36 research outputs found
Crystallographic Study Of The Phosphoethanolamine Transferase EptC required For Polymyxin Resistance And Motility In Campylobacter jejuni
The foodborne enteric pathogen Campylobacter jejuni decorates a variety of its cell-surface structures with phosphoethanolamine (pEtN). Modifying lipid A with pEtN promotes cationic antimicrobial peptide resistance, whereas post-translationally modifying the flagellar rod protein FlgG with pEtN promotes flagellar assembly and motility, which are processes that are important for intestinal colonization. EptC, the pEtN transferase required for all known pEtN cell-surface modifications in C. jejuni, is a predicted inner-membrane metalloenzyme with a five-helix N-terminal transmembrane domain followed by a soluble sulfatase-like catalytic domain in the periplasm. The atomic structure of the catalytic domain of EptC (cEptC) was crystallized and solved to a resolution of 2.40 angstrom. cEptC adopts the alpha/beta/alpha fold of the sulfatase protein family and harbors a zinc-binding site. A phosphorylated Thr266 residue was observed that was hypothesized to mimic a covalent pEtN-enzyme intermediate. The requirement for Thr266 as well as the nearby residues Asn308, Ser309, His358 and His440 was ascertained via in vivo activity assays on mutant strains. The results establish a basis for the design of pEtN transferase inhibitors.National Institutes of Health (grants AI064184, AI076322, GM106112Army Research Office (grantW911NF-12-1-0390)College of Natural SciencesOffice of the Executive Vice President and ProvostInstitute for Cellular and Molecular Biology at the University of Texas at AustinUS DOE DE-AC02-06CH11357National Institute of General Medical SciencesHoward Hughes Medical InstituteOffice of Science, Office of Basic Energy Sciences of the US Department of Energy DE-AC02-05CH11231Maria
Person and the Proteomics Facility at the University of Texas at Austin ES007784 (CRED) and
RP110782 (CPRIT)Molecular Bioscience
The Kalimantacin Polyketide Antibiotics Inhibit Fatty Acid Biosynthesis in Staphylococcus aureus by Targeting the Enoyl-Acyl Carrier Protein Binding Site of FabI
Crystal Structure of Bacillus subtilis Cysteine Desulfurase SufS and Its Dynamic Interaction with Frataxin and Scaffold Protein SufU.
The biosynthesis of iron sulfur (Fe-S) clusters in Bacillus subtilis is mediated by a SUF-type gene cluster, consisting of the cysteine desulfurase SufS, the scaffold protein SufU, and the putative chaperone complex SufB/SufC/SufD. Here, we present the high-resolution crystal structure of the SufS homodimer in its product-bound state (i.e., in complex with pyrodoxal-5'-phosphate, alanine, Cys361-persulfide). By performing hydrogen/deuterium exchange (H/DX) experiments, we characterized the interaction of SufS with SufU and demonstrate that SufU induces an opening of the active site pocket of SufS. Recent data indicate that frataxin could be involved in Fe-S cluster biosynthesis by facilitating iron incorporation. H/DX experiments show that frataxin indeed interacts with the SufS/SufU complex at the active site. Our findings deepen the current understanding of Fe-S cluster biosynthesis, a complex yet essential process, in the model organism B. subtilis
Native ESI-MS and Collision-Induced Unfolding (CIU) of the Complex between Bacterial Elongation Factor-Tu and the Antibiotic Enacyloxin IIa
Collision-induced unfolding (CIU) of protein ions, monitored by ion mobility-mass spectrometry, can be used to assess the stability of their compact gas-phase fold and hence provide structural information. The bacterial elongation factor EF-Tu, a key protein for mRNA translation in prokaryotes and hence a promising antibiotic target, has been studied by CIU. The major [M + 12H]12+ ion of EF-Tu unfolded in collision with Ar atoms between 40 and 50 V, corresponding to an Elab energy of 480â500 eV. Binding of the cofactor analogue GDPNP and the antibiotic enacyloxin IIa stabilized the compact fold of EF-Tu, although dissociation of the latter from the complex diminished its stabilizing effect at higher collision energies. Molecular dynamics simulations of the [M + 12H]12+ EF-Tu ion showed similar qualitative behavior to the experimental results
The ring residue proline 8 is crucial for the thermal stability of the lasso peptide caulosegnin II
Lasso peptides are fascinating natural products with a unique structural fold that can exhibit tremendous thermal stability.</p
Crystallographic study of the phosphoethanolamine transferase EptC required for polymyxin resistance and motility in Campylobacter jejuni
General rules of fragmentation evidencing lasso structures in CID and ETD
