Aug 2026· Open Biology· Vol 16 8· 0 citations· 77 references
Medicine
TL;DR
The 3.0 Å cryo-electron microscopy apo-structure of the PGT enzyme WbaP from Escherichia coli, a UDP-galactose:undecaprenolphosphate galactose-1-phosphoryl transferase, provides new insights into the PGT family of enzymes.
Abstract
Host-pathogen interactions frequently depend on key components of the bacterial cell surface, such as capsules and lipopolysaccharides in Gram-negative bacteria. The first step in the synthesis of lipid-linked polysaccharides is the substitution of a uridine diphosphate (UDP)-sugar by a lipid monophosphate catalysed by a phosphoglycosyl transferase (PGT). We report the 3.0 Å cryo-electron microscopy apo-structure of the PGT enzyme WbaP from Escherichia coli, a UDP-galactose:undecaprenolphosphate galactose-1-phosphoryl transferase. The structure is a dimer with each monomer formed of four N-terminal transmembrane helices, a small α/β domain with a distinctive β-hairpin that inserts into the other monomer, and a catalytic domain, which sits perpendicular to the transmembrane domain. A complex of WbaP with the UDP-galactose substrate shows binding of the UDP moiety by R319 and R377. Mutations of R319 and R377, along with K331 and R401, highlighted the essential nature of these residues for the catalytic activity of the protein, as confirmed by an in vivo functional assay. Our results provide new insights into the PGT family of enzymes.
In Staphylococcus aureus, over 50 different types of lipoproteins are tethered to the membrane surface through acylation of an N-terminal cysteine residue where they perform multiple cellular roles, from nutrient transport to displaying virulence factors. Lipoproteins are also key focal points for detection by the toll-like receptor 2 family of innate immunity. While all staphylococci initiate lipoprotein biosynthesis by attaching a thioether-linked diacyl glycerol moiety, subsequent α-amino tailoring occurs in a species dependent manner that attenuates toll-like receptor 2 signaling. A two-gene system encoding an NlpC/P60 superfamily enzyme (LnsA) and a CAAX protease/bacteriocin-processing family integral membrane protein (LnsB) is required for lipoprotein N-acylation in S. aureus, but little is known regarding the mechanism. Herein, we show that the LnsA N-terminal α-helix is a canonical cleaved signal peptide that is unnecessary for membrane retention. We use protein complex modeling with diacylated lipopeptide substrate and substituted cysteine crosslinking to demonstrate a key loop on LnsA interacts with LnsB in a multimeric complex. Using targeted site mutagenesis, a Cys-His catalytic dyad common to NlpC/P60 superfamily members is defined while no CAAX protease/bacteriocin-processing motif residues were essential. Reconstitution using recombinant LnsA and LnsB with lipopeptide substrate confirmed that both proteins are required for catalysis in vitro, and that the SN1 acyl chain of phosphatidylglycerol is the preferred acyl chain substrate donor. This work begins to define the LnsAB complex, and further underscores the rich source of unique acylation biochemistry that has evolved in bacterial lipoprotein N-terminal modification pathways.
John H. Gardiner, Gloria Komazin, Timothy C. Meredith· Journal of Biological Chemis...· 0 citations
Sortase A (SrtA) enzymes covalently anchor surface proteins to Gram-positive bacterial cell walls, promoting colonization and virulence. In Streptococcus pneumoniae, previous studies identified both a domain-swapped dimer and an active refolded monomer, but the active enzyme has not been characterized at the structural and residue-specific level. Here, we performed quantitative proteomic comparisons of wild-type and SrtA knockout strains that confirmed the loss of multiple LPxTG-containing virulence factors, including ZmpB, NanA, and IgA1 protease, consistent with an essential role for SrtA in surface protein anchoring. To enable mechanistic studies, we established a biochemical framework to produce monomeric Streptococcus pneumoniae SrtA by refolding and developed a gel-based assay using recombinant substrates to monitor catalytic activity. The refolded monomer, but not the swapped dimer, catalyzed cleavage and transpeptidation of a canonical LPxTG substrate in a metal-independent manner under the conditions examined. We further report high-resolution NMR backbone assignments for the active monomer and identify substrate-induced chemical shift perturbations that localize to the active site. Together, these findings provide an integrated proteomic, biochemical, and NMR characterization of monomeric, catalytically active Streptococcus pneumoniae SrtA and reveal residue-specific interactions with a canonical LPNTG recognition peptide.
Eunjeong Lee, Blaine H. Gordon, J. Redzic et al.· Biomolecules· 0 citations
Peptidoglycan biogenesis requires membrane flippases to translocate lipid-linked precursors across the cytoplasmic membrane for processing. This essential step is mediated by MurJ, the lipid II flippase conserved across all peptidoglycan-producing bacteria. While MurJ from diderm bacteria has been structurally resolved in multiple conformational states, its monoderm homolog remains uncharacterized. Monoderm MurJ homologs exhibit substantial sequence divergence yet retain the same lipid II flipping function and are promising antibiotic targets. Here we report structures of Staphylococcus aureus MurJ (SaMurJ) captured in both outward- and inward-facing conformations. These structures show that SaMurJ adopts the conserved MOP family fold and undergoes conformational transitions consistent with an alternating-access mechanism. Our findings reveal conserved and divergent features of MurJ between diderm and monoderm bacteria that are critical for lipid II flipping and provide a structural framework for probing substrate recognition and specific inhibition.
