High-resolution X-ray Structures of Human Apoferritin H-chain Mutants Correlated with Their Activity and Metal-binding Sites

2007 ◽  
Vol 365 (2) ◽  
pp. 440-452 ◽  
Author(s):  
Louise Toussaint ◽  
Luc Bertrand ◽  
Louis Hue ◽  
Robert R. Crichton ◽  
Jean-Paul Declercq
Keyword(s):  
High Resolution ◽  
Metal Binding ◽  
Binding Sites ◽  
X Ray ◽  
Metal Binding Sites

scholarly journals Analyses of Homing Endonucleases and Mechanism of Action of CRISPR-Cas9 HNH Endonucleases

2020 ◽  
pp. 1-25
Author(s):  
Peramachi Palanivelu
Keyword(s):  
Amino Acids ◽  
Mechanism Of Action ◽  
Metal Binding ◽  
Binding Sites ◽  
Proton Acceptor ◽  
Homing Endonucleases ◽  
Multiple Sequence ◽  
Conserved Motifs ◽  
X Ray ◽  
Metal Binding Sites

Aim: To analyze different HNH endonucleases from various sources including the HNH endonuclease regions of CRISPR-Cas9 proteins for their conserved motifs, metal-binding sites and catalytic amino acids and propose a plausible mechanism of action for HNH endonucleases, using CRISPR-Cas9 as the model enzyme. Study Design: Multiple sequence analysis (MSA) of homing endonucleases including the CRISPR-Cas9 using Clustal Omega was studied. Other biochemical, Site-directed mutagenesis (SDM) and X-ray crystallographic data were also analyzed. Place and Duration of Study: School of Biotechnology, Madurai Kamaraj University, Madurai, India, between 2007 and 2013. Methodology: Bioinformatics, Biochemical, SDM and X-ray crystallographic data of the HNH endonucleases from different organisms including CRISPR-Cas9 enzymes were analyzed. The advanced version of Clustal Omega was used for protein sequence analysis of different HNH endonucleases from various sources. The conserved motifs identified by the bioinformatics analysis were analyzed further with the data already available from biochemical and SDM and X-ray crystallographic analyses of this group of enzymes and to confirm the possible amino acids involved in the active sites and catalysis. Results: Different types of homing endonucleases from various sources including the HNH endonuclease regions of CRISPR-Cas9 enzymes exhibit different catalytic regions and metal-binding sites. However, the catalytic amino acid, i.e., the proton acceptor histidine (His), is completely conserved in all homing endonucleases analyzed. From these data, a plausible mechanism of action for HNH endonucleases, using CRISPR-Cas9 from Streptococcus pyogenes, as the model enzyme is proposed. Furthermore, multiple sequence alignment (MSA) of various homing endonucleases from different organisms showed many highly conserved motifs also among them. However, some of the HNH endonucleases showed consensus only around the active site regions. Possible catalytic amino acids identified among them belong to either -DH---N or -HH--N types. There are at least two types of metal-binding sites and bind Mg2+ or Zn2+ or both. The CRISPR-Cas9 enzyme from S. pyogenes belongs to the -DH- based HNH endonucleases and possesses –DxD- type metal-binding site where it possibly binds to a Mg2+ ion. The other HNH enzymes possess one or two invariant Zn binding CxxC/ CxxxC motifs. Conclusions: The CRISPR-Cas9 enzymes are found to be -DH- type where the first D is likely to involve in metal-binding and the second invariant H acts as the proton acceptor and the N in –HNH- Cas9 confers specificity by interacting with the nucleotide near the catalytic region. In this communication, a metal-bound water molecule is shown as the nucleophile initiating catalysis. Homing endonucleases may be used as novel DNA binding and cleaving reagents for a variety of genome editing applications and Zinc finger nucleases have already found applications in genome editing.

Characterisation of Metal Binding Sites for 8-Azaguanine and 8-Azahypoxanthine Derivatives Synthesis and X-Ray Structural Analysis of Methylmercury(II) and Zinc(II) Complexes

1986 ◽  
Vol 41 (9) ◽  
pp. 1117-1122 ◽  
Author(s):  
W. S. Sheldrick ◽  
P. Bell
Keyword(s):  
Structural Analysis ◽  
Binding Site ◽  
Metal Binding ◽  
Binding Sites ◽  
Antineoplastic Agent ◽  
X Ray ◽  
Metal Binding Sites ◽  
Ph Values ◽  
Ph Range

