tRNA Modification Enzymes GidA and MnmE: Potential Role in Virulence of Bacterial Pathogens

Transfer RNA (tRNA) is an RNA molecule that carries amino acids to the ribosomes for protein synthesis. These tRNAs function at the peptidyl (P) and aminoacyl (A) binding sites of the ribosome during translation, with each codon being recognized by a specific tRNA. Due to this specificity, tRNA modification is essential for translational efficiency. Many enzymes have been implicated in the modification of bacterial tRNAs, and these enzymes may complex with one another or interact individually with the tRNA. Approximately, 100 tRNA modification enzymes have been identified with glucose-inhibited division (GidA) protein and MnmE being two of the enzymes studied. In Escherichia coli and Salmonella, GidA and MnmE bind together to form a functional complex responsible for the proper biosynthesis of 5-methylaminomethyl-2-thiouridine (mnm⁵s²U34) of tRNAs. Studies have implicated this pathway in a major pathogenic regulatory mechanism as deletion of gidA and/or mnmE has attenuated several bacterial pathogens like Salmonella enterica serovar Typhimurium, Pseudomonas syringae, Aeromonas hydrophila, and many others. In this review, we summarize the potential role of the GidA/MnmE tRNA modification pathway in bacterial virulence, interactions with the host, and potential therapeutic strategies resulting from a greater understanding of this regulatory mechanism.     

Lack of Charge Interaction in the Ion Binding Site Determines Anion Selectivity in the Sodium Bicarbonate Cotransporter NBCe1

The Na/HCO₃ cotransporter NBCe1 is a member of SLC4A transporters that move HCO₃⁻ across cell membranes and regulate intracellular pH or transepithelial HCO₃ transport. NBCe1 is highly selective to HCO₃⁻ and does not transport other anions; the molecular mechanism of anion selectivity is presently unclear. We previously reported that replacing Asp⁵⁵⁵ with a Glu (D555E) in NBCe1 induces increased selectivity to other anions, including Cl⁻. This finding is unexpected because all SLC4A transporters contain either Asp or Glu at the corresponding position and maintain a high selectivity to HCO₃⁻. In this study, we tested whether the Cl⁻ transport in D555E is mediated by an interaction between residues in the ion binding site. Human NBCe1 and mutant transporters were expressed in Xenopus oocytes, and their ability to transport Cl⁻ was assessed by two-electrode voltage clamp. The results show that the Cl⁻ transport is induced by a charge interaction between Glu⁵⁵⁵ and Lys⁵⁵⁸. The bond length between the two residues is within the distance for a salt bridge, and the ionic strength experiments confirm an interaction. This finding indicates that the HCO₃⁻ selectivity in NBCe1 is established by avoiding a specific charge interaction in the ion binding site, rather than maintaining such an interaction.     

A Deeper Insight in Metal Binding to the hCtr1 N-terminus Fragment: Affinity,Speciation and Binding Mode of Binuclear Cu2+ and Mononuclear Ag+ Complex Species

Ctr1 regulates copper uptake and its intracellular distribution. The first 14 amino acid sequence of the Ctr1 ectodomain Ctr1_(1-14) encompasses the characteristic Amino Terminal Cu²⁺ and Ni²⁺ binding motif (ATCUN) as well as the bis-His binding motif (His5 and His6). We report a combined thermodynamic and spectroscopic (UV-vis, CD, EPR) study dealing with the formation of Cu²⁺ homobinuclear complexes with Ctr1_(1-14), the percentage of which is not negligible even in the presence of a small Cu²⁺ excess and clearly prevails at a M/L ratio of 1.9. Ascorbate fails to reduce Cu²⁺ when bound to the ATCUN motif, while it reduces Cu²⁺ when bound to the His5-His6 motif involved in the formation of binuclear species. The histidine diade characterizes the second binding site and is thought to be responsible for ascorbate oxidation. Binding constants and speciation of Ag⁺ complexes with Ctr1_(1-14), which are assumed to mimic Cu⁺ interaction with N-terminus of Ctr1_(1-14), were also determined. A preliminary immunoblot assay evidences that the anti-Ctr1 extracellular antibody recognizes Ctr1_(1-14) in a different way from the longer Ctr1_(1-25) that encompasses a second His and Met rich domain.     

