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- PDB-5ezu: Crystal structure of the N-terminal domain of vaccinia virus immu... -

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Basic information

Entry
Database: PDB / ID: 5ezu
TitleCrystal structure of the N-terminal domain of vaccinia virus immunomodulator A46 in complex with myristic acid.
ComponentsProtein A46
KeywordsVIRAL PROTEIN / immunomodulator / beta sheet / ab initio phasing / vaccinia virus / A46 / fatty acids / myristic acid
Function / homology
Function and homology information


extrinsic component of cytoplasmic side of plasma membrane / protein sequestering activity / symbiont-mediated suppression of host NF-kappaB cascade / symbiont-mediated suppression of host toll-like receptor signaling pathway / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF3 activity / virus-mediated perturbation of host defense response
Similarity search - Function
Poxvirus Bcl-2-like / Poxvirus domain superfamily / Poxvirus Bcl-2-like proteins
Similarity search - Domain/homology
MYRISTIC ACID / Protein OPG176
Similarity search - Component
Biological speciesVaccinia virus
MethodX-RAY DIFFRACTION / SYNCHROTRON / AB INITIO PHASING / Resolution: 1.55 Å
AuthorsFedosyuk, S. / Bezerra, G.A. / Sammito, M. / Uson, I. / Skern, T.
Funding support Austria, 1items
OrganizationGrant numberCountry
Austrian Science FundW1221 Austria
CitationJournal: PLoS Pathog / Year: 2016
Title: Vaccinia Virus Immunomodulator A46: A Lipid and Protein-Binding Scaffold for Sequestering Host TIR-Domain Proteins.
Authors: Sofiya Fedosyuk / Gustavo Arruda Bezerra / Katharina Radakovics / Terry K Smith / Massimo Sammito / Nina Bobik / Adam Round / Lynn F Ten Eyck / Kristina Djinović-Carugo / Isabel Usón / Tim Skern /
Abstract: Vaccinia virus interferes with early events of the activation pathway of the transcriptional factor NF-kB by binding to numerous host TIR-domain containing adaptor proteins. We have previously ...Vaccinia virus interferes with early events of the activation pathway of the transcriptional factor NF-kB by binding to numerous host TIR-domain containing adaptor proteins. We have previously determined the X-ray structure of the A46 C-terminal domain; however, the structure and function of the A46 N-terminal domain and its relationship to the C-terminal domain have remained unclear. Here, we biophysically characterize residues 1-83 of the N-terminal domain of A46 and present the X-ray structure at 1.55 Å. Crystallographic phases were obtained by a recently developed ab initio method entitled ARCIMBOLDO_BORGES that employs tertiary structure libraries extracted from the Protein Data Bank; data analysis revealed an all β-sheet structure. This is the first such structure solved by this method which should be applicable to any protein composed entirely of β-sheets. The A46(1-83) structure itself is a β-sandwich containing a co-purified molecule of myristic acid inside a hydrophobic pocket and represents a previously unknown lipid-binding fold. Mass spectrometry analysis confirmed the presence of long-chain fatty acids in both N-terminal and full-length A46; mutation of the hydrophobic pocket reduced the lipid content. Using a combination of high resolution X-ray structures of the N- and C-terminal domains and SAXS analysis of full-length protein A46(1-240), we present here a structural model of A46 in a tetrameric assembly. Integrating affinity measurements and structural data, we propose how A46 simultaneously interferes with several TIR-domain containing proteins to inhibit NF-κB activation and postulate that A46 employs a bipartite binding arrangement to sequester the host immune adaptors TRAM and MyD88.
History
DepositionNov 26, 2015Deposition site: RCSB / Processing site: PDBE
Revision 1.0Nov 30, 2016Provider: repository / Type: Initial release
Revision 1.1Dec 28, 2016Group: Database references
Revision 1.2Sep 6, 2017Group: Author supporting evidence / Refinement description / Category: pdbx_audit_support / software / Item: _pdbx_audit_support.funding_organization
Revision 1.3May 8, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Protein A46
B: Protein A46
hetero molecules


Theoretical massNumber of molelcules
Total (without water)20,3033
Polymers20,0742
Non-polymers2281
Water1,02757
1
A: Protein A46
B: Protein A46
hetero molecules

A: Protein A46
B: Protein A46
hetero molecules


Theoretical massNumber of molelcules
Total (without water)40,6056
Polymers40,1494
Non-polymers4572
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_658-x+1,y,-z+31
Buried area8610 Å2
ΔGint-15 kcal/mol
Surface area12900 Å2
MethodPISA
Unit cell
Length a, b, c (Å)65.787, 59.580, 47.257
Angle α, β, γ (deg.)90.000, 117.700, 90.000
Int Tables number5
Space group name H-MC121
Components on special symmetry positions
IDModelComponents
11A-230-

HOH

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Components

#1: Protein Protein A46


Mass: 10037.169 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus (strain Western Reserve)
Gene: VACWR172, A46R / Plasmid: pET-TRX / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P26672
#2: Chemical ChemComp-MYR / MYRISTIC ACID


Mass: 228.371 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C14H28O2
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 57 / Source method: isolated from a natural source / Formula: H2O

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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: 2.36 Å3/Da / Density % sol: 47.78 %
Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop / pH: 7
Details: PEG6000, sodium chloride, lithium chloride, ammonium chloride

