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- PDB-6w1a: Crystal structure of Streptococcus dysgalactiae SHP pheromone rec... -

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Basic information

Entry
Database: PDB / ID: 6w1a
TitleCrystal structure of Streptococcus dysgalactiae SHP pheromone receptor Rgg2 bound to DNA
Components
  • (DNA (30-MER)) x 2
  • Transcriptional regulatorTranscriptional regulation
KeywordsDNA BINDING PROTEIN/DNA / PHEROMONE BINDING / QUORUM SENSING / DNA BINDING PROTEIN / RRNPP / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


HTH-type transcriptional regulator Rgg, C-terminal domain / Transcription activator MutR, C-terminal / Helix-turn-helix / Helix-turn-helix XRE-family like proteins / Cro/C1-type HTH domain profile. / Cro/C1-type helix-turn-helix domain / Lambda repressor-like, DNA-binding domain superfamily
Similarity search - Domain/homology
PHOSPHATE ION / DNA / DNA (> 10) / Transcriptional regulator
Similarity search - Component
Biological speciesStreptococcus dysgalactiae (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.8 Å
AuthorsCapodagli, G.C. / Neiditch, M.B.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI125452 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: Structure-function studies of Rgg binding to pheromones and target promoters reveal a model of transcription factor interplay.
Authors: Glenn C Capodagli / Kaitlyn M Tylor / Jason T Kaelber / Vasileios I Petrou / Michael J Federle / Matthew B Neiditch /
Abstract: Regulator gene of glucosyltransferase (Rgg) family proteins, such as Rgg2 and Rgg3, have emerged as primary quorum-sensing regulated transcription factors in species, controlling virulence, ...Regulator gene of glucosyltransferase (Rgg) family proteins, such as Rgg2 and Rgg3, have emerged as primary quorum-sensing regulated transcription factors in species, controlling virulence, antimicrobial resistance, and biofilm formation. Rgg2 and Rgg3 function is regulated by their interaction with oligopeptide quorum-sensing signals called short hydrophobic peptides (SHPs). The molecular basis of Rgg-SHP and Rgg-target DNA promoter specificity was unknown. To close this gap, we determined the cryoelectron microscopy (cryo-EM) structure of Rgg3 bound to its quorum-sensing signal, SHP3, and the X-ray crystal structure of Rgg3 alone. Comparison of these structures with that of an Rgg in complex with cyclosporin A (CsA), an inhibitor of SHP-induced Rgg activity, reveals the molecular basis of CsA function. Furthermore, to determine how Rgg proteins recognize DNA promoters, we determined X-ray crystal structures of both Rgg2 and Rgg3 in complex with their target DNA promoters. The physiological importance of observed Rgg-DNA interactions was dissected using in vivo genetic experiments and in vitro biochemical assays. Based on these structure-function studies, we present a revised unifying model of Rgg regulatory interplay. In contrast to existing models, where Rgg2 proteins are transcriptional activators and Rgg3 proteins are transcriptional repressors, we propose that both are capable of transcriptional activation. However, when Rgg proteins with different activation requirements compete for the same DNA promoters, those with more stringent activation requirements function as repressors by blocking promoter access of SHP-bound conformationally active Rgg proteins. While a similar gene expression regulatory scenario has not been previously described, in all likelihood it is not unique to streptococci.
History
DepositionMar 3, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 21, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 16, 2022Group: Author supporting evidence / Database references / Category: database_2 / pdbx_audit_support
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_audit_support.funding_organization
Revision 1.2Oct 18, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model / struct_ncs_dom_lim
Item: _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id ..._struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Transcriptional regulator
B: Transcriptional regulator
E: DNA (30-MER)
F: DNA (30-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)85,9959
Polymers85,5294
Non-polymers4665
Water34219
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area11390 Å2
ΔGint-84 kcal/mol
Surface area33920 Å2
MethodPISA
Unit cell
Length a, b, c (Å)110.269, 110.269, 173.984
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number170
Space group name H-MP65
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11(chain A and (resid 4 through 185 or resid 187...
21(chain B and (resid 4 through 185 or resid 187...

