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- PDB-9b71: Cryo-EM structure of MraY in complex with analogue 3 -

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Basic information

Entry
Database: PDB / ID: 9b71
TitleCryo-EM structure of MraY in complex with analogue 3
Components
  • MraYAA Nanobody
  • Phospho-N-acetylmuramoyl-pentapeptide-transferase
KeywordsTRANSFERASE / inhibitor / antibiotics
Function / homology
Function and homology information


phospho-N-acetylmuramoyl-pentapeptide-transferase / phospho-N-acetylmuramoyl-pentapeptide-transferase activity / UDP-N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-2,6-diaminopimelyl-D-alanyl-D-alanine:undecaprenyl-phosphate transferase activity / cell wall macromolecule biosynthetic process / phosphotransferase activity, for other substituted phosphate groups / peptidoglycan biosynthetic process / cell wall organization / regulation of cell shape / cell division / metal ion binding ...phospho-N-acetylmuramoyl-pentapeptide-transferase / phospho-N-acetylmuramoyl-pentapeptide-transferase activity / UDP-N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-2,6-diaminopimelyl-D-alanyl-D-alanine:undecaprenyl-phosphate transferase activity / cell wall macromolecule biosynthetic process / phosphotransferase activity, for other substituted phosphate groups / peptidoglycan biosynthetic process / cell wall organization / regulation of cell shape / cell division / metal ion binding / identical protein binding / plasma membrane
Similarity search - Function
Phospho-N-acetylmuramoyl-pentapeptide transferase / Phospho-N-acetylmuramoyl-pentapeptide transferase, conserved site / Phospho-N-acetylmuramoyl-pentapeptide-transferase signature 1 / MraY family signature 1. / MraY family signature 2. / Glycosyl transferase, family 4 / Glycosyl transferase family 4
Similarity search - Domain/homology
: / Phospho-N-acetylmuramoyl-pentapeptide-transferase
Similarity search - Component
Biological speciesLama glama (llama)
Aquifex aeolicus VF5 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsHao, A. / Lee, S.-Y.
Funding support United States, 1items
OrganizationGrant numberCountry
Other privateDuke Science Technology Scholar Fund United States
CitationJournal: Nat Commun / Year: 2024
Title: Development of a natural product optimization strategy for inhibitors against MraY, a promising antibacterial target.
Authors: Kazuki Yamamoto / Toyotaka Sato / Aili Hao / Kenta Asao / Rintaro Kaguchi / Shintaro Kusaka / Radhakrishnam Raju Ruddarraju / Daichi Kazamori / Kiki Seo / Satoshi Takahashi / Motohiro ...Authors: Kazuki Yamamoto / Toyotaka Sato / Aili Hao / Kenta Asao / Rintaro Kaguchi / Shintaro Kusaka / Radhakrishnam Raju Ruddarraju / Daichi Kazamori / Kiki Seo / Satoshi Takahashi / Motohiro Horiuchi / Shin-Ichi Yokota / Seok-Yong Lee / Satoshi Ichikawa /
Abstract: MraY (phospho-N-acetylmuramoyl-pentapeptide-transferase) inhibitory natural products are attractive molecules as candidates for a new class of antibacterial agents to combat antimicrobial-resistant ...MraY (phospho-N-acetylmuramoyl-pentapeptide-transferase) inhibitory natural products are attractive molecules as candidates for a new class of antibacterial agents to combat antimicrobial-resistant bacteria. Structural optimization of these natural products is required to improve their drug-like properties for therapeutic use. However, chemical modifications of these natural products are painstaking tasks due to complex synthetic processes, which is a bottleneck in advancing natural products to the clinic. Here, we develop a strategy for a comprehensive in situ evaluation of the build-up library, which enables us to streamline the preparation of the analogue library and directly assess its biological activities. We apply this approach to a series of MraY inhibitory natural products. Through construction and evaluation of the 686-compound library, we identify promising analogues that exhibit potent and broad-spectrum antibacterial activity against highly drug-resistant strains in vitro as well as in vivo in an acute thigh infection model. Structures of the MraY-analogue complexes reveal distinct interaction patterns, suggesting that these analogues represent MraY inhibitors with unique binding modes. We further demonstrate the generality of our strategy by applying it to tubulin-binding natural products to modulate their tubulin polymerization activities.
History
DepositionMar 26, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 26, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
H: MraYAA Nanobody
A: Phospho-N-acetylmuramoyl-pentapeptide-transferase
G: MraYAA Nanobody
B: Phospho-N-acetylmuramoyl-pentapeptide-transferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)113,9496
Polymers112,0254
Non-polymers1,9242
Water1086
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Antibody MraYAA Nanobody


Mass: 15057.651 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#2: Protein Phospho-N-acetylmuramoyl-pentapeptide-transferase / UDP-MurNAc-pentapeptide phosphotransferase


Mass: 40954.684 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aquifex aeolicus VF5 (bacteria) / Gene: mraY, aq_053 / Production host: Escherichia coli (E. coli)
References: UniProt: O66465, phospho-N-acetylmuramoyl-pentapeptide-transferase
#3: Chemical ChemComp-A1AI2 / (2~{S},3~{S})-3-[(2~{S},3~{R},4~{S},5~{R})-5-(aminomethyl)-3,4-bis(oxidanyl)oxolan-2-yl]oxy-3-[(2~{S},3~{S},4~{R},5~{R})-5-[2,4-bis(oxidanylidene)pyrimidin-1-yl]-3,4-bis(oxidanyl)oxolan-2-yl]-2-[[4-[[[(2~{S})-5-carbamimidamido-2-(hexadecanoylamino)pentanoyl]amino]methyl]phenyl]methylamino]propanoic acid


Mass: 962.140 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C46H75N9O13 / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Complex of MraY AA dimer with nanobody bound / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.2 MDa / Experimental value: NO
Source (natural)Organism: Aquifex aeolicus VF5 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8 / Details: 20mM Tris-HCl, 150mM NaCl, 2mM DTT, 5mM DM
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris hydrochlorideTris-HCl1
2150 mMSodium chlorideNaCl1
35 mMdecyl-maltosideDM1
42 mMDithiothreitolDTT1
SpecimenConc.: 7.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4.6 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2087
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameCategory
2Latitudeimage acquisition
4cryoSPARCCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 68364 / Symmetry type: POINT
Atomic model buildingPDB-ID: 5CKR

5ckr


Accession code: 5CKR / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0047404
ELECTRON MICROSCOPYf_angle_d0.58210098
ELECTRON MICROSCOPYf_dihedral_angle_d12.372464
ELECTRON MICROSCOPYf_chiral_restr0.0441198
ELECTRON MICROSCOPYf_plane_restr0.0031224

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