Structural and functional characterization of FabG4 from Mycolicibacterium smegmatis.

The rise in antimicrobial resistance is a global health crisis and necessitates the development of novel strategies to treat infections. For example, in 2022 tuberculosis (TB) was the second leading infectious killer after COVID-19, with multi-drug-resistant strains of TB having an ∼40% fatality rate. Targeting essential biosynthetic pathways in pathogens has proven to be successful for the development of novel antimicrobial treatments. Fatty-acid synthesis (FAS) in bacteria proceeds via the type II pathway, which is substantially different from the type I pathway utilized in animals. This makes bacterial fatty-acid biosynthesis (Fab) enzymes appealing as drug targets. FabG is an essential FASII enzyme, and some bacteria, such as Mycobacterium tuberculosis, the causative agent of TB, harbor multiple homologs. FabG4 is a conserved, high-molecular-weight FabG (HMwFabG) that was first identified in M. tuberculosis and is distinct from the canonical low-molecular-weight FabG. Here, structural and functional analyses of Mycolicibacterium smegmatis FabG4, the third HMwFabG studied to date, are reported. Crystal structures of NAD+ and apo MsFabG4, along with kinetic analyses, show that MsFabG4 preferentially binds and uses NADH when reducing CoA substrates. As M. smegmatis is often used as a model organism for M. tuberculosis, these studies may aid the development of drugs to treat TB and add to the growing body of research that distinguish HMwFabGs from the archetypal low-molecular-weight FabG.

pSK41/pGO1-family conjugative plasmids of Staphylococcus aureus encode a cryptic repressor of replication.

The majority of large multiresistance plasmids of Staphylococcus aureus utilise a RepA_N-type replication initiation protein, the expression of which is regulated by a small antisense RNA (RNAI) that overlaps the rep mRNA leader. The pSK41/pGO1-family of conjugative plasmids additionally possess a small (86 codon) divergently transcribed ORF (orf86) located upstream of the rep locus. The product of pSK41 orf86 was predicted to have a helix-turn-helix motif suggestive of a likely function in transcriptional repression. In this study, we investigated the effect of Orf86 on transcription of thirteen pSK41 backbone promoters. We found that Orf86 only repressed transcription from the rep promoter, and hence now redesignate the product as Cop. Over-expression of Cop in trans reduced the copy number of pSK41 mini-replicons, both in the presence and absence of rnaI. in vitro protein-DNA binding experiments with purified 6 × His-Cop demonstrated specific DNA binding, adjacent to, and partially overlapping the -35 hexamer of the rep promoter. The crystal structure of Cop revealed a dimeric structure similar to other known transcriptional regulators. Cop mRNA was found to result from "read-through" transcription from the strong RNAI promoter that escapes the rnaI terminator. Thus, PrnaI is responsible for transcription of two distinct negative regulators of plasmid copy number; the antisense RNAI that primarily represses Rep translation, and Cop protein that can repress rep transcription. Deletion of cop in a native plasmid did not appear to impact copy number, indicating a cryptic auxiliary role.

Characterization of a family I inorganic pyrophosphatase from Legionella pneumophila Philadelphia 1.

Inorganic pyrophosphate (PPi) is generated as an intermediate or byproduct of many fundamental metabolic pathways, including DNA/RNA synthesis. The intracellular concentration of PPi must be regulated as buildup can inhibit many critical cellular processes. Inorganic pyrophosphatases (PPases) hydrolyze PPi into two orthophosphates (Pi), preventing the toxic accumulation of the PPi byproduct in cells and making Pi available for use in biosynthetic pathways. Here, the crystal structure of a family I inorganic pyrophosphatase from Legionella pneumophila is reported at 2.0 Šresolution. L. pneumophila PPase (LpPPase) adopts a homohexameric assembly and shares the oligonucleotide/oligosaccharide-binding (OB) ß-barrel core fold common to many other bacterial family I PPases. LpPPase demonstrated hydrolytic activity against a general substrate, with Mg2+ being the preferred metal cofactor for catalysis. Legionnaires' disease is a severe respiratory infection caused primarily by L. pneumophila, and thus increased characterization of the L. pneumophila proteome is of interest.

Crystal structure of acetoacetyl-CoA reductase from Rickettsia felis.

Rickettsia felis, a Gram-negative bacterium that causes spotted fever, is of increasing interest as an emerging human pathogen. R. felis and several other Rickettsia strains are classed as National Institute of Allergy and Infectious Diseases priority pathogens. In recent years, R. felis has been shown to be adaptable to a wide range of hosts, and many fevers of unknown origin are now being attributed to this infectious agent. Here, the structure of acetoacetyl-CoA reductase from R. felis is reported at a resolution of 2.0 Å. While R. felis acetoacetyl-CoA reductase shares less than 50% sequence identity with its closest homologs, it adopts a fold common to other short-chain dehydrogenase/reductase (SDR) family members, such as the fatty-acid synthesis II enzyme FabG from the prominent pathogens Staphylococcus aureus and Bacillus anthracis. Continued characterization of the Rickettsia proteome may prove to be an effective means of finding new avenues of treatment through comparative structural studies.

