
Water39622
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: SAXS, SAXS data indicates that BusR is a tetramer in solution.
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area20160 Å2
ΔGint-139 kcal/mol
Surface area38640 Å2
MethodPISA
Unit cell
Length a, b, c (Å)114.180, 114.180, 240.480
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number79
Space group name H-MI4
Components on special symmetry positions
IDModelComponents
11B-302-

HOH

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Components

#1: Protein
GntR family transcriptional regulator / Transcriptional regulator / GntR family


Mass: 24067.738 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: N-terminal GP residues derive from tag / Source: (gene. exp.) Streptococcus agalactiae (bacteria)
Gene: BM110_ORF1201, AX245_01365, C6N10_09995, F5043_05515, GD434_05225, RDF_1124
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: K0JNC6
#2: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 22 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: 4.1 Å3/Da / Density % sol: 70.02 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / Details: 150 mM Mg-acetate, 6% D+ Trehalose

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, EMBL c/o DESY / Beamline: P13 (MX1) / Wavelength: 0.9797 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Oct 22, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9797 Å / Relative weight: 1
ReflectionResolution: 2.8→48.22 Å / Num. obs: 74011 / % possible obs: 99.1 % / Redundancy: 6.9 % / CC1/2: 1 / Net I/σ(I): 19.4
Reflection shellResolution: 2.8→2.9 Å / Num. unique obs: 3694 / CC1/2: 0.615

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Processing

Software
NameVersionClassification
PHENIX1.18.2_3874refinement
XDSdata reduction
XSCALEdata scaling
PDB_EXTRACT3.27data extraction
SHELXDEphasing
RefinementMethod to determine structure: SAD / Resolution: 2.8→48.22 Å / SU ML: 0.48 / Cross valid method: FREE R-VALUE / σ(F): 1.3 / Phase error: 41.19 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2689 3628 4.9 %random selection
Rwork0.2573 70359 --
obs0.2579 73987 99.08 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 297.19 Å2 / Biso mean: 127.6763 Å2 / Biso min: 61.18 Å2
Refinement stepCycle: final / Resolution: 2.8→48.22 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6506 0 0 22 6528
Biso mean---97.54 -
Num. residues----828
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 26

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.8-2.840.4668880.42892807289598
2.84-2.880.47471230.40122564268797
2.88-2.920.36451400.39412745288599
2.92-2.960.41531160.37962686280298
2.96-3.010.40231080.36862772288098
3.01-3.060.38981380.37452616275497
3.06-3.110.32351200.38222690281099
3.11-3.170.4241520.37212716286899
3.17-3.230.45091200.37052688280899
3.23-3.290.40191240.35692749287399
3.29-3.370.38861540.33482701285599
3.37-3.440.42821100.32322734284499
3.44-3.530.36621540.31382640279499
3.53-3.630.28361500.303227012851100
3.63-3.730.3641620.30532725288799
3.73-3.850.32061400.30592685282599
3.85-3.990.28391520.289327122864100
3.99-4.150.29841360.264627412877100
4.15-4.340.31311640.257426872851100
4.34-4.570.25771680.23922688285699
4.57-4.850.22161440.235427102854100
4.85-5.230.23691460.234427322878100
5.23-5.750.2921240.257527642888100
5.76-6.580.24971660.26626972863100
6.59-8.290.21731680.213827052873100
8.3-48.220.17711610.17082704286599
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
12.5205-0.52440.49863.6157-0.1108-0.1072-0.36790.5267-0.48050.26150.04470.10610.0931-0.2354-0.00690.85570.20980.15710.81020.06131.019530.039618.642131.3574
2-1.31750.97160.1254-0.54770.69040.57430.09030.2081-0.2036-0.5243-0.03180.5361-0.1742-1.446501.45140.0887-0.08961.3902-0.17571.054741.889811.0561-15.235
35.7842-2.2901-1.00063.2268-0.40911.0063-0.23970.13470.5259-0.24780.31090.1962-0.18280.235601.3733-0.0987-0.23681.2308-0.1061.037154.118328.4402-41.3974
42.9393-0.35810.0951.10450.86360.7428-0.0307-0.3494-0.3238-0.2820.1193-0.0791.02220.6973-01.771-0.04690.08081.4433-0.16621.030667.965110.8905-49.1794
5-1.4891-0.92280.7335-1.4494-1.46651.5639-0.48220.1402-0.3711-0.288-1.1212-0.06710.22570.394401.2659-0.00110.07431.4028-0.06040.967851.19659.1848-3.2095
64.9414-0.8813-0.28790.4568-0.1810.60230.05620.4830.24740.48520.17610.2099-0.4932-0.24490.00250.95850.110.02940.57280.10670.880447.247430.026823.0188
72.52471.2765-0.92461.0437-0.04360.4507-0.12940.2221-0.1755-0.1887-0.0650.32580.5474-0.340601.51350.0129-0.2891.3742-0.20661.123524.845523.1381-48.2852
8-0.02-0.5921-0.3495-0.4594-0.12830.9565-0.3767-1.08230.33420.4302-0.1742-0.0585-1.5035-3.45290.00081.02210.0398-0.1511.23290.05531.122643.40920.3375-7.9337
95.02130.2795-0.26123.6441.13233.88130.0712-0.94150.31651.2363-0.01990.0051-0.6789-0.138601.02790.1459-0.01870.44580.05620.787152.485221.740440.2811
105.41222.3571-0.20885.4018-0.55722.315-0.03720.0678-0.52920.15780.1681-0.3854-0.18350.03470.29890.7893-0.0484-0.02390.56860.01510.961169.729911.253328.1034
110.0802-0.5960.48790.90170.57860.8155-0.11320.53250.4333-1.2212-0.5595-0.26570.2872-0.083401.18610.1943-0.10911.327-0.00510.950353.73618.0936-9.8833
121.8844-0.0448-0.8421-0.1446-0.39862.4552-0.28830.62210.1173-0.58490.216-0.09810.40130.6214-01.4737-0.1787-0.13161.3319-0.07071.062745.840322.5547-58.1759
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 0 through 94 )A0 - 94
2X-RAY DIFFRACTION2chain 'A' and (resid 95 through 131 )A95 - 131
3X-RAY DIFFRACTION3chain 'A' and (resid 132 through 210 )A132 - 210
4X-RAY DIFFRACTION4chain 'B' and (resid 8 through 94 )B8 - 94
5X-RAY DIFFRACTION5chain 'B' and (resid 95 through 131 )B95 - 131
6X-RAY DIFFRACTION6chain 'B' and (resid 132 through 210 )B132 - 210
7X-RAY DIFFRACTION7chain 'D' and (resid 11 through 94 )D11 - 94
8X-RAY DIFFRACTION8chain 'D' and (resid 95 through 125 )D95 - 125
9X-RAY DIFFRACTION9chain 'D' and (resid 126 through 210 )D126 - 210
10X-RAY DIFFRACTION10chain 'C' and (resid 1 through 94 )C1 - 94
11X-RAY DIFFRACTION11chain 'C' and (resid 95 through 124 )C95 - 124
12X-RAY DIFFRACTION12chain 'C' and (resid 125 through 210 )C125 - 210

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