Conformational Dynamics of metallo-β-lactamase CcrA during Catalysis Investigated by Using DEER Spectroscopy

Previous crystallographic and mutagenesis studies have implicated the role of a position-conserved hairpin loop in the metallo-β-lactamases in substrate binding and catalysis. In an effort to probe the motion of that loop during catalysis, rapid-freeze-quench double electron–electron resonance (RFQ-DEER) spectroscopy was used to interrogate metallo-β-lactamase CcrA, which had a spin label at position 49 on the loop and spin labels (at positions 82, 126, or 233) 20–35 Å away from residue 49, during catalysis. At 10 ms after mixing, the DEER spectra show distance increases of 7, 10, and 13 Å between the spin label at position 49 and the spin labels at positions 82, 126, and 233, respectively. In contrast to previous hypotheses, these data suggest that the loop moves nearly 10 Å away from the metal center during catalysis and that the loop does not clamp down on the substrate during catalysis. This study demonstrates that loop motion during catalysis can be interrogated on the millisecond time scale

Dilution of Dipolar Interactions in a Spin-labeled, Multimeric Metalloenzyme for DEER Studies

The metallo-β-lactamases (MβLs), which require one or two Zn(II) ions in their active sites for activity, hydrolyze the amide bond in β-lactam-containing antibiotics, and render the antibiotics inactive. All known MβLs contain a mobile element near their active sites, and these mobile elements have been implicated in the catalytic mechanisms of these enzymes. However little is known about the dynamics of these elements. In this study, we prepared a site-specific, double spin-labeled analog of homotetrameric MβL L1 with spin labels at positions 163 and 286 and analyzed the sample with DEER (double electron electron resonance) spectroscopy. Four unique distances were observed in the DEER distance distribution, and these distances were assigned to the desired intramolecular dipolar coupling (between spin labels at positions 163 and 286 in one subunit) and to intermolecular dipolar couplings. To rid the spin-labeled analog of L1 of the intermolecular couplings, spin-labeled L1 was “diluted” by unfolding/refolding the spin-labeled enzyme in the presence of excess wild-type L1. DEER spectra of the resulting, spin-diluted enzyme revealed a single distance corresponding to the desire intramolecular dipolar coupling

Structure–activity relationship of ipglycermide binding to phosphoglycerate mutases

Catalysis of human phosphoglycerate mutase is dependent on a 2,3-bisphosphoglycerate cofactor (dPGM), whereas the nonhomologous isozyme in many parasitic species is cofactor independent (iPGM). This mechanistic and phylogenetic diversity offers an opportunity for selective pharmacologic targeting of glycolysis in disease-causing organisms. We previously discovered ipglycermide, a potent inhibitor of iPGM, from a large combinatorial cyclic peptide library. To fully delineate the ipglycermide pharmacophore, herein we construct a detailed structure–activity relationship using 280 substituted ipglycermide analogs. Binding affinities of these analogs to immobilized Caenorhabditis elegans iPGM, measured as fold enrichment relative to the index residue by deep sequencing of an mRNA display library, illuminated the significance of each amino acid to the pharmacophore. Using cocrystal structures and binding kinetics, we show that the high affinity of ipglycermide for iPGM orthologs, from Brugia malayi, Onchocerca volvulus, Dirofilaria immitis, and Escherichia coli, is achieved by a codependence between (1) the off-rate mediated by the macrocycle Cys14 thiolate coordination to an active-site Zn2+ in the iPGM phosphatase domain and (2) shape complementarity surrounding the macrocyclic core at the phosphotransferase–phosphatase domain interface. Our results show that the high-affinity binding of ipglycermide to iPGMs freezes these structurally dynamic enzymes into an inactive, stable complex

A general reaction mechanism for carbapenem hydrolysis by mononuclear and binuclear metallo-β-lactamases