International audienceLasso peptides constitute a structurally unique class of ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by a mechanically interlocked structure in which the C-terminal tail of the peptide is threaded and trapped within an N-terminal macrolactam ring. Tandem mass spectrometry using collision induced dissociation (CID) and electron capture dissociation (ECD) have shown previously different fragmentation patterns for capistruin, microcin J25 and their corresponding branched-cyclic forms in which the C-terminal tail is unthreaded. In order to develop general rules that unambiguously discriminate the lasso and branched-cyclic topologies, this report presents experimental evidence for a set of twenty-one lasso peptides analyzed by CID and electron transfer dissociation (ETD). CID experiments on lasso peptides specifically yielded mechanically interlocked species with associated bi and yj fragments. For class II lasso peptides, these lasso-specific fragments were observed only for peptides in which the loop, located above the macrolactam ring, was strictly longer than four amino acid residues. For class I and III lasso peptides, part of the C-terminal tail remains covalently linked to the macrolactam ring by disulfide bonds; associated bi and yj fragments therefore do not clearly constitute a signature of the lasso topology. ETD experiments of lasso peptides showed a significant increase of hydrogen migration events in the loop region when compared to their branched-cyclic topoisomers, leading to the formation of specific ciË/zâČj fragments for all lasso peptides, regardless of their class and loop size. Our experiments enabled us to establish general rules for obtaining structural details from CID and ETD fragmentation patterns, obviating the need for structure determination by NMR or X-ray crystallography
The Missing Linker: A Dimerization Motif Located within Polyketide Synthase Modules
The
dimerization of multimodular polyketide synthases is essential
for their function. Motifs that supplement the contacts made by dimeric
polyketide synthase enzymes have previously been characterized outside
the boundaries of modules, at the N- and C-terminal ends of polyketide
synthase subunits. Here we describe a heretofore uncharacterized dimerization
motif located within modules. The dimeric state of this dimerization
element was elucidated through the 2.6 Ă
resolution crystal structure
of a fragment containing a dimerization element and a ketoreductase.
The solution structure of a standalone dimerization element was revealed
by nuclear magnetic resonance spectroscopy to be consistent with that
of the crystal structure, and its dimerization constant was measured
through analytical ultracentrifugation to be âŒ20 ÎŒM.
The dimer buries âŒ990 Ă
<sup>2</sup> at its interface,
and its C-terminal helices rigidly connect to ketoreductase domains
to constrain their locations within a module. These structural restraints
permitted the construction of a common type of polyketide synthase
module
The Missing Linker: A Dimerization Motif Located within Polyketide Synthase Modules
The
dimerization of multimodular polyketide synthases is essential
for their function. Motifs that supplement the contacts made by dimeric
polyketide synthase enzymes have previously been characterized outside
the boundaries of modules, at the N- and C-terminal ends of polyketide
synthase subunits. Here we describe a heretofore uncharacterized dimerization
motif located within modules. The dimeric state of this dimerization
element was elucidated through the 2.6 Ă
resolution crystal structure
of a fragment containing a dimerization element and a ketoreductase.
The solution structure of a standalone dimerization element was revealed
by nuclear magnetic resonance spectroscopy to be consistent with that
of the crystal structure, and its dimerization constant was measured
through analytical ultracentrifugation to be âŒ20 ÎŒM.
The dimer buries âŒ990 Ă
<sup>2</sup> at its interface,
and its C-terminal helices rigidly connect to ketoreductase domains
to constrain their locations within a module. These structural restraints
permitted the construction of a common type of polyketide synthase
module
The Missing Linker: A Dimerization Motif Located within Polyketide Synthase Modules
The
dimerization of multimodular polyketide synthases is essential
for their function. Motifs that supplement the contacts made by dimeric
polyketide synthase enzymes have previously been characterized outside
the boundaries of modules, at the N- and C-terminal ends of polyketide
synthase subunits. Here we describe a heretofore uncharacterized dimerization
motif located within modules. The dimeric state of this dimerization
element was elucidated through the 2.6 Ă
resolution crystal structure
of a fragment containing a dimerization element and a ketoreductase.
The solution structure of a standalone dimerization element was revealed
by nuclear magnetic resonance spectroscopy to be consistent with that
of the crystal structure, and its dimerization constant was measured
through analytical ultracentrifugation to be âŒ20 ÎŒM.
The dimer buries âŒ990 Ă
<sup>2</sup> at its interface,
and its C-terminal helices rigidly connect to ketoreductase domains
to constrain their locations within a module. These structural restraints
permitted the construction of a common type of polyketide synthase
module