ABSTRACT
The peptide antibiotic albicidin is a DNA topoisomerase inhibitor with low-nanomolar bactericidal activity towards fluoroquinolone-resistant Gram-negative pathogens. However, its mode of action is poorly understood. We determined a 2.6 Å resolution cryoelectron microscopy structure of a ternary complex between Escherichia coli topoisomerase DNA gyrase, a 217 bp double-stranded DNA fragment and albicidin. Albicidin employs a dual binding mechanism where one end of the molecule obstructs the crucial gyrase dimer interface, while the other intercalates between the fragments of cleaved DNA substrate. Thus, albicidin efficiently locks DNA gyrase, preventing it from religating DNA and completing its catalytic cycle. Two additional structures of this trapped state were determined using synthetic albicidin analogues that demonstrate improved solubility, and activity against a range of gyrase variants and E. coli topoisomerase IV. The extraordinary promiscuity of the DNA-intercalating region of albicidins and their excellent performance against fluoroquinolone-resistant bacteria holds great promise for the development of last-resort antibiotics.
ABSTRACT
Isobutanol is deemed to be a next-generation biofuel and a renewable platform chemical.1 Non-natural biosynthetic pathways for isobutanol production have been implemented in cell-based and in vitro systems with Bacillus subtilis acetolactate synthase (AlsS) as key biocatalyst.2-6 AlsS catalyzes the condensation of two pyruvate molecules to acetolactate with thiamine diphosphate and Mg(2+) as cofactors. AlsS also catalyzes the conversion of 2-ketoisovalerate into isobutyraldehyde, the immediate precursor of isobutanol. Our phylogenetic analysis suggests that the ALS enzyme family forms a distinct subgroup of ThDP-dependent enzymes. To unravel catalytically relevant structure-function relationships, we solved the AlsS crystal structure at 2.3 Å in the presence of ThDP, Mg(2+) and in a transition state with a 2-lactyl moiety bound to ThDP. We supplemented our structural data by point mutations in the active site to identify catalytically important residues.
Subject(s)
Acetolactate Synthase/chemistry , Bacillus subtilis/chemistry , Bacterial Proteins/chemistry , Butanols/chemistry , Acetolactate Synthase/genetics , Acetolactate Synthase/metabolism , Aldehydes/chemistry , Aldehydes/metabolism , Bacillus subtilis/classification , Bacillus subtilis/enzymology , Bacillus subtilis/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biocatalysis , Biofuels , Butanols/metabolism , Catalytic Domain , Cations, Divalent , Coenzymes/chemistry , Coenzymes/metabolism , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Hemiterpenes , Keto Acids/chemistry , Keto Acids/metabolism , Lactates/chemistry , Lactates/metabolism , Magnesium/chemistry , Magnesium/metabolism , Models, Molecular , Phylogeny , Point Mutation , Pyruvic Acid/chemistry , Pyruvic Acid/metabolism , Structure-Activity Relationship , Thiamine Pyrophosphate/chemistry , Thiamine Pyrophosphate/metabolismABSTRACT
Instead of a classical single-stranded deoxyribonuleic acid (DNA)-binding protein (SSB), some hyperthermophilic crenarchaea harbor a non-canonical SSB termed ThermoDBP. Two related but poorly characterized groups of proteins, which share the ThermoDBP N-terminal DNA-binding domain, have a broader phylogenetic distribution and co-exist with ThermoDBPs and/or other SSBs. We have investigated the nucleic acid binding properties and crystal structures of representatives of these groups of ThermoDBP-related proteins (ThermoDBP-RPs) 1 and 2. ThermoDBP-RP 1 and 2 oligomerize by different mechanisms and only ThermoDBP-RP2 exhibits strong single-stranded DNA affinity in vitro. A crystal structure of ThermoDBP-RP2 in complex with DNA reveals how the NTD common to ThermoDBPs and ThermoDBP-RPs can contact the nucleic acid in a manner that allows a symmetric homotetrameric protein complex to bind single-stranded DNA molecules asymmetrically. While single-stranded DNA wraps around the surface or binds along channels of previously investigated SSBs, it traverses an internal, intersubunit tunnel system of a ThermoDBP-RP2 tetramer. Our results indicate that some archaea have acquired special SSBs for genome maintenance in particularly challenging environments.
Subject(s)
Archaeal Proteins/chemistry , DNA, Single-Stranded/chemistry , DNA-Binding Proteins/chemistry , Archaeal Proteins/metabolism , Binding Sites , DNA/chemistry , DNA/metabolism , DNA-Binding Proteins/metabolism , Models, Molecular , Protein Binding , Pyrococcus furiosus , RNA/metabolismABSTRACT
In metazoans, replication-dependent histone mRNAs end in a stem-loop structure instead of the poly(A) tail characteristic of all other mature mRNAs. This specialized 3' end is bound by stem-loop binding protein (SLBP), a protein that participates in the nuclear export and translation of histone mRNAs. The translational activity of SLBP is mediated by interaction with SLIP1, a middle domain of initiation factor 4G (MIF4G)-like protein that connects to translation initiation. We determined the 2.5 Å resolution crystal structure of zebrafish SLIP1 bound to the translation-activation domain of SLBP and identified the determinants of the recognition. We discovered a SLIP1-binding motif (SBM) in two additional proteins: the translation initiation factor eIF3g and the mRNA-export factor DBP5. We confirmed the binding of SLIP1 to DBP5 and eIF3g by pull-down assays and determined the 3.25 Å resolution structure of SLIP1 bound to the DBP5 SBM. The SBM-binding and homodimerization residues of SLIP1 are conserved in the MIF4G domain of CBP80/20-dependent translation initiation factor (CTIF). The results suggest how the SLIP1 homodimer or a SLIP1-CTIF heterodimer can function as platforms to bridge SLBP with SBM-containing proteins involved in different steps of mRNA metabolism.