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: MASSIF-1 / Wavelength: 0.979 Å
DetectorType: DECTRIS PILATUS 2M / Detector: PIXEL / Date: Jul 25, 2014
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.979 Å / Relative weight: 1
ReflectionResolution: 1.55→41.84 Å / Num. obs: 23425 / % possible obs: 99.6 % / Redundancy: 1 % / Biso Wilson estimate: 23.99 Å2 / Rmerge(I) obs: 0.036 / Net I/σ(I): 12.8 / Num. measured all: 116682
Reflection shellResolution: 1.55→1.65 Å / Redundancy: 1.93 % / Rmerge(I) obs: 0.54 / Mean I/σ(I) obs: 1.53 / % possible all: 98.9

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Processing

Software
NameVersionClassification
PHENIXdev_1938refinement
PDB_EXTRACT3.15data extraction
XPREPdata reduction
Arcimboldophasing
RefinementMethod to determine structure: AB INITIO PHASING / Resolution: 1.55→41.842 Å / SU ML: 0.23 / Cross valid method: FREE R-VALUE / σ(F): 1.37 / Phase error: 33.04 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2418 1101 4.71 %
Rwork0.1997 22288 -
obs0.2017 23389 99.41 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 106.08 Å2 / Biso mean: 40.9362 Å2 / Biso min: 17.31 Å2
Refinement stepCycle: final / Resolution: 1.55→41.842 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1150 0 16 57 1223
Biso mean--56.1 49.38 -
Num. residues----143
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0061247
X-RAY DIFFRACTIONf_angle_d1.0251703
X-RAY DIFFRACTIONf_chiral_restr0.056199
X-RAY DIFFRACTIONf_plane_restr0.004222
X-RAY DIFFRACTIONf_dihedral_angle_d13.585468
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 8

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.55-1.62050.39371010.35552776287798
1.6205-1.7060.34911490.32472768291799
1.706-1.81290.30531410.28492749289099
1.8129-1.95280.28281430.22762759290299
1.9528-2.14940.24361320.200327972929100
2.1494-2.46040.24091660.192527652931100
2.4604-3.09960.25251510.216227972948100
3.0996-41.8570.2071180.165628772995100
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)
12.077-0.3659-3.02514.71311.15145.00870.27260.74130.1057-0.7259-0.08610.2089-0.0799-0.4466-0.22660.33360.0729-0.04070.36530.00010.2301-7.7194-10.846442.4799
27.3811-2.2457-3.00465.11761.22466.1498-0.4063-0.20311.21510.17510.3714-0.1433-0.3505-0.30190.04480.29040.1307-0.10110.2586-0.07360.3606-12.98654.268650.2152
34.77750.054-0.88954.6346-1.70739.4883-0.03960.2890.27150.05140.02260.1413-0.3237-0.8270.24590.24430.0718-0.03580.3389-0.05080.285-15.8255-0.8548.3491
42.9773-2.6660.08034.78860.92911.3758-0.05760.0268-0.1618-0.0182-0.04940.3430.1281-0.4850.13670.22190.00530.02280.3628-0.05140.215-11.7634-12.043845.823
56.2172-0.77390.38314.35055.73738.0003-0.16850.04640.44820.254-0.4111-0.1129-0.69310.18880.18830.40770.0214-0.09660.2335-0.00580.25082.3567-7.310857.1114
63.3675-2.3906-0.93487.02592.35812.6286-0.4437-1.00440.06440.7520.34460.45530.0312-0.44640.19270.3150.17110.04170.5057-0.14940.2382-10.9229-6.601760.1362
78.0924-6.4572-3.46834.84793.57893.646-0.1758-0.18820.31241.0687-0.1754-0.1965-0.1833-0.73570.21220.33720.0627-0.07160.2954-0.05670.1836-8.6073-6.025661.4989
86.7487-3.71071.05132.3489-1.35585.95340.2152-0.0998-0.115-0.1597-0.41020.40870.0728-0.10240.25080.3503-0.0006-0.07680.2273-0.00140.2342-4.8199-7.179763.6723
93.4644-2.6158-0.33964.70393.15454.4345-0.3753-0.20691.62881.24860.39050.62-0.9291-0.2376-0.17790.74410.1313-0.13890.3101-0.11430.8224-8.67278.103957.7078
105.5949-3.00491.09096.4207-0.0992.76560.22060.47540.1899-0.3164-0.3838-0.8143-0.03010.46270.14080.17590.03210.05090.27410.00920.29164.0413-11.68147.8113
116.5947-4.9134-2.76174.83424.4484.811-0.16850.1621.02620.58920.1686-1.3771-0.30650.32650.06910.28910.0096-0.10020.2623-0.00340.35671.8061-4.15452.5798
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 1 through 13 )A1 - 13
2X-RAY DIFFRACTION2chain 'A' and (resid 14 through 32 )A14 - 32
3X-RAY DIFFRACTION3chain 'A' and (resid 33 through 47 )A33 - 47
4X-RAY DIFFRACTION4chain 'A' and (resid 48 through 76 )A48 - 76
5X-RAY DIFFRACTION5chain 'B' and (resid 1 through 8 )B1 - 8
6X-RAY DIFFRACTION6chain 'B' and (resid 9 through 24 )B9 - 24
7X-RAY DIFFRACTION7chain 'B' and (resid 25 through 32 )B25 - 32
8X-RAY DIFFRACTION8chain 'B' and (resid 33 through 42 )B33 - 42
9X-RAY DIFFRACTION9chain 'B' and (resid 43 through 47 )B43 - 47
10X-RAY DIFFRACTION10chain 'B' and (resid 48 through 58 )B48 - 58
11X-RAY DIFFRACTION11chain 'B' and (resid 59 through 67 )B59 - 67

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