A Single Serine Residue Determines Selectivity to Monovalent Metal Ions in Metalloregulators of the MerR Family

ABSTRACTMerR metalloregulators alleviate toxicity caused by an excess of metal ions, such as copper, zinc, mercury, lead, cadmium, silver, or gold, by triggering the expression of specific efflux or detoxification systems upon metal detection. The sensor protein binds the inducer metal ion by using two conserved cysteine residues at the C-terminal metal-binding loop (MBL). Divalent metal ion sensors, such as MerR and ZntR, require a third cysteine residue, located at the beginning of the dimerization (α5) helix, for metal coordination, while monovalent metal ion sensors, such as CueR and GolS, have a serine residue at this position. This serine residue was proposed to provide hydrophobic and steric restrictions to privilege the binding of monovalent metal ions. Here we show that the presence of alanine at this position does not modify the activation pattern of monovalent metal sensors. In contrast, GolS or CueR mutant sensors with a substitution of cysteine for the serine residue respond to monovalent metal ions or Hg(II) with high sensitivities. Furthermore, in a mutant deleted of the Zn(II) exporter ZntA, they also trigger the expression of their target genes in response to either Zn(II), Cd(II), Pb(II), or Co(II).IMPORTANCESpecificity in a stressor's recognition is essential for mounting an appropriate response. MerR metalloregulators trigger the expression of specific resistance systems upon detection of heavy metal ions. Two groups of these metalloregulators can be distinguished, recognizing either +1 or +2 metal ions, depending on the presence of a conserved serine in the former or a cysteine in the latter. Here we demonstrate that the serine residue in monovalent metal ion sensors excludes divalent metal ion detection, as its replacement by cysteine renders a pan-metal ion sensor. Our results indicate that the spectrum of signals detected by these sensors is determined not only by the metal-binding ligand availability but also by the metal-binding cavity flexibility.

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HK97 gp74 Possesses an α-Helical Insertion in the ββα Fold That Affects Its Metal Binding, cos Site Digestion, and In Vivo Activities

Journal of Bacteriology ◽

10.1128/jb.00644-19 ◽

2020 ◽

Vol 202 (8) ◽

Author(s):

Sasha A. Weiditch ◽

Sarah C. Bickers ◽

Diane Bona ◽

Karen L. Maxwell ◽

Voula Kanelis

Keyword(s):

Metal Binding ◽

Metal Ion ◽

Divalent Metal ◽

Metal Ion Binding ◽

Divalent Metal Ions ◽

Nmr Studies ◽

Phage Dna ◽

Divalent Metal Ion ◽

Phage Morphogenesis

ABSTRACT The last gene in the genome of the bacteriophage HK97 encodes gp74, an HNH endonuclease. HNH motifs contain two conserved His residues and an invariant Asn residue, and they adopt a ββα structure. gp74 is essential for phage head morphogenesis, likely because gp74 enhances the specific endonuclease activity of the HK97 terminase complex. Notably, the ability of gp74 to enhance the terminase-mediated cleavage of the phage cos site requires an intact HNH motif in gp74. Mutation of H82, the conserved metal-binding His residue in the HNH motif, to Ala abrogates gp74-mediated stimulation of terminase activity. Here, we present nuclear magnetic resonance (NMR) studies demonstrating that gp74 contains an α-helical insertion in the Ω-loop, which connects the two β-strands of the ββα fold, and a disordered C-terminal tail. NMR data indicate that the Ω-loop insert makes contacts to the ββα fold and influences the ability of gp74 to bind divalent metal ions. Further, the Ω-loop insert and C-terminal tail contribute to gp74-mediated DNA digestion and to gp74 activity in phage morphogenesis. The data presented here enrich our molecular-level understanding of how HNH endonucleases enhance terminase-mediated digestion of the cos site and contribute to the phage replication cycle. IMPORTANCE This study demonstrates that residues outside the canonical ββα fold, namely, the Ω-loop α-helical insert and a disordered C-terminal tail, regulate the activity of the HNH endonuclease gp74. The increased divalent metal ion binding when the Ω-loop insert is removed compared to reduced cos site digestion and phage formation indicates that the Ω-loop insert plays multiple regulatory roles. The data presented here provide insights into the molecular basis of the involvement of HNH proteins in phage DNA packing.