Carbapenem-resistant Enterobacteriaceae threaten human health, since carbapenems are last resort drugs for infections by such organisms. Metallo-β-lactamases (MβLs) are the main mechanism of resistance against carbapenems. Clinically approved inhibitors of MBLs are currently unavailable as design has been limited by the incomplete knowledge of their mechanism. Here, we report a biochemical and biophysical study of carbapenem hydrolysis by the B1 enzymes NDM-1 and BcII in the bi-Zn(II) form, the mono-Zn(II) B2 Sfh-I and the mono-Zn(II) B3 GOB-18. These MβLs hydrolyse carbapenems via a similar mechanism, with accumulation of the same anionic intermediates. We characterize the Michaelis complex formed by mono-Zn(II) enzymes, and we identify all intermediate species, enabling us to propose a chemical mechanism for mono and binuclear MβLs. This common mechanism open avenues for rationally designed inhibitors of all MβLs, notwithstanding the profound differences between these enzymes' active site structure, β-lactam specificity and metal content.Fil: Lisa, María Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Instituto Pasteur de Montevideo; UruguayFil: Palacios, Antonela Rocio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Aitha, Mahesh. Miami University; Estados UnidosFil: Gonzalez, Mariano Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Moreno, Diego Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; ArgentinaFil: Crowder, Michael W.. Miami University; Estados UnidosFil: Bonomo, Robert A.. Case Western Reserve University; Estados Unidos. Louis Stokes Cleveland Department of Veterans Affairs Medical Center; Estados UnidosFil: Spencer, James. University Walk; Reino Unido. University of Bristol; Reino UnidoFil: Tierney, David L.. Miami University; Estados UnidosFil: Llarrull, Leticia Irene. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química Biológica. Área Biofísica; ArgentinaFil: Vila, Alejandro Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química Biológica. Área Biofísica; Argentina. Case Western Reserve University; Estados Unido

Spectroscopic and Mechanistic Studies of Heterodimetallic Forms of Metallo-β-lactamase NDM-1

In an effort to characterize the roles of each metal ion in metallo-β-lactamase NDM-1, heterodimetallic analogues (CoCo-, ZnCo-, and CoCd-) of the enzyme were generated and characterized. UV–vis, 1H NMR, EPR, and EXAFS spectroscopies were used to confirm the fidelity of the metal substitutions, including the presence of a homogeneous, heterodimetallic cluster, with a single-atom bridge. This marks the first preparation of a metallo-β-lactamase selectively substituted with a paramagnetic metal ion, Co(II), either in the Zn1 (CoCd-NDM-1) or in the Zn2 site (ZnCo-NDM-1), as well as both (CoCo-NDM-1). We then used these metal-substituted forms of the enzyme to probe the reaction mechanism, using steady-state and stopped-flow kinetics, stopped-flow fluorescence, and rapid-freeze-quench EPR. Both metal sites show significant effects on the kinetic constants, and both paramagnetic variants (CoCd- and ZnCo-NDM-1) showed significant structural changes on reaction with substrate. These changes are discussed in terms of a minimal kinetic mechanism that incorporates all of the data

Interactions of DNA bases with selected metal ions

Several authors have investigated the interactions between DNA and metal ions to understand how metal ions affect the structure of DNA leading to the some types of cancers and neurodegenerative diseases. But these studies on interaction between DNA and metal ions have led to conflicting results, probably because DNA is studied as a macromolecule. The study of the larger bio-molecules, such as DNA and proteins, could be accounted by examining the smaller fragments of which they are composed. Our study was designed to examine the interactions of two DNA bases (thymine and cytosine) with various metal ions (Al, Ca, Ce, Cu, Fe, Hg, Mg, and Tl) using UV visible, infrared and Raman techniques. The study was carried out in aqueous solutions at different pH values and metal ion/ligand concentrations. UV visible spectra of thymine/cytosine and their metal complexes have shown that all metal ions cited above interact with thymine/cytosine, but the extent of the interaction depends on the nature of the metal ion, molar ratio, and pH value. The stronger interaction was observed between trivalent metal ions (Al, Ce, and Tl) and thymine/cytosine. Infrared and Raman spectra have shown some marker bands useful to identify the sites involved in metal chelation to the thymine and cytosine. Particularly, preferred binding sites on thymine and cytosine are N and O atoms for the all metal ions. For thymine, the preferred binding sites are N and O atoms for the divalent and trivalent metal ions, and only N atom for the monovalent metal ions. For cytosine, the preferred binding sites are N and O atoms for the divalent and trivalent metal ions, and N atom for the monovalent metal ions. Infrared and Raman spectroscopes are powerful tools for investigating the preferred binding sites of the metal ions on a molecule

Monitoring the Retention of Human Proliferating Cell Nuclear Antigen at Primer/Template Junctions by Proteins That Bind Single-Stranded DNA