Yan-Cheng E. Li, Grace F. Baron, W. Clemons· Journal of Biological Chemis...· 0 citations
Gram‐negative bacteria are protected by a three‐layered cell envelope and must tightly coordinate the biosynthesis of phospholipids (PL), peptidoglycan (PG) and lipopolysaccharides (LPS) to maintain envelope integrity. In Escherichia coli, the inner membrane protein LapB (YciM) plays a critical dual role in LPS homeostasis by acting as a scaffold for cytoplasmic LPS and PL biosynthesis enzymes and by promoting FtsH‐dependent proteolysis of the key enzyme LpxC. Because LPS and PG biosynthesis compete for the shared precursor UDP‐GlcNAc, we investigated whether MurA, catalyzing the first committed step of PG biosynthesis, is an integral component of the LapB complex. Using bacterial two‐hybrid analysis, pull‐down assays and microscale thermophoresis, we demonstrate that MurA directly interacts with LapB, LpxA, LpxC, LpxD, and FabZ. Together, these findings support a model in which PL, PG, and LPS biosynthesis enzymes are engaged in a protein–protein interaction hub that synchronizes the production of all three layers of the Gram‐negative cell envelope in E. coli.
Isoaspartate (isoAsp) formation is typically viewed as a “molecular clock” through nonenzymatic degradation of aspartate or asparagine during protein aging. Here we report a nearly universal enzymatic pathway for the formation of a conserved isoAsp in the bacterial ribosomal protein uS11. Proteome-wide protein-protein interaction scans using AlphaFold3 identified YbeY as a candidate enzyme from Escherichia coli. NMR spectroscopy supported a stable YbeY–uS11 complex from Thermotoga maritima. Biochemical assays indicated that EcYbeY catalysis is zinc-dependent and prefers the conserved Asn-Gly motif for isoAsp formation. A high-resolution cryo-electron microscopy structure of the 70S ribosome from E. coli ΔybeY revealed that loss of isoAsp alters contacts with the 16S rRNA groove and bS21. Phylogenetic analysis indicated that YbeY is present in almost all bacteria, and its absence is correlated to changes in the Asn-Gly motif of uS11. Additionally, our structural analyses implicate Fap7 as the functional counterpart in archaea and eukaryotes. Table of Contents
Yanqing Xue, Chandrima Majumdar, Salimat O. Sofela et al.· bioRxiv· 0 citations
ABSTRACT Coliphage N4 employs a unique infection and transcription strategy in which early gene expression is driven by a virion-encapsidated RNA polymerase (vRNAP) that is injected into the host cytoplasm upon infection. Despite extensive biochemical and crystallographic studies of the polymerase domain of vRNAP, the structural organization and regulatory roles of the N-terminal domain (NTD) and C-terminal domain (CTD) regions of the 3,500-residue-long whole enzyme have remained unresolved. Here, we report the cryo-electron microscopy (cryo-EM) structures of full-length N4 vRNAP in its apo state and in a transcription initiation complex (TIC) with promoter DNA and initiating nucleotides. The apo structure reveals a modular architecture in which an α-helical CTD packs against the Pol domain to stabilize an autoinhibited conformation characterized by occlusion of the nucleotide-binding site through tight contact between the plug module and motif B loop. In contrast, promoter binding induces conformational rearrangements that displace the motif B loop from the active site and separate the CTD from the Pol domain. The NTD is unresolved in both states, consistent with substantial intrinsic flexibility, and supporting its proposed role in membrane association and genome injection. Structural modeling suggests that domain segmentation and conformational plasticity may enable translocation of vRNAP through the ~30 Å wide phage tail channel during infection. Together, these results define the molecular architecture of full-length vRNAP and establish a structural framework for understanding how the conformational transition of vRNAP is coupled to its ejection, DNA injection, and early gene expression. IMPORTANCE This study investigates the structure of full-length bacteriophage N4 virion RNA polymerase (vRNAP), one of the largest known single-subunit RNA polymerases. The functions of its extensive N- and C-terminal regions remained unknown. Our work uncovers how the C-terminal domain regulates polymerase activity through a structural “switch” that locks the enzyme in an inactive state until it recognizes its promoter DNA. These findings explain how the phage prevents premature transcription and ensures precise control of early gene expression during infection. By integrating structures with the architecture of the N4 phage particle, we propose a mechanism by which this vRNAP is transported through the narrow phage tail into the host cell. Together, this work provides fundamental insight into phage transcription and viral gene regulation. This study investigates the structure of full-length bacteriophage N4 virion RNA polymerase (vRNAP), one of the largest known single-subunit RNA polymerases. The functions of its extensive N- and C-terminal regions remained unknown. Our work uncovers how the C-terminal domain regulates polymerase activity through a structural “switch” that locks the enzyme in an inactive state until it recognizes its promoter DNA. These findings explain how the phage prevents premature transcription and ensures precise control of early gene expression during infection. By integrating structures with the architecture of the N4 phage particle, we propose a mechanism by which this vRNAP is transported through the narrow phage tail into the host cell. Together, this work provides fundamental insight into phage transcription and viral gene regulation.
M. Narwal, Y. Shin, Katsuhiko S. Murakami· Journal of Bacteriology· 0 citations
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