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- PDB-6x3j: Crystal structure of streptogramin A acetyltransferase VatA from ... -

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Basic information

Entry
Database: PDB / ID: 6x3j
TitleCrystal structure of streptogramin A acetyltransferase VatA from Staphylococcus aureus in complex with streptogramin analog F0224 (46)
ComponentsVirginiamycin A acetyltransferase,VatA
KeywordsTRANSFERASE / Acetyltransferase
Function / homology
Function and homology information


acyltransferase activity / Transferases; Acyltransferases; Transferring groups other than aminoacyl groups / response to antibiotic
Similarity search - Function
Hexapeptide transferase, conserved site / Hexapeptide-repeat containing-transferases signature. / Hexapeptide repeat / Bacterial transferase hexapeptide (six repeats) / Trimeric LpxA-like superfamily
Similarity search - Domain/homology
Chem-O7V / PHOSPHATE ION / Chem-SXA / Virginiamycin A acetyltransferase
Similarity search - Component
Biological speciesStaphylococcus aureus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.7 Å
AuthorsChaires, H.A. / Fraser, J.S.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM128656 United States
CitationJournal: Nature / Year: 2020
Title: Synthetic group A streptogramin antibiotics that overcome Vat resistance.
Authors: Qi Li / Jenna Pellegrino / D John Lee / Arthur A Tran / Hector A Chaires / Ruoxi Wang / Jesslyn E Park / Kaijie Ji / David Chow / Na Zhang / Axel F Brilot / Justin T Biel / Gydo van Zundert ...Authors: Qi Li / Jenna Pellegrino / D John Lee / Arthur A Tran / Hector A Chaires / Ruoxi Wang / Jesslyn E Park / Kaijie Ji / David Chow / Na Zhang / Axel F Brilot / Justin T Biel / Gydo van Zundert / Kenneth Borrelli / Dean Shinabarger / Cindy Wolfe / Beverly Murray / Matthew P Jacobson / Estelle Mühle / Olivier Chesneau / James S Fraser / Ian B Seiple /
Abstract: Natural products serve as chemical blueprints for most antibiotics in clinical use. The evolutionary process by which these molecules arise is inherently accompanied by the co-evolution of resistance ...Natural products serve as chemical blueprints for most antibiotics in clinical use. The evolutionary process by which these molecules arise is inherently accompanied by the co-evolution of resistance mechanisms that shorten the clinical lifetime of any given class of antibiotics. Virginiamycin acetyltransferase (Vat) enzymes are resistance proteins that provide protection against streptogramins, potent antibiotics against Gram-positive bacteria that inhibit the bacterial ribosome. Owing to the challenge of selectively modifying the chemically complex, 23-membered macrocyclic scaffold of group A streptogramins, analogues that overcome the resistance conferred by Vat enzymes have not been previously developed. Here we report the design, synthesis, and antibacterial evaluation of group A streptogramin antibiotics with extensive structural variability. Using cryo-electron microscopy and forcefield-based refinement, we characterize the binding of eight analogues to the bacterial ribosome at high resolution, revealing binding interactions that extend into the peptidyl tRNA-binding site and towards synergistic binders that occupy the nascent peptide exit tunnel. One of these analogues has excellent activity against several streptogramin-resistant strains of Staphylococcus aureus, exhibits decreased rates of acetylation in vitro, and is effective at lowering bacterial load in a mouse model of infection. Our results demonstrate that the combination of rational design and modular chemical synthesis can revitalize classes of antibiotics that are limited by naturally arising resistance mechanisms.
History
DepositionMay 21, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 18, 2020Provider: repository / Type: Initial release
Revision 1.1Oct 18, 2023Group: Advisory / Data collection ...Advisory / Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_unobs_or_zero_occ_atoms
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Virginiamycin A acetyltransferase,VatA
B: Virginiamycin A acetyltransferase,VatA
C: Virginiamycin A acetyltransferase,VatA
D: Virginiamycin A acetyltransferase,VatA
E: Virginiamycin A acetyltransferase,VatA
F: Virginiamycin A acetyltransferase,VatA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)150,22533
Polymers142,8876
Non-polymers7,33827
Water3,747208
1
A: Virginiamycin A acetyltransferase,VatA
B: Virginiamycin A acetyltransferase,VatA
D: Virginiamycin A acetyltransferase,VatA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)74,98813
Polymers71,4433
Non-polymers3,54510
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area10320 Å2
ΔGint-87 kcal/mol
Surface area26190 Å2
MethodPISA
2
C: Virginiamycin A acetyltransferase,VatA
E: Virginiamycin A acetyltransferase,VatA
F: Virginiamycin A acetyltransferase,VatA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)75,23620
Polymers71,4433
Non-polymers3,79317
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area10670 Å2
ΔGint-141 kcal/mol
Surface area26160 Å2
MethodPISA
Unit cell
Length a, b, c (Å)104.800, 109.220, 173.030
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2