Structure-guided U2AF65 variant improves recognition and splicing of a defective pre-mRNA.

Purine interruptions of polypyrimidine (Py) tract splice site signals contribute to human genetic diseases. The essential splicing factor U2AF(65) normally recognizes a Py tract consensus sequence preceding the major class of 3' splice sites. We found that neurofibromatosis- or retinitis pigmentosa-causing mutations in the 5' regions of Py tracts severely reduce U2AF(65) affinity. Conversely, we identified a preferred binding site of U2AF(65) for purine substitutions in the 3' regions of Py tracts. Based on a comparison of new U2AF(65) structures bound to either A- or G-containing Py tracts with previously identified pyrimidine-containing structures, we expected to find that a D231V amino acid change in U2AF(65) would specify U over other nucleotides. We found that the crystal structure of the U2AF(65)-D231V variant confirms favorable packing between the engineered valine and a target uracil base. The D231V amino acid change restores U2AF(65) affinity for two mutated splice sites that cause human genetic diseases and successfully promotes splicing of a defective retinitis pigmentosa-causing transcript. We conclude that reduced U2AF(65) binding is a molecular consequence of disease-relevant mutations, and that a structure-guided U2AF(65) variant is capable of manipulating gene expression in eukaryotic cells.

Unique helicase determinants in the essential conjugative TraI factor from Salmonella enterica serovar Typhimurium plasmid pCU1.

The widespread development of multidrug-resistant bacteria is a major health emergency. Conjugative DNA plasmids, which harbor a wide range of antibiotic resistance genes, also encode the protein factors necessary to orchestrate the propagation of plasmid DNA between bacterial cells through conjugative transfer. Successful conjugative DNA transfer depends on key catalytic components to nick one strand of the duplex DNA plasmid and separate the DNA strands while cell-to-cell transfer occurs. The TraI protein from the conjugative Salmonella plasmid pCU1 fulfills these key catalytic roles, as it contains both single-stranded DNA-nicking relaxase and ATP-dependent helicase domains within a single, 1,078-residue polypeptide. In this work, we unraveled the helicase determinants of Salmonella pCU1 TraI through DNA binding, ATPase, and DNA strand separation assays. TraI binds DNA substrates with high affinity in a manner influenced by nucleic acid length and the presence of a DNA hairpin structure adjacent to the nick site. TraI selectively hydrolyzes ATP, and mutations in conserved helicase motifs eliminate ATPase activity. Surprisingly, the absence of a relatively short (144-residue) domain at the extreme C terminus of the protein severely diminishes ATP-dependent strand separation. Collectively, these data define the helicase motifs of the conjugative factor TraI from Salmonella pCU1 and reveal a previously uncharacterized C-terminal functional domain that uncouples ATP hydrolysis from strand separation activity.

Large favorable enthalpy changes drive specific RNA recognition by RNA recognition motif proteins.

The RNA recognition motif (RRM) is a prevalent class of RNA binding domains. Although a number of RRM/RNA structures have been determined, thermodynamic analyses are relatively uncommon. Here, we use isothermal titration calorimetry to characterize single-stranded (ss)RNA binding by four representative RRM-containing proteins: (i) U2AF(65), (ii) SXL, (iii) TIA-1, and (iv) PAB. In all cases, ssRNA binding is accompanied by remarkably large favorable enthalpy changes (-30 to -60 kcal mol(-1)) and unfavorable entropy changes. Alterations of key RRM residues and binding sites indicate that under the nearly physiological conditions of these studies, large thermodynamic changes represent a signature of specific ssRNA recognition by RRMs.

Structural basis for NADH/NAD+ redox sensing by a Rex family repressor.

Nicotinamide adenine dinucleotides have emerged as key signals of the cellular redox state. Yet the structural basis for allosteric gene regulation by the ratio of reduced NADH to oxidized NAD(+) is poorly understood. A key sensor among Gram-positive bacteria, Rex represses alternative respiratory gene expression until a limited oxygen supply elevates the intracellular NADH:NAD(+) ratio. Here we investigate the molecular mechanism for NADH/NAD(+) sensing among Rex family members by determining structures of Thermus aquaticus Rex bound to (1) NAD(+), (2) DNA operator, and (3) without ligand. Comparison with the Rex/NADH complex reveals that NADH releases Rex from the DNA site following a 40 degrees closure between the dimeric subunits. Complementary site-directed mutagenesis experiments implicate highly conserved residues in NAD-responsive DNA-binding activity. These rare views of a redox sensor in action establish a means for slight differences in the nicotinamide charge, pucker, and orientation to signal the redox state of the cell.