NCS domain segments:

Ens-ID: 1

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11GLUGLUTHRTHR(chain A and (resid 4 through 185 or resid 187...AA4 - 1854 - 185
12LEULEUASPASP(chain A and (resid 4 through 185 or resid 187...AA187 - 197187 - 197
13SERSERILEILE(chain A and (resid 4 through 185 or resid 187...AA199 - 211199 - 211
14PHEPHETYRTYR(chain A and (resid 4 through 185 or resid 187...AA213 - 263213 - 263
15SERSERGLYGLY(chain A and (resid 4 through 185 or resid 187...AA265 - 276265 - 276
21GLUGLUTHRTHR(chain B and (resid 4 through 185 or resid 187...BB4 - 1854 - 185
22LEULEUASPASP(chain B and (resid 4 through 185 or resid 187...BB187 - 197187 - 197
23SERSERSERSER(chain B and (resid 4 through 185 or resid 187...BB199199
24PHEPHETYRTYR(chain B and (resid 4 through 185 or resid 187...BB213 - 263213 - 263
25SERSERGLYGLY(chain B and (resid 4 through 185 or resid 187...BB265 - 276265 - 276

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein Transcriptional regulator / Transcriptional regulation


Mass: 33543.980 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus dysgalactiae (bacteria) / Gene: mutR / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0J9X288

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DNA chain , 2 types, 2 molecules EF

#2: DNA chain DNA (30-MER)


Mass: 9044.882 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus dysgalactiae (bacteria)
#3: DNA chain DNA (30-MER)


Mass: 9396.084 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus dysgalactiae (bacteria)

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Non-polymers , 3 types, 24 molecules

#4: Chemical ChemComp-PO4 / PHOSPHATE ION / Phosphate


Mass: 94.971 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: PO4
#5: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C3H8O3
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 19 / Source method: isolated from a natural source / Formula: H2O

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Details

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: 3.57 Å3/Da / Density % sol: 65.55 %
Crystal growTemperature: 298 K / Method: vapor diffusion / Details: 100 mM trilithium citrate and 24% PEG 3,350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL14-1 / Wavelength: 1.18076 Å
DetectorType: MARMOSAIC 325 mm CCD / Detector: CCD / Date: Aug 5, 2015
RadiationMonochromator: Si 111 / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.18076 Å / Relative weight: 1
ReflectionResolution: 2.8→50 Å / Num. obs: 29582 / % possible obs: 99.9 % / Redundancy: 8.3 % / CC1/2: 0.999 / Rmerge(I) obs: 0.102 / Rpim(I) all: 0.038 / Rrim(I) all: 0.109 / Χ2: 1.751 / Net I/av σ(I): 33.1 / Net I/σ(I): 10.5
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2.8-2.858.31.51814680.580.5631.6191.286100
2.85-2.98.31.21115080.6890.4481.2911.289100
2.9-2.968.31.07414560.750.3981.1461.304100
2.96-3.028.31.03114570.7820.3811.11.316100
3.02-3.088.30.75314940.8640.2790.8031.315100
3.08-3.158.30.55214750.9190.2040.5881.36100
3.15-3.238.30.41614700.9490.1540.4441.427100
3.23-3.328.30.34714740.9610.1290.3711.601100
3.32-3.428.40.29314700.9740.1080.3121.646100
3.42-3.538.30.24114670.9790.090.2571.962100
3.53-3.658.40.18714830.9860.0690.1992.047100
3.65-3.88.30.17314790.9860.0650.1852.24100
3.8-3.978.30.15414750.9880.0570.1641.725100
3.97-4.188.30.12614680.9910.0470.1352.561100
4.18-4.448.40.11814910.9870.0440.1262.38100
4.44-4.798.40.10615000.9930.0390.1132.145100
4.79-5.278.40.09914600.9940.0370.1051.945100
5.27-6.038.40.08914900.9950.0330.0952.06100
6.03-7.598.40.0714900.9970.0260.0751.98100
7.59-508.10.03815070.9990.0140.0411.39499