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Target transcription binding sites differentiate two groups of MerR-monovalent metal ion sensors

Molecular Microbiology ◽

10.1111/j.1365-2958.2010.07370.x ◽

2010 ◽

Vol 78 (4) ◽

pp. 853-865 ◽

Cited By ~ 24

Author(s):

María E. Pérez Audero ◽

Brenda M. Podoroska ◽

María M. Ibáñez ◽

Ana Cauerhff ◽

Susana K. Checa ◽

...

Keyword(s):

Binding Sites ◽

Metal Ion ◽

Ion Sensors ◽

Monovalent Metal ◽

Transcription Binding Sites ◽

Metal Ion Sensors

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Structure of New Binary and Ternary DNA Polymerase Complexes From Bacteriophage RB69

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.704813 ◽

2021 ◽

Vol 8 ◽

Author(s):

Jongseo Park ◽

Hyung-Seop Youn ◽

Jun Yop An ◽

Youngjin Lee ◽

Soo Hyun Eom ◽

...

Keyword(s):

Metal Ions ◽

Dna Polymerase ◽

Metal Ion ◽

Ternary Complex ◽

Critical Role ◽

Divalent Metal ◽

Binary Complex ◽

Divalent Metal Ions ◽

Divalent Metal Ion ◽

Initial Binding

DNA polymerase plays a critical role in passing the genetic information of any living organism to its offspring. DNA polymerase from enterobacteria phage RB69 (RB69pol) has both polymerization and exonuclease activities and has been extensively studied as a model system for B-family DNA polymerases. Many binary and ternary complex structures of RB69pol are known, and they all contain a single polymerase-primer/template (P/T) DNA complex. Here, we report a crystal structure of the exonuclease-deficient RB69pol with the P/T duplex in a dimeric form at a resolution of 2.2 Å. The structure includes one new closed ternary complex with a single divalent metal ion bound and one new open binary complex in the pre-insertion state with a vacant dNTP-binding pocket. These complexes suggest that initial binding of the correct dNTP in the open state is much weaker than expected and that initial binding of the second divalent metal ion in the closed state is also much weaker than measured. Additional conformational changes are required to convert these complexes to high-affinity states. Thus, the measured affinities for the correct incoming dNTP and divalent metal ions are average values from many conformationally distinctive states. Our structure provides new insights into the order of the complex assembly involving two divalent metal ions. The biological relevance of specific interactions observed between one RB69pol and the P/T duplex bound to the second RB69pol observed within this dimeric complex is discussed.

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Activation of Bovine Factor X in the Presence of Calcium, Magnesium, Barium or Manganese ion

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648669 ◽

1978 ◽

Vol 40 (02) ◽

pp. 358-367 ◽

Cited By ~ 2

Author(s):

Robert H Yue ◽

Menard M Gertler

Keyword(s):