Spectrofluorometric and Molecular Docking Studies on the Binding of Curcumenol and Curcumenone to Human Serum Albumin

Curcumenol and curcumenone are two major constituents of the plants of medicinally important genus of Curcuma, and often govern the pharmacological effect of these plant extracts. These two compounds, isolated from C. zedoaria rhizomes were studied for their binding to human serum albumin (HSA) using the fluorescence quench titration method. Molecular docking was also performed to get a more detailed insight into their interaction with HSA at the binding site. Additions of these sesquiterpenes to HSA produced significant fluorescence quenching and blue shifts in the emission spectra of HSA. Analysis of the fluorescence data pointed toward moderate binding affinity between the ligands and HSA, with curcumenone showing a relatively higher binding constant (2.46 × 10⁵ M⁻¹) in comparison to curcumenol (1.97 × 10⁴ M⁻¹). Cluster analyses revealed that site I is the preferred binding site for both molecules with a minimum binding energy of −6.77 kcal·mol⁻¹. However, binding of these two molecules to site II cannot be ruled out as the binding energies were found to be −5.72 and −5.74 kcal·mol⁻¹ for curcumenol and curcumenone, respectively. The interactions of both ligands with HSA involved hydrophobic interactions as well as hydrogen bonding.     

Using 15N‐Ammonium to Characterise and Map Potassium Binding Sites in Proteins by NMR Spectroscopy

A variety of enzymes are activated by the binding of potassium ions. The potassium binding sites of these enzymes are very specific, but ammonium ions can often replace potassium ions in vitro because of their similar ionic radii. In these cases, ammonium can be used as a proxy for potassium to characterise potassium binding sites in enzymes: the ¹H,¹⁵N spin‐pair of enzyme‐bound ¹⁵NH₄⁺ can be probed by ¹⁵N‐edited heteronuclear NMR experiments. Here, we demonstrate the use of NMR spectroscopy to characterise binding of ammonium ions to two different enzymes: human histone deacetylase 8 (HDAC8), which is activated allosterically by potassium, and the bacterial Hsp70 homologue DnaK, for which potassium is an integral part of the active site. Ammonium activates both enzymes in a similar way to potassium, thus supporting this non‐invasive approach. Furthermore, we present an approach to map the observed binding site onto the structure of HDAC8. Our method for mapping the binding site is general and does not require chemical shift assignment of the enzyme resonances.     

Ions in the Deep Subsurface of Earth,Mars, and Icy Moons: Their Effects in Combination with Temperature and Pressure on tRNA–Ligand Binding

The interactions of ligands with nucleic acids are central to numerous reactions in the biological cell. How such reactions are affected by harsh environmental conditions such as low temperatures, high pressures, and high concentrations of destructive ions is still largely unknown. To elucidate the ions’ role in shaping habitability in extraterrestrial environments and the deep subsurface of Earth with respect to fundamental biochemical processes, we investigated the effect of selected salts (MgCl₂, MgSO₄, and Mg(ClO₄)₂) and high hydrostatic pressure (relevant for the subsurface of that planet) on the complex formation between tRNA and the ligand ThT. The results show that Mg²⁺ salts reduce the binding tendency of ThT to tRNA. This effect is largely due to the interaction of ThT with the salt anions, which leads to a strong decrease in the activity of the ligand. However, at mM concentrations, binding is still favored. The ions alter the thermodynamics of binding, rendering complex formation that is more entropy driven. Remarkably, the pressure favors ligand binding regardless of the type of salt. Although the binding constant is reduced, the harsh conditions in the subsurface of Earth, Mars, and icy moons do not necessarily preclude nucleic acid–ligand interactions of the type studied here.     

