Intrinsic disorder and metal binding in UreG proteins from Archae hyperthermophiles: GTPase enzymes involved in the activation of Ni(II) dependent urease

Urease is a Ni(II) enzyme present in every domain of life, in charge for nitrogen recycling through urea hydrolysis. Its activity requires the presence of two Ni(II) ions in the active site. These are delivered by the concerted action of four accessory proteins, named UreD, UreF, UreG and UreE. This process requires protein flexibility at different levels and some disorder-to-order transition events that coordinate the mechanism of protein-protein interaction. In particular, UreG, the GTPase in charge of nucleotide hydrolysis required for urease activation, presents a significant degree of intrinsic disorder, existing as a conformational ensemble featuring characteristics that recall a molten globule. Here, the folding properties of UreG were explored in Archaea hyperthermophiles, known to generally feature significantly low level of structural disorder in their proteome. UreG proteins from Methanocaldococcus jannaschii (Mj) and Metallosphaera sedula (Ms) were structurally and functionally analyzed by integrating circular dichroism, NMR, light scattering and enzymatic assays. Metal-binding properties were studied using isothermal titration calorimetry. The results indicate that, as the mesophilic counterparts, both proteins contain a significant amount of secondary structure but maintain a flexible fold and a low GTPase activity. As opposed to other UreGs, secondary structure is lost at high temperatures (68 and 75 °C, respectively) with an apparent two-state mechanism. Both proteins bind Zn(II) and Ni(II), with affinities two orders of magnitude higher for Zn(II) than for Ni(II). No major modifications of the average conformational ensemble are observed, but binding of Zn(II) yields a more compact dimeric form in MsUreG

Copper ions and coordination complexes as novel carbapenem adjuvants

Carbapenem-resistant Enterobacteriaceae are an urgent threat to global human health. These organisms produce β-lactamases with carbapenemase activity, such as the metallo-β-lactamase NDM-1, which is notable due to its association with mobile genetic elements and the lack of a clinically useful inhibitor. Here we examined the ability of copper to inhibit the activity of NDM-1 and explored the potential of a copper coordination complex as a mechanism to efficiently deliver copper as an adjuvant in clinical therapeutics. An NDM-positive Escherichia coli isolate, MS6192, was cultured from the urine of a patient with urinary tract infection. MS6192 was resistant to antibiotics from multiple classes, including diverse β-lactams (penicillins, cephalosporins, and carbapenems), aminoglycosides and fluoroquinolones. However, in the presence of copper (range 0-2 mM), the susceptibility of MS6192 to the carbapenems ertapenem and meropenem increased significantly. In standard checkerboard assays, copper decreased the MIC of ertapenem and meropenem against MS6192 in a dose-dependent manner, suggesting a synergistic mode of action. To examine the inhibitory effect of copper in the absence of other β-lactamases, the blaNDM-1 gene from MS6192 was cloned and expressed in a recombinant E. coli K-12 strain. Analysis of cell-free extracts prepared from this strain revealed copper directly inhibits NDM-1 activity, and this was further confirmed using purified recombinant NDM-1. Finally, delivery of copper at a low concentration of 10 μM using the FDA-approved coordination complex copper-pyrithione sensitised MS6192 to ertapenem and meropenem in a synergistic manner. Overall, this work demonstrates the potential use of copper-coordination complexes as novel carbapenemase adjuvants

Intrinsic disorder and metal binding in UreG proteins from Archae hyperthermophiles: GTPase enzymes involved in the activation of Ni(II) dependent urease

Urease is a Ni(II) enzyme present in every domain of life, in charge for nitrogen recycling through urea hydrolysis. Its activity requires the presence of two Ni(II) ions in the active site. These are delivered by the concerted action of four accessory proteins, named UreD, UreF, UreG and UreE. This process requires protein flexibility at different levels and some disorder-to-order transition events that coordinate the mechanism of protein-protein interaction. In particular, UreG, the GTPase in charge of nucleotide hydrolysis required for urease activation, presents a significant degree of intrinsic disorder, existing as a conformational ensemble featuring characteristics that recall a molten globule. Here, the folding properties of UreG were explored in Archaea hyperthermophiles, known to generally feature significantly low level of structural disorder in their proteome. UreG proteins from Methanocaldococcus jannaschii (Mj) and Metallosphaera sedula (Ms) were structurally and functionally analyzed by integrating circular dichroism, NMR, light scattering and enzymatic assays. Metal-binding properties were studied using isothermal titration calorimetry. The results indicate that, as the mesophilic counterparts, both proteins contain a significant amount of secondary structure but maintain a flexible fold and a low GTPase activity. As opposed to other UreGs, secondary structure is lost at high temperatures (68 and 75 °C, respectively) with an apparent two-state mechanism. Both proteins bind Zn(II) and Ni(II), with affinities two orders of magnitude higher for Zn(II) than for Ni(II). No major modifications of the average conformational ensemble are observed, but binding of Zn(II) yields a more compact dimeric form in MsUreG

Identification and characterization of an unusual metallo-beta-lactamase from Serratia proteamaculans

Metallo-beta-lactamases (MBLs) are a family of metalloenzymes that are capable of hydrolyzing beta-lactam antibiotics and are an important means by which bacterial pathogens use to inactivate antibiotics. A database search of the available amino acid sequences from Serratia proteamaculans indicates the presence of an unusual MBL. A full length amino acid sequence alignment indicates overall homology to B3-type MBLs, but also suggests considerable variations in the active site, notably among residues that are relevant to metal ion binding. Steady-state kinetic measurements further indicate functional differences and identify two relevant pK (a) values for catalysis (3.8 for the enzyme-substrate complex and 7.8 for the free enzyme) and a preference for penams with modest reactivity towards some cephalosporins. An analysis of the metal ion content indicates the presence of only one zinc ion per active site in the resting enzyme. In contrast, kinetic data suggest that the enzyme may operate as a binuclear enzyme, and it is thus proposed that a catalytically active di-Zn2+ center is formed only once the substrate is present