Metal Ions ◽

Binding Site ◽

Metal Ion ◽

Factor Xa ◽

Divalent Metal ◽

Activation Process ◽

Factor X ◽

Divalent Metal Ion ◽

Russell’S Viper ◽

Bovine Factor

SummaryThe binding of divalent metal ions to bovine factor X, factor Xa and the coagulant protein in Russell’s viper venom was studied by the technique of fluorescence quenching. Titration of factor X with Ca+2, Mg+2 or Ba+2 revealed that these metal ions can bind to factor X. A tightly binding site(s) was observed with Kd of 79 and 98 μM for Ca+2 and Mg+2 respectively. A loosely binding site(s) was evident with Kd of 0.55, 0.50 and 0.35 mM for Ca+2, Mg+2 and Ba+2 respectively. The quenching phenomenon was also observed when Mn+2 was used as titrant but factor X precipitated out when the concentration of Mn+2 was 10 mM. The binding of Ca+2, Mg+2, Ba+2 or Mn+2 to bovine factor Xa or to the purified coagulant fraction of Russell’s viper venom was very weak in each case.In the absence of Ca+2, the coagulation fraction of Russell’s viper venom could not activate bovine factor X. Activation of factor X was achieved when Ca+2 was replaced by either Mg+2, Ba+2 or Mn+2. When the concentration of these ions were 5 mM, the efficiency of factor Xa generation was estimated to be: Ca+2> Mg+2> Ba+2> Mn+2. Higher concentration of Mg+2, Ba+2, or Mn+2 retarded the activation process. However, Ca+2, Mg+2, Ba+2 or Mn+2 has little or no influence on the esterase activity of factor Xa or purified Rusell’s viper venom.The results suggest that complexation of divalent metal ion with factor X is prerequisite in the activation process. The binding of Mg+2, Ba+2 or Mn+2 to these loosely binding sites might have altered the geometrical configuration as well as the electrostatic environment on factor X significantly. Thus, it is more difficult to form the binary complex and a slower generation of factor Xa results. Therefore, divalent metal ion serves as a dual role in the activation of factor X to factor Xa depending upon the ionic concentration.

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Multiple Metal Binding Domains Enhance the Zn(II) Selectivity of the Divalent Metal Ion Transporter AztA†

Biochemistry ◽

10.1021/bi7006367 ◽

2007 ◽

Vol 46 (39) ◽

pp. 11057-11068 ◽

Cited By ~ 13

Author(s):

Tong Liu ◽

Hermes Reyes-Caballero ◽

Chenxi Li ◽

Robert A. Scott ◽

David P. Giedroc

Keyword(s):

Metal Binding ◽

Metal Ion ◽

Divalent Metal ◽

Ion Transporter ◽

Binding Domains ◽

Divalent Metal Ion

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Divalent metal ion-mediated assembly of spherical nucleic acids: the case study of Cu2+

Physical Chemistry Chemical Physics ◽

10.1039/c5cp05202e ◽

2015 ◽

Vol 17 (45) ◽

pp. 30292-30299 ◽

Cited By ~ 4

Author(s):

Jang Ho Joo ◽

Jae-Seung Lee

Keyword(s):

Nucleic Acids ◽

Metal Ions ◽

Metal Ion ◽

Divalent Metal ◽

Divalent Metal Ions ◽

New Strategy ◽

Divalent Metal Ion ◽

Spherical Nucleic Acids

A new strategy for reversibly assembling spherical nucleic acids (SNAs) is demonstrated based on the coordinative binding of divalent metal ions, particularly Cu2+, to nucleobases.

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Effect of divalent metal ions on annexin-mediated aggregation of asolectin liposomes.

Acta Biochimica Polonica ◽

10.18388/abp.2000_3988 ◽

2000 ◽

Vol 47 (3) ◽

pp. 675-683 ◽

Cited By ~ 1

Author(s):

V I Mel'gunov ◽

E I Akimova ◽

K S Krasavchenko

Keyword(s):

Metal Ions ◽

Metal Ion ◽

Relative Concentration ◽

Toxic Metal ◽

Transition Metal Ions ◽

Divalent Metal ◽

Divalent Metal Ions ◽

Large Unilamellar Vesicles ◽

Protein Molecules ◽

Divalent Metal Ion

Annexins belong to a family of Ca2+- and phospholipid-binding proteins that can mediate the aggregation of granules and vesicles in the presence of Ca2+. We have studied the effects of different divalent metal ions on annexin-mediated aggregation of liposomes using annexins isolated from rabbit liver and large unilamellar vesicles prepared from soybean asolectin II-S. In the course of these studies, we have found that annexin-mediated aggregation of liposomes can be driven by various earth and transition metal ions other than Ca2+. The ability of metal ions to induce annexin-mediated aggregation decreases in the order: Cd2+ > Ba2+, Sr2+ > Ca2+ > Mn2+ > Ni2+ > Co2+. Annexin-mediated aggregation of vesicles is more selective to metal ions than the binding of annexins to membranes. We speculate that not every type of divalent metal ion can induce conformational change sufficient to promote the interaction of annexins either with two opposing membranes or with opposing protein molecules. Relative concentration ratios of metal ions in the intimate environment may be crucial for the functioning of annexins within specialized tissues and after treatment with toxic metal ions.

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