2-Aminopurine Fluorescence Spectroscopy for Probing a Glucose Binding Aptamer

Glucose is the most important analyte for biosensors. Recently a DNA aptamer was reported allowing binding-based detection. However, due to a relatively weak binding affinity, it is difficult to perform binding assays to understand the property of this aptamer. In this work, we replaced the only adenine base in the aptamer binding pocket with a 2-aminopurine (2AP) and used fluorescence spectroscopy to study glucose binding. In the selection buffer, glucose increased the 2AP fluorescence with a K_d of 15.0 mM glucose, which was comparable with the 10 mM K_d previously reported using the strand displacement assay. The binding required two Na⁺ ions or one Mg²⁺ that cannot be replaced by Li⁺ or K⁺. The binding was weaker at higher temperature and its van't Hoff plot indicated enthalpy-driven binding. While other monosaccharides failed to achieve saturated binding even at high concentrations, two glucose-containing disaccharides, namely trehalose and sucrose, reached a similar fluorescence level as glucose although with over 10-fold higher K_d values. Detection limits in both the selection buffer (0.9 mM) and in artificial interstitial fluids (6.0 mM) were measured.     

Binding of Sulpiride to Seric Albumins

The aim of this work was to study the interaction of sulpiride with human serum albumin (HSA) and bovine serum albumin (BSA) through the fluorescence quenching technique. As sulpiride molecules emit fluorescence, we have developed a simple mathematical model to discriminate the quencher fluorescence from the albumin fluorescence in the solution where they interact. Sulpiride is an antipsychotic used in the treatment of several psychiatric disorders. We selectively excited the fluorescence of tryptophan residues with 290 nm wavelength and observed the quenching by titrating HSA and BSA solutions with sulpiride. Stern-Volmer graphs were plotted and quenching constants were estimated. Results showed that sulpiride form complexes with both albumins. Estimated association constants for the interaction sulpiride–HSA were 2.20 (±0.08) × 10⁴ M⁻¹, at 37 °C, and 5.46 (±0.20) × 10⁴ M⁻¹, at 25 °C. Those for the interaction sulpiride-BSA are 0.44 (±0.01) × 10⁴ M⁻¹, at 37 °C and 2.17 (±0.04) × 10⁴ M⁻¹, at 25 °C. The quenching intensity of BSA, which contains two tryptophan residues in the peptide chain, was found to be higher than that of HSA, what suggests that the primary binding site for sulpiride in albumin should be located next to the sub domain IB of the protein structure.     

Synthesis and characterization of cellulose ethers/Eu(III)

The coordination complexes of the crystalline structure of cellulose ethers/Eu(III) with fluorescence emission, viz CMC/Eu(III), MC/Eu(III), and HEC/Eu(III), were synthesized and characterized. Results showed the emission spectra of Eu³⁺ ions in these coordination compounds, which originates from electric dipole transition. The main emission peak at 615 nm generated from ⁵D₀→⁷F₂ transition of Eu³⁺ ions. Their absorption and excitation spectra were different, because the effect of the high polarity of water and having both hydrogen bond donor and acceptor properties on the excited molecule is different from the effect on the ground state of the molecule. Our study demonstrated that the Degree of Substitute (DS) of CMC could influence the fluorescence intensity (FI) of CMC/Eu(III). The emission intensity of CMC/Eu(III) varies with the DS of CMC. For example, when the DS of CMC was 0.89, the FS (fluorescent spectra) of solid CMC/Eu(III) displayed three emission peaks Eu(III): the strongest emission peak at 615 nm (⁵D₀→⁷F₂ transition) and other two weaker peaks at 583 nm (⁵D₀→⁷F₁ transition) and at 652 nm (⁵D₀→⁷F₃ transition), respectively. The concentration of Eu(III) could also affect the FI of these coordination complexes. The FI of the coordination complexes peaked at 615 nm all reached maximum when Eu³⁺ concentration was at 5% (wt/wt). © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 95: 743–747, 2005     

