Characterisation of two calmodulin-like proteins from the liver fluke, Fasciola hepatica

2007 ◽  
Vol 388 (6) ◽  
pp. 593-599 ◽  
Author(s):  
Sean L. Russell ◽  
Neil V. McFerran ◽  
Elizabeth M. Hoey ◽  
Alan Trudgett ◽  
David J. Timson
Keyword(s):  
Metal Binding ◽  
Metal Ion ◽  
Liver Fluke ◽  
Fasciola Hepatica ◽  
Expression Vectors ◽  
Metal Ion Binding ◽  
Ion Sensing ◽  
Calcium Magnesium ◽  
Ion Binding Sites ◽  
Liver Flukes

Abstract Calmodulin is a calcium ion-sensing signalling protein found in eukaryotics. Two calmodulin-like gene sequences were identified in an EST library from adult liver flukes. One codes for a protein (FhCaM1) homologous to mammalian calmodulins (98% identity), whereas the other protein (FhCaM2) has only 41% identity. These genes were cloned into expression vectors and the recombinant proteins were expressed in Escherichia coli. Gel shift assays showed that both proteins bind to calcium, magnesium and zinc ions. Homology models have been built for both proteins. As expected, FhCaM1 has a highly similar structure to other calmodulins. Although FhCaM2 has a similar fold, its surface charge is higher than FhCaM1. One of the potential metal ion-binding sites has lower metal-ion co-ordination capability, while another has an adjacent lysine residue, both of which may decrease the metal-binding affinity. These differences may reflect a specialised role for FhCaM2 in the liver fluke.

scholarly journals Crystallographic and X-ray absorption spectroscopic characterization of Helicobacter pylori UreE bound to Ni2+ and Zn2+ reveals a role for the disordered C-terminal arm in metal trafficking

2012 ◽  
Vol 441 (3) ◽  
pp. 1017-1035 ◽  
Author(s):  
Katarzyna Banaszak ◽  
Vlad Martin-Diaconescu ◽  
Matteo Bellucci ◽  
Barbara Zambelli ◽  
Wojciech Rypniewski ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Metal Ions ◽  
Metal Binding ◽  
Metal Ion ◽  
Mutual Influence ◽  
Accurate Information ◽  
Metal Ion Binding ◽  
X Ray ◽  
X Ray Absorption

The survival and growth of the pathogen Helicobacter pylori in the gastric acidic environment is ensured by the activity of urease, an enzyme containing two essential Ni2+ ions in the active site. The metallo-chaperone UreE facilitates in vivo Ni2+ insertion into the apoenzyme. Crystals of apo-HpUreE (H. pylori UreE) and its Ni2+- and Zn2+-bound forms were obtained from protein solutions in the absence and presence of the metal ions. The crystal structures of the homodimeric protein, determined at 2.00 Å (apo), 1.59 Å (Ni2+) and 2.52 Å (Zn2+) resolution, show the conserved proximal and solvent-exposed His102 residues from two adjacent monomers invariably involved in metal binding. The C-terminal regions of the apoprotein are disordered in the crystal, but acquire significant ordering in the presence of the metal ions due to the binding of His152. The analysis of X-ray absorption spectral data obtained using solutions of Ni2+- and Zn2+-bound HpUreE provided accurate information of the metal-ion environment in the absence of solid-state effects. These results reveal the role of the histidine residues at the protein C-terminus in metal-ion binding, and the mutual influence of protein framework and metal-ion stereo-electronic properties in establishing co-ordination number and geometry leading to metal selectivity.

scholarly journals Analysis of the HindIII-catalyzed reaction by time-resolved crystallography

2015 ◽  
Vol 71 (2) ◽  
pp. 256-265 ◽  
Author(s):  
Takashi Kawamura ◽  
Tomoki Kobayashi ◽  
Nobuhisa Watanabe
Keyword(s):  
Electron Density ◽  
Active Site ◽  
Dna Cleavage ◽  
Metal Ion ◽  
Manganese Ions ◽  
Metal Ion Binding ◽  
Time Resolved ◽  
Manganese Ion ◽  
Ion Binding Sites ◽  
Metal Ion Binding Sites

In order to investigate the mechanism of the reaction catalyzed by HindIII, structures of HindIII–DNA complexes with varying durations of soaking time in cryoprotectant buffer containing manganese ions were determined by the freeze-trap method. In the crystal structures of the complexes obtained after soaking for a longer duration, two manganese ions, indicated by relatively higher electron density, are clearly observed at the two metal ion-binding sites in the active site of HindIII. The increase in the electron density of the two metal-ion peaks followed distinct pathways with increasing soaking times, suggesting variation in the binding rate constant for the two metal sites. DNA cleavage is observed when the second manganese ion appears, suggesting that HindIII uses the two-metal-ion mechanism, or alternatively that its reactivity is enhanced by the binding of the second metal ion. In addition, conformational change in a loop near the active site accompanies the catalytic reaction.

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

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.

scholarly journals Structural basis of ion transport and inhibition in ferroportin

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Yaping Pan ◽  
Zhenning Ren ◽  
Shuai Gao ◽  
Jiemin Shen ◽  
Lie Wang ◽  
...  
Keyword(s):  
Binding Sites ◽  
Metal Ion ◽  
Peptide Hormone ◽  
Structural Basis ◽  
Deficiency Anemia ◽  
Pharmacological Intervention ◽  
Ion Binding ◽  
Coupled Transport ◽  
Metal Ion Binding ◽  
Ion Binding Sites

Abstract Ferroportin is an iron exporter essential for releasing cellular iron into circulation. Ferroportin is inhibited by a peptide hormone, hepcidin. In humans, mutations in ferroportin lead to ferroportin diseases that are often associated with accumulation of iron in macrophages and symptoms of iron deficiency anemia. Here we present the structures of the ferroportin from the primate Philippine tarsier (TsFpn) in the presence and absence of hepcidin solved by cryo-electron microscopy. TsFpn is composed of two domains resembling a clamshell and the structure defines two metal ion binding sites, one in each domain. Both structures are in an outward-facing conformation, and hepcidin binds between the two domains and reaches one of the ion binding sites. Functional studies show that TsFpn is an electroneutral H+/Fe2+ antiporter so that transport of each Fe2+ is coupled to transport of two H+ in the opposite direction. Perturbing either of the ion binding sites compromises the coupled transport of H+ and Fe2+. These results establish the structural basis of metal ion binding, transport and inhibition in ferroportin and provide a blueprint for targeting ferroportin in pharmacological intervention of ferroportin diseases.

scholarly journals Specific metal-ion binding sites in a model of the P4-P6 triple-helical domain of a group I intron

RNA ◽  
2001 ◽  
Vol 7 (8) ◽  
pp. 1115-1125 ◽  
Author(s):  
MARTINA LINDQVIST ◽  
KARIN SANDSTRÖM ◽  
VILNIS LIEPINS ◽  
ROGER STRÖMBERG ◽  
ASTRID GRÄSLUND
Keyword(s):  
Binding Sites ◽  
Metal Ion ◽  
Group I Intron ◽  
Ion Binding ◽  
Metal Ion Binding ◽  
Group I ◽  
Ion Binding Sites ◽  
Triple Helical Domain ◽  
Metal Ion Binding Sites ◽  
Specific Metal
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