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- PDB-6x3c: Crystal structure of streptogramin A acetyltransferase VatA from ... -

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Basic information

Entry
Database: PDB / ID: 6x3c
TitleCrystal structure of streptogramin A acetyltransferase VatA from Staphylococcus aureus in complex with streptogramin analog F1037 (47)
ComponentsVirginiamycin A acetyltransferase
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-O7S / PHOSPHATE ION / Chem-SXA / Virginiamycin A acetyltransferase
Similarity search - Component
Biological speciesStaphylococcus aureus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.05 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: 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
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
B: Virginiamycin A acetyltransferase
C: Virginiamycin A acetyltransferase
D: Virginiamycin A acetyltransferase
E: Virginiamycin A acetyltransferase
F: Virginiamycin A acetyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)149,15644
Polymers141,4336
Non-polymers7,72338
Water79344
1
A: Virginiamycin A acetyltransferase
E: Virginiamycin A acetyltransferase
F: Virginiamycin A acetyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)74,51822
Polymers70,7163
Non-polymers3,80119
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area11400 Å2
ΔGint-139 kcal/mol
Surface area25730 Å2
MethodPISA
2
B: Virginiamycin A acetyltransferase
C: Virginiamycin A acetyltransferase
D: Virginiamycin A acetyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)74,63922
Polymers70,7163
Non-polymers3,92219
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area11140 Å2
ΔGint-141 kcal/mol
Surface area25880 Å2
MethodPISA
Unit cell
Length a, b, c (Å)105.540, 105.620, 178.200
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


Mass: 23572.156 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 , 7 types, 82 molecules

#2: 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
#3: Chemical
ChemComp-O7S / (3R,4R,5E,10E,12E,14S,16R,26aR)-16-fluoro-14-hydroxy-12-methyl-3-(propan-2-yl)-4-(prop-2-en-1-yl)-3,4,8,9,14,15,16,17,24,25,26,26a-dodecahydro-1H,7H,22H-21,18-(azeno)pyrrolo[2,1-c][1,8,4,19]dioxadiazacyclotetracosine-1,7,22-trione


Mass: 557.654 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C30H40FN3O6 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-PO4 / PHOSPHATE ION / Phosphate


Mass: 94.971 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: PO4
#5: Chemical
ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: SO4
#6: Chemical
ChemComp-CL / CHLORIDE ION / Chloride


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


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


Mass: 18.015 Da / Num. of mol.: 44 / 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: 3.31 Å3/Da / Density % sol: 62.82 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop
Details: 0.2 M (NH4)2SO4, 0.1 M phosphate-citrate pH 4.2, 20 %v/v PEG 300; 10 % glycerol

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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: 3.05→89.138 Å / Num. obs: 38654 / % possible obs: 99.92 % / Redundancy: 13.2 % / Biso Wilson estimate: 65.64 Å2 / CC1/2: 0.989 / CC star: 0.997 / Rrim(I) all: 0.3773 / Net I/σ(I): 6.67
Reflection shellResolution: 3.05→3.159 Å / Redundancy: 12.8 % / Num. unique obs: 3766 / CC1/2: 0.575 / CC star: 0.855 / % possible all: 99.92

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Processing

Software
NameVersionClassification
PHENIX1.17.1_3660refinement
PHENIX1.17.1_3660phasing
xia2data reduction
xia2data scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4HUR

4hur


Resolution: 3.05→89.138 Å / Cross valid method: FREE R-VALUE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2662 --
Rwork0.2335 --
obs-38637 99.92 %
Displacement parametersBiso mean: 64.13 Å2
Refinement stepCycle: LAST / Resolution: 3.05→89.138 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10207 0 98 44 10349
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.009110537
X-RAY DIFFRACTIONf_angle_d1.102314276
X-RAY DIFFRACTIONf_chiral_restr0.06521501
X-RAY DIFFRACTIONf_plane_restr0.00711794
X-RAY DIFFRACTIONf_dihedral_angle_d14.67396060

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