Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis

Area of Science:

• Structural Biology
• Microbiology
• Biochemistry

Background:

• Shape, elongation, division, and sporulation (SEDS) proteins are essential transmembrane enzymes involved in bacterial cell wall biology.
• Recent studies identified RodA, a prototypical SEDS protein, as a peptidoglycan polymerase, challenging previous attributions to penicillin-binding proteins.
• SEDS proteins, including RodA, are emerging as potential targets for novel antibiotics, yet their molecular mechanisms remain largely unelucidated.

Purpose of the Study:

• To determine the high-resolution crystal structure of the Thermus thermophilus RodA protein.
• To investigate the molecular basis of SEDS protein function and identify catalytically important regions.
• To provide a structural framework for understanding bacterial cell wall synthesis and developing new therapeutic strategies.

Main Methods:

• Crystal structure determination of Thermus thermophilus RodA at 2.9 Å resolution using evolutionary covariance-based fold prediction for molecular replacement.
• Analysis of the transmembrane fold, extracellular loops, and conserved cavities within the RodA structure.
• In vitro and in vivo mutagenesis experiments in Bacillus subtilis and Escherichia coli to assess the functional importance of the identified cavity.

Main Results:

• The crystal structure reveals a ten-pass transmembrane fold with significant extracellular loop structures.
• A highly conserved cavity was identified within the transmembrane domain, structurally analogous to ligand-binding sites in transmembrane receptors.
• Perturbation of this conserved cavity through mutagenesis abolished RodA function in both in vitro enzymatic assays and in vivo cellular studies.

Conclusions:

• The determined structure of RodA provides unprecedented molecular insights into the SEDS protein family.
• The identified transmembrane cavity is essential for RodA's peptidoglycan polymerase activity and bacterial cell wall synthesis.
• These findings offer a structural basis for understanding bacterial cell envelope biogenesis and guide the development of novel antibiotics targeting SEDS proteins.