The interaction between bovine serum albumin and the self-aggregated nanoparticles of cholesterol-modified O-carboxymethyl chitosan

The interaction between bovine serum albumin (BSA) and self-aggregated nanoparticles of cholesterol-modified O-carboxymethyl chitosan (CCMC) with different degrees of substitution (DS) of cholesterol moiety was studied by transmission electron microscopy (TEM), fluorescence quenching method and circular dichroism (CD) measurement. This interaction was started at the disaggregation of CCMC self-aggregated nanoparticles and reached equilibrium after 3-4 h. The apparent quenching constant (K_q) between BSA and CCMC self-aggregated nanoparticles calculated by the modified Stern-Volmer plot increased from 4.14 × 10⁴ to 1.95 × 10⁵ M⁻¹ with DS of cholesterol moiety increasing from 3.2% to 9.8%, whereas the fraction of tryptophan residues in BSA molecule involved in the interaction decreased at the same time. Compared with free BSA, the relative α-helix content of BSA decreased and the unfolding of BSA by a denaturant such as urea was largely suppressed upon interaction with CCMC self-aggregated nanoparticles. DS of cholesterol moiety significantly affected the interaction between BSA and CCMC self-aggregated nanoparticles.     

Investigating the Role of Sulfate Groups for the Binding of Gd3+ Ions to Glycosaminoglycans with NMR Relaxometry

Glycosaminoglycans (GAGs) are highly negatively charged macromolecules with a large cation binding capacity, but their interaction potential with exogeneous Gd³⁺ ions is under-investigated. These might be released from chelates used as Gadolinium-based contrast agents (GBCAs) for clinical MR imaging due to transmetallation with endogenous cations like Zn²⁺. Recent studies have quantified how an endogenous GAG sequesters released Gd³⁺ ions and impacts the thermodynamic and kinetic stability of some GBCAs. In this study, we investigate and compare the chelation ability of two important GAGs (heparin and chondroitin sulfate), as well as the homopolysaccharides dextran and dextran sulfate that are used as models for alternative macromolecular chelators. Our combined approach of MRI-based relaxometry and isothermal titration calorimetry shows that the chelation process of Gd³⁺ into GAGs is not just a long-range electrostatic interaction as proposed for the Manning model, but presumably a site-specific binding. Furthermore, our results highlight the crucial role of sulfate groups in this process and indicate that the potential of a specific GAG to engage in this mechanism increases with its degree of sulfation. The transchelation of Gd³⁺ ions from GBCAs to sulfated GAGs should thus be considered as one possible explanation for the observed long-term deposition of Gd³⁺ in vivo and related observations of long-term signal enhancements on T₁-weighted MR images.     

Binding of hirudin to meizothrombin

Prothrombin (coagulation factor II) is the inactive precursor molecule of thrombin (coagulation factor IIa). Proteolytic cleavage of the peptide bond Arg320-Ile321 converts prothrombin into the two-chain thrombin precursor meizothrombin. Meizothrombin hydrolyses peptidyl substrates, but cleavage of fibrinogen is poor. Unfortunately, meizothrombin exhibits a significant autocatalytic activity and thus is not structurally stable in solution. Hirudin, the 65-residue peptide anticoagulant from the salivary gland of the European leech Hirudo medicinalis, is a highly specific and effective thrombin inhibitor. To study the interactions of meizothrombin and hirudin, recombinant prothrombin with active site Asp419 replaced by Asn (D419N-prothrombin) was produced in CHO cells and transformed into D419N-meizothrombin in vitro. D419N-meizothrombin exhibited no proteolytic and autocatalytic activity. D419N-meizothrombin was affinity purified at an immobilized C- terminal hirudin-derived peptide demonstrating the presence and activity of the anion binding exosite. D419N-meizothrombin exhibited binding activity to hirudin immobilized at the solid phase in an ELISA. Incubation of D419N-meizothrombin with hirudin resulted in a significant increase of intrinsic fluorescence. Fluorescence titration of D419N-meizothrombin with hirudin produced a sharp break in the titration curve at the molar equivalence point and a total fluorescence enhancement of 24%. However, the titration curve did not reflect a simple binding mechanism. Incubation of D419N-meizothrombin with fibrinopeptide A and C-terminal hirudin peptide 54-65 did not change fluorescence emission. Trp468 located in the gamma-loop of thrombin was replaced by Phe in the double-mutant D419N/W468F-thrombin. Similar to D419N-thrombin and D419N-meizothrombin, formation of the D419N/W468F- thrombin/hirudin complex resulted a significant increase in intrinsic fluorescence. Apparently, the binding of hirudin induces similar structural changes in both meizothrombin and thrombin. The structural change does not involve the flexible gamma-loop. The results suggest that meizothrombin binds hirudin similar to thrombin.      