Subject(s)
DEAD-box RNA Helicases/chemistry , Nucleocytoplasmic Transport Proteins/chemistry , RNA-Binding Proteins/chemistry , Zebrafish Proteins/chemistry , Amino Acid Sequence , Animals , DEAD-box RNA Helicases/metabolism , Eukaryotic Initiation Factor-3/chemistry , Eukaryotic Initiation Factor-3/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Nucleocytoplasmic Transport Proteins/metabolism , Protein Interaction Domains and Motifs , Protein Multimerization , RNA-Binding Proteins/metabolism , Sequence Alignment , Zebrafish Proteins/metabolismABSTRACT
The DEAD-box protein DBP5 is essential for mRNA export in both yeast and humans. It binds RNA and is concentrated and locally activated at the cytoplasmic side of the nuclear pore complex. We have determined the crystal structures of human DBP5 bound to RNA and AMPPNP, and bound to the cytoplasmic nucleoporin NUP214. The structures reveal that binding of DBP5 to nucleic acid and to NUP214 is mutually exclusive. Using in vitro assays, we demonstrate that NUP214 decreases both the RNA binding and ATPase activities of DBP5. The interactions are mediated by conserved residues, implying a conserved recognition mechanism. These results suggest a framework for the consecutive steps leading to the release of mRNA at the final stages of nuclear export. More generally, they provide a paradigm for how binding of regulators can specifically inhibit DEAD-box proteins.
Subject(s)
DEAD-box RNA Helicases/chemistry , DEAD-box RNA Helicases/metabolism , Nuclear Pore Complex Proteins/chemistry , Nuclear Pore Complex Proteins/metabolism , Nucleocytoplasmic Transport Proteins/chemistry , Nucleocytoplasmic Transport Proteins/metabolism , Protein Structure, Quaternary , RNA, Messenger/metabolism , Adenylyl Imidodiphosphate/chemistry , Adenylyl Imidodiphosphate/metabolism , Crystallography, X-Ray , Humans , Models, Molecular , Protein BindingABSTRACT
Bacterial bile salt hydrolases catalyze the degradation of conjugated bile acids in the mammalian gut. The crystal structures of conjugated bile acid hydrolase (CBAH) from Clostridium perfringens as apoenzyme and in complex with taurodeoxycholate that was hydrolyzed to the reaction products taurine and deoxycholate are described here at 2.1 and 1.7 A resolution, respectively. The crystal structures reveal close relationship between CBAH and penicillin V acylase from Bacillus sphaericus. This similarity together with the N-terminal cysteine classifies CBAH as a member of the N-terminal nucleophile (Ntn) hydrolase superfamily. Both crystal structures show an identical homotetrameric organization with dihedral (D(2) or 222) point group symmetry. The structure analysis of C. perfringens CBAH identifies critical residues in catalysis, substrate recognition, and tetramer formation which may serve in further biochemical characterization of bile acid hydrolases.
Subject(s)
Amidohydrolases/chemistry , Amidohydrolases/metabolism , Bile Acids and Salts/metabolism , Amidohydrolases/genetics , Amino Acid Sequence , Apoenzymes/chemistry , Apoenzymes/genetics , Apoenzymes/metabolism , Bile Acids and Salts/chemistry , Catalytic Domain , Clostridium perfringens/enzymology , Clostridium perfringens/genetics , Crystallography, X-Ray , Deoxycholic Acid/metabolism , Models, Molecular , Molecular Sequence Data , Protein Structure, Quaternary , Protein Subunits , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Substrate Specificity , Taurine/metabolismABSTRACT
Chitin hydrolases have been identified in a variety of organisms ranging from bacteria to eukaryotes. They have been proposed to be possible targets for the design of novel chemotherapeutics against human pathogens such as fungi and protozoan parasites as mammals were not thought to possess chitin-processing enzymes. Recently, a human chitotriosidase was described as a marker for Gaucher disease with plasma levels of the enzyme elevated up to 2 orders of magnitude. The chitotriosidase was shown to be active against colloidal chitin and is inhibited by the family 18 chitinase inhibitor allosamidin. Here, the crystal structure of the human chitotriosidase and complexes with a chitooligosaccharide and allosamidin are described. The structures reveal an elongated active site cleft, compatible with the binding of long chitin polymers, and explain the inactivation of the enzyme through an inherited genetic deficiency. Comparison with YM1 and HCgp-39 shows how the chitinase has evolved into these mammalian lectins by the mutation of key residues in the active site, tuning the substrate binding specificity. The soaking experiments with allosamidin and chitooligosaccharides give insight into ligand binding properties and allow the evaluation of differential binding and design of species-selective chitinase inhibitors.