National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R35 GM132120
United States
Citation
Journal: Nat Commun / Year: 2023 Title: Structural basis of peptidoglycan synthesis by E. coli RodA-PBP2 complex. Authors: Rie Nygaard / Chris L B Graham / Meagan Belcher Dufrisne / Jonathan D Colburn / Joseph Pepe / Molly A Hydorn / Silvia Corradi / Chelsea M Brown / Khuram U Ashraf / Owen N Vickery / Nicholas ...Authors: Rie Nygaard / Chris L B Graham / Meagan Belcher Dufrisne / Jonathan D Colburn / Joseph Pepe / Molly A Hydorn / Silvia Corradi / Chelsea M Brown / Khuram U Ashraf / Owen N Vickery / Nicholas S Briggs / John J Deering / Brian Kloss / Bruno Botta / Oliver B Clarke / Linda Columbus / Jonathan Dworkin / Phillip J Stansfeld / David I Roper / Filippo Mancia / Abstract: Peptidoglycan (PG) is an essential structural component of the bacterial cell wall that is synthetized during cell division and elongation. PG forms an extracellular polymer crucial for cellular ...Peptidoglycan (PG) is an essential structural component of the bacterial cell wall that is synthetized during cell division and elongation. PG forms an extracellular polymer crucial for cellular viability, the synthesis of which is the target of many antibiotics. PG assembly requires a glycosyltransferase (GT) to generate a glycan polymer using a Lipid II substrate, which is then crosslinked to the existing PG via a transpeptidase (TP) reaction. A Shape, Elongation, Division and Sporulation (SEDS) GT enzyme and a Class B Penicillin Binding Protein (PBP) form the core of the multi-protein complex required for PG assembly. Here we used single particle cryo-electron microscopy to determine the structure of a cell elongation-specific E. coli RodA-PBP2 complex. We combine this information with biochemical, genetic, spectroscopic, and computational analyses to identify the Lipid II binding sites and propose a mechanism for Lipid II polymerization. Our data suggest a hypothesis for the movement of the glycan strand from the Lipid II polymerization site of RodA towards the TP site of PBP2, functionally linking these two central enzymatic activities required for cell wall peptidoglycan biosynthesis.
Name: RodA-PBP2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 / Details: Nanodisc were formed using MSP1E3D1 and POPG lipid
Source (natural)
Organism: Escherichia coli (E. coli)
Molecular weight
Theoretical: 111.803 KDa
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
-
Sample preparation
Concentration
0.66 mg/mL
Buffer
pH: 7 Component:
Concentration
Name
Formula
20.0 mM
HEPES
150.0 mM
Sodium Chloride
NaCl
1.0 mM
TCEP
tris(2-carboxyethyl)phosphine
Vitrification
Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
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Electron microscopy
Microscope
FEI TITAN
Image recording
Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 11120 / Average exposure time: 2.5 sec. / Average electron dose: 58.5 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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