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Components

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Protein , 1 types, 6 molecules ABCDEF

#1: Protein
Virginiamycin A acetyltransferase,VatA


Mass: 23814.494 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria) / Gene: vat / Production host: Escherichia coli (E. coli)
References: UniProt: P26839, Transferases; Acyltransferases; Transferring groups other than aminoacyl groups

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Non-polymers , 6 types, 235 molecules

#2: Chemical
ChemComp-O7V / (2R)-2-[(3S,4R,5E,10E,12E,14S,16R,26aR)-16-fluoro-14-hydroxy-4,12-dimethyl-1,7,22-trioxo-4,7,8,9,14,15,16,17,24,25,26,26a-dodecahydro-1H,3H,22H-21,18-(azeno)pyrrolo[2,1-c][1,8,4,19]dioxadiazacyclotetracosin-3-yl]propyl isoquinolin-3-ylcarbamate


Mass: 717.783 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C38H44FN5O8 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-PO4 / PHOSPHATE ION / Phosphate


Mass: 94.971 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: PO4
#4: Chemical
ChemComp-SXA / THIOACETIC ACID S-{2-[3-(2-HYDROXY-3,3-DIMETHYL-4-PHOSPHONOOXY-BUTYRYLAMINO)-PROPIONYLAMINO]-ETHYL} ESTER


Mass: 400.385 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C13H25N2O8PS
#5: Chemical
ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: Cl
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#7: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 208 / Source method: isolated from a natural source / Formula: H2O

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Details

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: 3.3 Å3/Da / Density % sol: 62.71 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / Details: 1 M LiCl, 0.1 M BICINE pH 9, and 10 %w/v PEG 6K

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Data collection

DiffractionMean temperature: 92 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 8.3.1 / Wavelength: 1.11583 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Aug 30, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.11583 Å / Relative weight: 1
ReflectionResolution: 2.7→86.51 Å / Num. obs: 55239 / % possible obs: 99.96 % / Redundancy: 29.6 % / Biso Wilson estimate: 57.72 Å2 / CC1/2: 0.995 / CC star: 0.999 / Net I/σ(I): 9.41
Reflection shellResolution: 2.7→2.797 Å / Num. unique obs: 5440 / CC1/2: 0.404 / CC star: 0.758 / % possible all: 99.96

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Processing

Software
NameVersionClassification
PHENIX1.17.1_3660refinement
xia2data reduction
xia2data scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4HUR

4hur


Resolution: 2.7→86.51 Å / Cross valid method: FREE R-VALUE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.271 --
Rwork0.2337 --
obs-55232 99.96 %
Displacement parametersBiso mean: 57.72 Å2
Refinement stepCycle: LAST / Resolution: 2.7→86.51 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9976 0 403 208 10587
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.005710690
X-RAY DIFFRACTIONf_angle_d0.929914474
X-RAY DIFFRACTIONf_chiral_restr0.05721516
X-RAY DIFFRACTIONf_plane_restr0.01011822
X-RAY DIFFRACTIONf_dihedral_angle_d16.42356141

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