DNA Binding Mode Analysis of a Core-Extended Naphthalene Diimide as a Conformation-Sensitive Fluorescent Probe of G-Quadruplex Structures

G-quadruplex existence was proved in cells by using both antibodies and small molecule fluorescent probes. However, the G-quadruplex probes designed thus far are structure- but not conformation-specific. Recently, a core-extended naphthalene diimide (c_ex-NDI) was designed and found to provide fluorescent signals of markedly different intensities when bound to G-quadruplexes of different conformations or duplexes. Aiming at evaluating how the fluorescence behaviour of this compound is associated with specific binding modes to the different DNA targets, c_ex-NDI was here studied in its interaction with hybrid G-quadruplex, parallel G-quadruplex, and B-DNA duplex models by biophysical techniques, molecular docking, and biological assays. c_ex-NDI showed different binding modes associated with different amounts of stacking interactions with the three DNA targets. The preferential binding sites were the groove, outer quartet, or intercalative site of the hybrid G-quadruplex, parallel G-quadruplex, and B-DNA duplex, respectively. Interestingly, our data show that the fluorescence intensity of DNA-bound c_ex-NDI correlates with the amount of stacking interactions formed by the ligand with each DNA target, thus providing the rationale behind the conformation-sensitive properties of c_ex-NDI and supporting its use as a fluorescent probe of G-quadruplex structures. Notably, biological assays proved that c_ex-NDI mainly localizes in the G-quadruplex-rich nuclei of cancer cells.     

Conformation-switched chemosensor for selective detection of Hg2+ in aqueous media

A conformation flexible chemosensor for selective detection of Hg²⁺ in aqueous media was achieved by incorporating two well-known rhodamine-6G dyes and a ferrocene group within one molecule. Distinguished from the monosubstituted ferrocene derivative which is previously reported a lack of interaction with Hg²⁺, the title compound was characteristic of two-armed bidendate binding unit. The Hg²⁺ sensing behavior can be switched via the conformation flexibility. The 1:1 sensor/Hg²⁺ binding mode was proposed and supported by the titration experiment and ESI mass spectrum. The fluorescent sensor can display a highly selective response of fluorescence enhancement toward Hg²⁺ and detect the parts per billion (ppb) level of Hg²⁺ in aqueous environment.     

Salt-Toggled Capture Selection of Uric Acid Binding Aptamers

Uric acid is the end-product of purine metabolism in humans and an important biomarker for many diseases. To achieve the detection of uric acid without using enzymes, we previously selected a DNA aptamer for uric acid with a K_d of 1 μM but the aptamer required multiple Na⁺ ions for binding. Saturated binding was achieved with around 700 mM Na⁺ and the binding at the physiological condition was much weaker. In this work, a new selection was performed by alternating Mg²⁺-containing buffers with Na⁺ and Li⁺. After 13 rounds of selection, a new aptamer sequence named UA-Mg-1 was obtained. Isothermal titration calorimetry confirmed aptamer binding in both selection buffers, and the K_d was around 8 μM. The binding of UA-Mg-1 to UA required only Mg²⁺. This is an indicator of successful switching of metal dependency via the salt-toggled selection method. The UA-Mg-1 aptamer was engineered into a fluorescent biosensor based on the strand-displacement assay with a limit of detection of 0.5 μM uric acid in the selection buffer. Finally, comparison with the previously reported Na⁺-dependent aptamer and a xanthine/uric acid riboswitch was also made.     

