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- PDB-6w1e: Crystal structure of Streptococcus thermophilus SHP pheromone rec... -

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Basic information

Entry
Database: PDB / ID: 6w1e
TitleCrystal structure of Streptococcus thermophilus SHP pheromone receptor Rgg3
ComponentsPositive transcriptional regulator MutR family
KeywordsDNA BINDING PROTEIN / PHEROMONE BINDING / QUORUM SENSING / RRNPP
Function / homologyTranscription activator MutR, C-terminal / HTH-type transcriptional regulator Rgg, C-terminal domain / : / Cro/C1-type HTH domain profile. / Cro/C1-type helix-turn-helix domain / Lambda repressor-like, DNA-binding domain superfamily / Tetratricopeptide-like helical domain superfamily / DNA binding / Positive transcriptional regulator MutR family
Function and homology information
Biological speciesStreptococcus thermophilus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.202 Å
AuthorsNeiditch, M.B. / Capodagli, G.C.
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 4, 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 / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Positive transcriptional regulator MutR family
hetero molecules


Theoretical massNumber of molelcules
Total (without water)33,4343
Polymers33,2421
Non-polymers1922
Water43224
1
A: Positive transcriptional regulator MutR family
hetero molecules

A: Positive transcriptional regulator MutR family
hetero molecules


Theoretical massNumber of molelcules
Total (without water)66,8686
Polymers66,4842
Non-polymers3844
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation5_554x-y,-y,-z-2/31
Buried area2860 Å2
ΔGint-64 kcal/mol
Surface area20800 Å2
MethodPISA
Unit cell
Length a, b, c (Å)110.360, 110.360, 54.933
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number154
Space group name H-MP3221

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Components

#1: Protein Positive transcriptional regulator MutR family


Mass: 33242.078 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus (strain ATCC BAA-250 / LMG 18311) (bacteria)
Strain: ATCC BAA-250 / LMG 18311 / Gene: stu1044 / Production host: Escherichia coli (E. coli) / References: UniProt: Q5M4D0
#2: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: SO4
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 24 / 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: 2.91 Å3/Da / Density % sol: 57.66 %
Crystal growTemperature: 298 K / Method: vapor diffusion / pH: 5.2
Details: 100 mM sodium citrate, 300 mM Na/K tartrate, and 1.4 M ammonium sulfate

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL9-2 / Wavelength: 0.9795 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Apr 12, 2018
RadiationMonochromator: Si 111 / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 2.2→50 Å / Num. obs: 19396 / % possible obs: 98 % / Redundancy: 9.4 % / Rmerge(I) obs: 0.081 / Rpim(I) all: 0.028 / Rrim(I) all: 0.086 / Χ2: 1.718 / Net I/av σ(I): 42.6 / Net I/σ(I): 8.5
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2.2-2.247.50.4718260.9160.1720.5030.59586
2.24-2.287.70.388710.9330.1390.4060.6887.6
2.28-2.327.60.3888870.9440.1420.4150.64592.6
2.32-2.378.70.3199660.9660.1090.3380.74498.1
2.37-2.429.20.2959660.9740.10.3110.81799.8
2.42-2.489.90.2989830.9820.0990.3140.767100
2.48-2.549.90.2629680.9830.0870.2760.86399.9
2.54-2.61100.2479900.9830.0820.2610.83399.7
2.61-2.699.90.2189800.9870.0720.231.03499.4
2.69-2.779.30.1939690.9860.0660.2041.16199.8
2.77-2.879.30.1659840.990.0570.1751.29799.6
2.87-2.9910.40.1469920.9930.0480.1541.49999.9
2.99-3.1210.20.1319700.9920.0430.1381.88999.6
3.12-3.29100.1189950.9940.040.1252.34599.9
3.29-3.499.90.1079900.9910.0360.1132.63899.5
3.49-3.7690.0969750.9950.0340.1023.06698.9
3.76-4.1410.20.08510090.9970.0280.0893.371100
4.14-4.749.90.07510020.9960.0250.0793.36999.8
4.74-5.979.20.06710140.9980.0230.0712.83299.4
5.97-509.10.05110590.9980.0180.0542.54499.4

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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: 2.202→47.787 Å / SU ML: 0.28 / Cross valid method: THROUGHOUT / σ(F): 1.38 / Phase error: 36.01
RfactorNum. reflection% reflection
Rfree0.3086 1929 9.96 %
Rwork0.2651 --
obs0.2695 19374 97.9 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 167.21 Å2 / Biso mean: 78.6552 Å2 / Biso min: 37.55 Å2
Refinement stepCycle: final / Resolution: 2.202→47.787 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1678 0 10 24 1712
Biso mean--131.94 69.38 -
Num. residues----202
LS refinement shell

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

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.202-2.25680.37741130.3441108386
2.2568-2.31780.34481250.3198111590
2.3178-2.3860.31181370.3034123998
2.386-2.4630.34251400.27561257100
2.463-2.55110.29171350.27691263100
2.5511-2.65320.32681400.2839125399
2.6532-2.77390.33971430.29121257100
2.7739-2.92020.31491430.28381262100
2.9202-3.10310.27931390.28051267100
3.1031-3.34260.29781420.25751266100
3.3426-3.67890.29331400.2464127599
3.6789-4.2110.26291400.22221278100
4.211-5.30430.28931450.23011284100
5.3043-47.7870.34791470.3011134699
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)
12.1670.68280.52360.8242-0.23830.3794-0.1142-1.5141.14152.018-0.73560.61-0.23260.2796-0.08681.4527-0.37860.14771.3257-0.17870.855541.387-19.8422-6.3223
23.02912.66031.63471.06341.02442.30910.4246-0.42040.287-0.3518-0.38950.12850.2213-0.37670.02540.5181-0.1308-0.01340.4992-0.050.543942.2114-18.2529-24.0492
31.82231.42631.36932.9302-0.4222.0235-0.38380.1256-0.099-0.2860.3428-0.1173-0.20880.203-00.5167-0.050.0740.41010.01020.443660.0255-9.7668-28.1017
40.60680.41720.32780.27590.1579-0.0378-0.3088-0.4464-0.25730.63560.2384-0.0740.06170.4194-0.00020.57230.09970.08870.40230.04010.64365.5729-14.9836-16.3575
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 78 through 108 )A78 - 108
2X-RAY DIFFRACTION2chain 'A' and (resid 109 through 203 )A109 - 203
3X-RAY DIFFRACTION3chain 'A' and (resid 204 through 263 )A204 - 263
4X-RAY DIFFRACTION4chain 'A' and (resid 264 through 284 )A264 - 284

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