AsIII Selectively Induces a Disorder-to-Order Transition in the Metalloid Binding Region of the AfArsR Protein.

Arsenic is highly toxic and a significant threat to human health, but certain bacteria have developed defense mechanisms initiated by AsIII binding to AsIII-sensing proteins of the ArsR family. The transcriptional regulator AfArsR responds to AsIII and SbIII by coordinating the metalloids with three cysteines, located in a short sequence of the same monomer chain. Here, we characterize the binding of AsIII and HgII to a model peptide encompassing this fragment of the protein via solution equilibrium and spectroscopic/spectrometric techniques (pH potentiometry, UV, CD, NMR, PAC, EXAFS, and ESI-MS) combined with DFT calculations and MD simulations. Coordination of AsIII changes the peptide structure from a random-coil to a well-defined structure of the complex. A trigonal pyramidal AsS3 binding site is formed with almost exactly the same structure as observed in the crystal structure of the native protein, implying that the peptide possesses all of the features required to mimic the AsIII recognition and response selectivity of AfArsR. Contrary to this, binding of HgII to the peptide does not lead to a well-defined structure of the peptide, and the atoms near the metal binding site are displaced and reoriented in the HgII model. Our model study suggests that structural organization of the metal site by the inducer ion is a key element in the mechanism of the metalloid-selective recognition of this protein.

Revisiting the hydrolysis of ampicillin catalyzed by Temoneira-1 ß-lactamase, and the effect of Ni(II), Cd(II) and Hg(II).

ß-Lactamases grant resistance to bacteria against ß-lactam antibiotics. The active center of TEM-1 ß-lactamase accommodates a Ser-Xaa-Xaa-Lys motif. TEM-1 ß-lactamase is not a metalloenzyme but it possesses several putative metal ion binding sites. The sites composed of His residue pairs chelate borderline transition metal ions such as Ni(II). In addition, there are many sulfur-containing donor groups that can coordinate soft metal ions such as Hg(II). Cd(II) may bind to both types of the above listed donor groups. No significant change was observed in the circular dichroism spectra of TEM-1 ß-lactamase on increasing the metal ion content of the samples, with the exception of Hg(II) inducing a small change in the secondary structure of the protein. A weak nonspecific binding of Hg(II) was proven by mass spectrometry and 119m Hg perturbed angular correlation spectroscopy. The hydrolytic process of ampicillin catalyzed by TEM-1 ß-lactamase was described by the kinetic analysis of the set of full catalytic progress curves, where the slow, yet observable conversion of the primary reaction product into a second one, identified as ampilloic acid by mass spectrometry, needed also to be considered in the applied model. Ni(II) and Cd(II) slightly promoted the catalytic activity of the enzyme while Hg(II) exerted a noticeable inhibitory effect. Hg(II) and Ni(II), applied at 10 µM concentration, inhibited the growth of E. coli BL21(DE3) in M9 minimal medium in the absence of ampicillin, but addition of the antibiotic could neutralize this toxic effect by complexing the metal ions.

Hydrolytic Mechanism of a Metalloenzyme Is Modified by the Nature of the Coordinated Metal Ion.

The nuclease domain of colicin E7 cleaves double-strand DNA non-specifically. Zn2+ ion was shown to be coordinated by the purified NColE7 as its native metal ion. Here, we study the structural and catalytic aspects of the interaction with Ni2+, Cu2+ and Cd2+ non-endogenous metal ions and the consequences of their competition with Zn2+ ions, using circular dichroism spectroscopy and intact protein mass spectrometry. An R447G mutant exerting decreased activity allowed for the detection of nuclease action against pUC119 plasmid DNA via agarose gel electrophoresis in the presence of comparable metal ion concentrations. It was shown that all of the added metal ions could bind to the apoprotein, resulting in a minor secondary structure change, but drastically shifting the charge distribution of the protein. Zn2+ ions could not be replaced by Ni2+, Cu2+ and Cd2+. The nuclease activity of the Ni2+-bound enzyme was extremely high in comparison with the other metal-bound forms, and could not be inhibited by the excess of Ni2+ ions. At the same time, this activity was significantly decreased in the presence of equivalent Zn2+, independent of the order of addition of each component of the mixture. We concluded that the Ni2+ ions promoted the DNA cleavage of the enzyme through a more efficient mechanism than the native Zn2+ ions, as they directly generate the nucleophilic OH- ion.

Temoneira-1 ß-lactamase is not a metalloenzyme, but its native metal ion binding sites allow for purification by immobilized metal ion affinity chromatography.

ß-lactamases protect bacteria from ß-lactam antibiotics. Temoneira (TEM) is a class A serine ß-lactamase and its coding sequence is designed into DNA vectors, such as pET-21a (+), to provide antibiotic resistance. TEM-1 ß-lactamase was overexpressed efficiently from this vector upon inducing protein expression by IPTG in BL21(DE3) cells. Immobilized metal ion affinity chromatography (IMAC) was used based on the three native putative metal ion binding sites of TEM-1 ß-lactamase, each consisting of a pair of histidine sidechains. Elution was achieved at low concentrations of imidazole (∼15-200 mM). Two steps of IMAC and a subsequent anion exchange purification produced highly pure TEM-1 ß-lactamase with a yield of 1.9 mg/g of wet bacterial pellet weight. Mass spectrometry revealed that the mature form of ß-lactamase (without the signal sequence) was obtained. The secondary structure composition, calculated from the circular dichroism spectrum, showed that the target protein was folded similar to the published crystal structure. Ni(II) binding to the enzyme was also investigated. Increasing amounts of Ni(II) ions had only a small effect on the protein structure. Mass spectrometry detected up to three bound metal ions at 10:1 Ni(II):protein molar ratio, but the major peak was assigned to the monometallated ß-lactamase indicating the presence of a paramount metal ion binding site formed by the H151/H156 pair.