Conjugation of a Dipicolyl Chelate to Polypeptide Conjugates Increases Binding Affinities for Human Serum Albumin and Survival Times in Human Serum

The affinity for human serum albumin (HSA) of a series of 2–5 kDa peptides covalently linked to 3,5‐bis[[bis(2‐pyridylmethyl)amino]methyl]benzoic acid, a dipicolyl chelator with micromolar affinity for Zn²⁺, was found by surface plasmon resonance to increase in the presence of 1 μm ZnCl₂ at physiological pH. The dependence on polypeptide hydrophobicity was found to be minor, thus suggesting that the conjugates bound to the metal‐binding site and not to the fatty‐acid‐binding site. The affinity of the conjugates increased strongly with the positive charge of the polypeptides, thus implicating the negatively charged protein surface surrounding the metal‐binding site. The survival times of the peptides in human serum were extended as a consequence of stronger binding to HSA, thus suggesting that Zn²⁺‐chelating agents might provide a general route to increased survival time of peptides in serum in therapeutic and diagnostic applications without significantly increasing their molecular weights.     

Residual Helicity at the Active Site of the Histidine Phosphocarrier,HPr, Modulates Binding Affinity to Its Natural Partners

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) modulates the preferential use of sugars in bacteria. The first proteins in the cascade are common to all organisms (EI and HPr). The active site of HPr involves a histidine (His15) located immediately before the beginning of the first α-helix. The regulator of sigma D (Rsd) protein also binds to HPr. The region of HPr comprising residues Gly9-Ala30 (HPr^9–30), involving the first α-helix (Ala16-Thr27) and the preceding active site loop, binds to both the N-terminal region of EI and intact Rsd. HPr^9–30 is mainly disordered. We attempted to improve the affinity of HPr^9–30 to both proteins by mutating its sequence to increase its helicity. We designed peptides that led to a marginally larger population in solution of the helical structure of HPr^9–30. Molecular simulations also suggested a modest increment in the helical population of mutants, when compared to the wild-type. The mutants, however, were bound with a less favorable affinity than the wild-type to both the N-terminal of EI (EIN) or Rsd, as tested by isothermal titration calorimetry and fluorescence. Furthermore, mutants showed lower antibacterial properties against Staphylococcus aureus than the wild-type peptide. Therefore, we concluded that in HPr, a compromise between binding to its partners and residual structure at the active site must exist to carry out its function.     

Studies on Interaction of Polysaccharide-Templated Silver Nanoparticles with Bovine Serum Albumin

In the present work, the interaction between bovine serum albumin (BSA) and as-synthesized silver nanoparticles (Oc-AgNPs) by using 4-acetamido-TEMPO-oxidized curdlan (Oc), as a reducing and stabilizing agent, was studied through fluorescence quenching method, ultraviolet visible spectrum (UV–Vis), and circular dichroism measurement. The results presented clearly indicate that the intrinsic fluorescence of BSA molecule was effectively quenched after the interaction with Oc-AgNPs by a static mechanism, which is further confirmed by UV–Vis analysis. The apparent binding constant (K), number of binding sites (n), and dissociation equilibrium constant (K_D) were calculated to be 7.5 × 10⁵ M^?1, 1.03, and 3.0 ± 0.6 μM, respectively. Furthermore, a conformational change of BSA was also observed when the Oc-AgNPs–BSA interactant formed.