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Processing

Software
NameVersionClassification
PHENIX1.15.2_3472refinement
SCALEPACKdata scaling
PDB_EXTRACT3.25data extraction
HKL-3000data reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4YV6

4yv6


Resolution: 2.8→49.57 Å / SU ML: 0.46 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 32.07
RfactorNum. reflection% reflection
Rfree0.2643 2002 6.78 %
Rwork0.2335 --
obs0.2356 29523 99.89 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 202.29 Å2 / Biso mean: 99.1391 Å2 / Biso min: 47.19 Å2
Refinement stepCycle: final / Resolution: 2.8→49.57 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4555 1230 28 19 5832
Biso mean--119.31 68.65 -
Num. residues----607
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A1610X-RAY DIFFRACTION8.492TORSIONAL
12B1610X-RAY DIFFRACTION8.492TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.8-2.86820.35541400.35751948100
2.8682-2.94580.33471450.34661990100
2.9458-3.03240.43011420.34311955100
3.0324-3.13030.35481420.33881953100
3.1303-3.24210.3761420.31281973100
3.2421-3.37190.33551390.32091946100
3.3719-3.52540.29941440.30571973100
3.5254-3.71120.26561410.27291954100
3.7112-3.94360.34911410.25351979100
3.9436-4.24790.25321430.22371979100
4.2479-4.67510.2221450.20561956100
4.6751-5.35090.24211450.21041953100
5.3509-6.73890.27581490.23511971100
6.7389-49.570.19931440.1608199199
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)
10.54210.1220.24790.38410.62642.1316-0.16430.16450.9177-0.06790.06020.70130.09150.4227-0.0070.5384-0.0086-0.06390.67770.23661.1907-50.848453.9718-15.482
21.18940.65930.13270.49660.28740.6758-0.46031.34280.3734-0.56750.30910.1998-0.39720.1583-0.06770.6643-0.10530.04730.97980.01260.3491-19.495744.4837-17.2843
31.19910.2230.49631.09330.54851.91070.1867-0.07180.08220.3542-0.1792-0.02810.11370.12380.00010.5598-0.0129-0.03090.58760.07060.3385-21.109939.87335.1918
40.129-0.17720.12220.2436-0.17990.1137-0.64190.5277-0.7544-0.2487-0.1877-0.94860.41541.2533-0.01290.89780.1552-0.08111.05920.00130.6977-16.368122.89591.4754
50.15370.0722-0.13070.14030.2270.20710.14430.24141.2959-0.1957-0.11640.055-0.43191.2856-0.00070.5992-0.1309-0.11880.81990.03871.1241-31.848465.931-7.275
60.75590.1839-1.48510.4596-0.49873.2736-0.71890.48460.4134-0.21390.4060.14960.6608-0.5372-0.29480.48360.0061-0.16830.62290.25721.1334-38.288562.2793-7.3192
71.84981.43450.52021.9921-0.30411.31250.086-0.13181.70040.2899-0.21431.67040.3553-0.04660.11260.6912-0.05820.51210.6115-0.32360.7944-59.331238.62572.4227
82.18080.13160.57070.86850.09111.020.46910.07090.01050.0911-0.44560.13150.4440.0145-0.00020.8757-0.0334-0.00240.5306-0.0250.4039-43.350221.3178-1.8354
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 3 through 71 )A3 - 71
2X-RAY DIFFRACTION2chain 'A' and (resid 72 through 115 )A72 - 115
3X-RAY DIFFRACTION3chain 'A' and (resid 116 through 256 )A116 - 256
4X-RAY DIFFRACTION4chain 'A' and (resid 257 through 276 )A257 - 276
5X-RAY DIFFRACTION5chain 'B' and (resid 4 through 29 )B4 - 29
6X-RAY DIFFRACTION6chain 'B' and (resid 30 through 71 )B30 - 71
7X-RAY DIFFRACTION7chain 'B' and (resid 72 through 156 )B72 - 156
8X-RAY DIFFRACTION8chain 'B' and (resid 157 through 276 )B157 - 276

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