In humans, proliferating cell nuclear antigen (PCNA) sliding clamps encircling DNA coordinate various aspects of DNA metabolism throughout the cell cycle. A critical aspect of this is restricting PCNA to the vicinity of its DNA target site. For example, PCNA must be maintained at or near primer/template (P/T) junctions during DNA synthesis. With a diverse array of cellular factors implicated, many of which interact with PCNA, DNA, or both, it is unknown how this critical feat is achieved. Furthermore, current biochemical assays that examine the retention of PCNA near P/T junctions are inefficient, discontinuous, and qualitative and significantly deviate from physiologically relevant conditions. To overcome these challenges and limitations, we recently developed a novel and convenient Förster resonance energy transfer (FRET) assay that directly and continuously monitors the retention of human PCNA at a P/T junction. Here we describe in detail the design, methodology, interpretation, and limitations of this quantitative FRET assay using the single-stranded DNA-binding protein, SSB, from <i>Escherichia coli</i> as an example. This powerful tool is broadly applicable to any single-stranded DNA-binding protein and may be utilized and/or expanded upon to dissect DNA metabolic pathways that are dependent upon PCNA

Reactions of Zn2+, Cd2+ and Hg2+ with Free Adenine

We are reporting the fluorescence quenching, IR, Raman, 1H-NMR, and potentiometric studies for the Zn2+:adenine and the Cd2+:adenine systems under ambient conditions. IR and Raman spectra suggest that Zn2+ and Cd2+ interact with adenine but the modes of interaction differ. Fluorescence spectra indicate that the interaction involving Zn2+ is more favorable than that of Cd2+, and this effect is due to the difference in ionic radii. 1H-NMR, potentiometry, and speciation diagrams indicate the formation of strong metal ion adenine complexes. Potentiometric titrations of the heavier member of group 12 metals (Hg2+) show similar results to that of Zn2+ and Cd2+. Some differences in the 1H-NMR experiments appeared between both (Zn2+ and Cd2+) compared to that of Hg2+. Due to the fluorescence quenching of adenine, adenine can be used as a sensor of Zn2+ and Cd2+

Metal Ion Dependence of the Matrix Metalloproteinase‑1 Mechanism

Matrix metalloproteinase-1 (MMP-1) plays crucial roles in disease-related physiologies and pathological processes in the human body. We report here solution studies of MMP-1, including characterization of a series of mutants designed to bind metal in either the catalytic site or the structural site (but not both). Circular dichroism and fluorescence spectroscopy of the mutants demonstrate the importance of the structural Zn­(II) in maintaining both secondary and tertiary structure, while UV–visible, nuclear magnetic resonance, electron paramagnetic resonance, and extended X-ray absorption fine structure show its presence influences the catalytic metal ion’s coordination number. The mutants allow us to demonstrate convincingly the preparation of a mixed-metal analogue, Co_CZn_S-MMP-1, with Zn­(II) in the structural site and Co­(II) in the catalytic site. Stopped-flow fluorescence of the native form, Zn_CZn_S-MMP-1, and the mixed-metal Co_CZn_S-MMP-1 analogue shows that the internal fluorescence of a nearby Trp residue is modulated with catalysis and can be used to monitor reactivity under a number of conditions, opening the door to substrate profiling

Mechanistic and Spectroscopic Studies of Metallo-β-lactamase NDM-1

In an effort to biochemically characterize metallo-β-lactamase NDM-1, we cloned, overexpressed, purified, and characterized several maltose binding protein (MBP)–NDM-1 fusion proteins with different N-termini (full-length, Δ6, Δ21, and Δ36). All MBP–NDM-1 fusion proteins were soluble; however, only one, MBP–NDM-1Δ36, exhibited high activity and bound 2 equiv of Zn­(II). Thrombin cleavage of this fusion protein resulted in the truncated NDM-1Δ36 variant, which exhibited a <i>k</i>_cat of 16 s^–1 and a <i>K</i>_m of 1.1 μM when using nitrocefin as a substrate, bound 2 equiv of Zn­(II), and was monomeric in solution. Extended X-ray absorption fine structure studies of the NDM-1Δ36 variant indicate the average metal binding site for Zn­(II) in this variant consists of four N/O donors (two of which are histidines) and 0.5 sulfur donor per zinc, with a Zn–Zn distance of 3.38 Å. This metal binding site is very similar to those of other metallo-β-lactamases that belong to the B1 subclass. Pre-steady-state kinetic studies using nitrocefin and chromacef and the NDM-1Δ36 variant indicate that the enzyme utilizes a kinetic mechanism similar to that used by metallo-β-lactamases L1 and CcrA, in which a reactive nitrogen anion is stabilized and its protonation is rate-limiting. While they are very different in terms of amino acid sequence, these studies demonstrate that NDM-1 is structurally and mechanistically very similar to metallo-β-lactamase CcrA