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- PDB-8w7a: Cryo-EM structure of ClassIII Lanthipeptide modification enzyme P... -

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Basic information

Entry
Database: PDB / ID: 8w7a
TitleCryo-EM structure of ClassIII Lanthipeptide modification enzyme PneKC in the presence of GTP.
ComponentsProtein kinase domain-containing protein
KeywordsANTIMICROBIAL PROTEIN / Lanthibiotic / RiPPs / LanKC / CryoEM / Antimicrobial peptides / Antiviral peptides.
Function / homology
Function and homology information


peptide modification / protein kinase activity / ATP binding
Similarity search - Function
Lanthionine synthetase C-like protein / Lanthionine synthetase C-like / : / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
GUANOSINE-5'-TRIPHOSPHATE / Protein kinase domain-containing protein
Similarity search - Component
Biological speciesStreptococcus pneumoniae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.77 Å
AuthorsLi, Y. / Luo, M. / Shao, K. / Li, J.
Funding support Singapore, 1items
OrganizationGrant numberCountry
Ministry of Education (MoE, Singapore) Singapore
CitationJournal: Nat Commun / Year: 2024
Title: Mechanistic insights into lanthipeptide modification by a distinct subclass of LanKC enzyme that forms dimers.
Authors: Yifan Li / Kai Shao / Zhaoxing Li / Kongfu Zhu / Bee Koon Gan / Jian Shi / Yibei Xiao / Min Luo /
Abstract: Naturally occurring lanthipeptides, peptides post-translationally modified by various enzymes, hold significant promise as antibiotics. Despite extensive biochemical and structural studies, the ...Naturally occurring lanthipeptides, peptides post-translationally modified by various enzymes, hold significant promise as antibiotics. Despite extensive biochemical and structural studies, the events preceding peptide modification remain poorly understood. Here, we identify a distinct subclass of lanthionine synthetase KC (LanKC) enzymes with distinct structural and functional characteristics. We show that PneKC, a member of this subclass, forms a dimer and possesses GTPase activity. Through three cryo-EM structures of PneKC, we illustrate different stages of peptide PneA binding, from initial recognition to full binding. Our structures show the kinase domain complexed with the PneA core peptide and GTPγS, a phosphate-bound lyase domain, and an unconventional cyclase domain. The leader peptide of PneA interact with a gate loop, transitioning from an extended to a helical conformation. We identify a dimerization hot spot and propose a "negative cooperativity" mechanism toggling the enzyme between tense and relaxed conformation. Additionally, we identify an important salt bridge in the cyclase domain, differing from those in in conventional cyclase domains. These residues are highly conserved in the LanKC subclass and are part of two signature motifs. These results unveil potential differences in lanthipeptide modification enzymes assembly and deepen our understanding of allostery in these multifunctional enzymes.
History
DepositionAug 30, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 28, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Protein kinase domain-containing protein
B: Protein kinase domain-containing protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)200,8673
Polymers200,3442
Non-polymers5231
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Protein kinase domain-containing protein


Mass: 100171.750 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pneumoniae (bacteria) / Gene: SPRM200_1177 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A2U3S0J5
#2: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM
Has ligand of interestY

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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: Dimeric structure of Class III lanthipeptide modification PneKC with GTP bound to one protomer.
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.21 MDa / Experimental value: YES
Source (natural)Organism: Streptococcus pneumoniae (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4 / Details: 25mM Tris, 200mM NaCl
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMsodium chlorideNaCl1
225 mMTris(HOCH2)3CNH21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 32 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.77 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 257035 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00613331
ELECTRON MICROSCOPYf_angle_d1.28818034
ELECTRON MICROSCOPYf_dihedral_angle_d8.4841739
ELECTRON MICROSCOPYf_chiral_restr0.0681939
ELECTRON MICROSCOPYf_plane_restr0.0092281

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