Abstract The complexes [(CH3Hg)AGuaH ] (1) and [(CH3Hg)2AGua] • H2O (2) have been isolated from aqueous 1:1 and 2:1 solutions of CH3HgOH and 8 -azaguanine (AGuaH2) at respective pH values of 5 and 9. Only one CH3Hg+ complex of 8 -azahypoxanthine (AHxH2), namely [(CH3Hg)2AHx] (3), could be isolated under analogous conditions. X-ray structural analyses established N1 and N9 as metal binding sites in 3 and N9 as the coordination position in [Zn(H2O)4(AHxH)2] (4). With 8-aza-9-benzylhypoxanthine (9-BzAHxH) only one CH3Hg+ complex [(CH3Hg)9-BzAHx] (5) could be isolated in the pH range 2-10. N1 was established by X-ray structural analysis as the binding site. The relevance o f these findings to an understanding of ligand behaviour of the antineoplastic agent 8 -azaguanine is discussed.

X-ray structure of fumarylacetoacetate hydrolase family member Homo sapiens FLJ36880

2004 ◽  
Vol 385 (10) ◽  
pp. 935-942 ◽  
Author(s):  
Babu A. Manjasetty ◽  
Frank H. Niesen ◽  
Heinrich Delbrück ◽  
Frank Götz ◽  
Volker Sievert ◽  
...  
Keyword(s):  
Metal Binding ◽  
Binding Sites ◽  
Homo Sapiens ◽  
Point Mutations ◽  
Aromatic Amino Acids ◽  
Type I ◽  
X Ray ◽  
Metal Binding Sites ◽  
Fumarylacetoacetate Hydrolase ◽  
Hydrolase Family

Abstract The human protein FLJ36880 belongs to the fumarylacetoacetate hydrolase family. The X-ray structure of FLJ36880 has been determined to 2.2 Å resolution employing the semi-automated high-throughput structural genomics approach of the Protein Structure Factory. FLJ36880 adopts a mixed β-sandwich roll fold and forms homodimers in crystals as well as in solution. One Mg2+ ion is bound to each subunit of the dimeric protein by coordination to three carboxylate oxygens and three water molecules. These metal binding sites are accessible from the same surface of the dimer, partly due to the disorder of the undecapeptide stretch D29 to L39. The overall structure and metal binding site of FLJ36880 bear clear similarities to the C-terminal domain of the bifunctional enzyme HpcE from Escherichia coli C, fumarylacetoacetate hydrolase from Mus musculus and to YcgM (Apc5008) from E. coli 1262. These similarities provide a framework for suggesting biochemical functions and evolutionary relationships of FLJ36880. It appears highly probable that the metal binding sites are involved in an enzymatic activity related to the catabolism of aromatic amino acids. Two point mutations in the active-site of FAH, responsible for the metabolic disease hereditary tyrosinemia type I (HTI) in humans, affect residues that are structurally conserved in FLJ36880 and located in the putative catalytic site.

scholarly journals Studies on Synthetic and Natural Melanin and Its Affinity for Fe(III) Ion

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
T. G. Costa ◽  
R. Younger ◽  
C. Poe ◽  
P. J. Farmer ◽  
B. Szpoganicz
Keyword(s):  
Metal Binding ◽  
Binding Sites ◽  
Quinone Imine ◽  
Scanning Electronic Microscopy ◽  
Electronic Microscopy ◽  
X Ray ◽  
Metal Binding Sites ◽  
Ph Values ◽  
Eds Analysis ◽  
New Interactions

In this work, we measured the metal-binding sites of natural and synthetic dihydroxyindole (DHI) melanins and their respective interactions with Fe(III) ions. Besides the two acid groups detected for the DHI system: catechol (Cat) and quinone-imine (QI), acetate groups were detected in the natural oligomer by potentiometric titrations. At acidic pH values, Fe(III) complexation with synthetic melanin was detected in an Fe(OH)(CatH2Cat) interaction. With an increase of pH, three new interactions occurred: dihydroxide diprotonated catechol, Fe(OH)2(CatH2Cat)−, dihydroxide monoprotonated catechol, [Fe(OH)2(CatHCat)]2−, and an interaction resulting from the association of one quinone-imine and a catechol group, [Fe(OH)2(Qi−)(CatHCat)]3−. In the natural melanin system, we detected the same interactions involving catechol and quinone-imine groups but also the metal interacts with acetate group at pH values lower than 4.0. Furthermore, interactions in the synthetic system were also characterized by infrared spectroscopy by using the characteristic vibrations of catechol and quinone-imine groups. Finally, scanning electronic microscopy (SEM) and energy-dispersive X-ray (EDS) analysis were used to examine the differences in morphology of these two systems in the absence and presence of Fe(III) ions. The mole ratio of metal and donor atoms was obtained by the EDS analysis.

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