Conserved amino acids in metal-binding motifs of PDE3A are involved in substrate and inhibitor binding

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3380-3386 ◽  
Author(s):  
Wei Zhang ◽  
Robert W. Colman
Keyword(s):  
Amino Acids ◽  
Metal Binding ◽  
Binding Sites ◽  
Metal Ion ◽  
Divalent Cations ◽  
Binding Motif ◽  
Inhibitor Binding ◽  
Binding Motifs ◽  
Mutant Proteins ◽  
Conserved Amino Acids

Abstract The activity of phosphodiesterase (PDE)3A requires divalent cations. Putative metal-binding sites are expected at 2 highly conserved metal-binding motifs, HXXXH(X)25E. A functional truncated recombinant PDE3A containing the catalytic domain (PDE3A▵1) and mutant proteins were expressed in a baculovirus/Sf9 cell system. All the mutant proteins had decreased catalytic efficiency (kcat/Km). Mutants H752A, H756A, and E825A had kcat of less than 0.0008 s−1 to 0.0475 s−1 compared to PDE3A▵1, with 1.86 second−1, with unchanged Km. Although E866A had a kcat of 0.235 s−1, the Kmfor cyclic adenosine monophosphate (cAMP) was increased 11-fold and the Ki for cyclic guanosine monophosphate (cGMP) was 27-fold higher than PDE3A▵1. The Ki of H836A for cGMP was 177-fold higher than that of PDE3A▵1. The Km for E971A was 5-fold higher than PDE3A▵1. These results suggest that the cAMP and cGMP binding sites are overlapping, but not identical, involving both common and different amino acids. Mutants E825A, H836A, and E866A showed low activity in a metal ion-free assay; however, their enzymatic activities were increased 4- to 10-fold in buffers containing Mn2+, Mg2+, or Co2+. This observation indicates that conserved amino acids in the second metal-binding motif might not be involved in binding divalent cations but may serve other functions such as substrate or inhibitor binding in PDE3A.

Conserved amino acids in metal-binding motifs of PDE3A are involved in substrate and inhibitor binding

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3380-3386 ◽  
Author(s):  
Wei Zhang ◽  
Robert W. Colman
Keyword(s):  
Amino Acids ◽  
Metal Binding ◽  
Binding Sites ◽  
Metal Ion ◽  
Divalent Cations ◽  
Binding Motif ◽  
Inhibitor Binding ◽  
Binding Motifs ◽  
Mutant Proteins ◽  
Conserved Amino Acids

The activity of phosphodiesterase (PDE)3A requires divalent cations. Putative metal-binding sites are expected at 2 highly conserved metal-binding motifs, HXXXH(X)25E. A functional truncated recombinant PDE3A containing the catalytic domain (PDE3A▵1) and mutant proteins were expressed in a baculovirus/Sf9 cell system. All the mutant proteins had decreased catalytic efficiency (kcat/Km). Mutants H752A, H756A, and E825A had kcat of less than 0.0008 s−1 to 0.0475 s−1 compared to PDE3A▵1, with 1.86 second−1, with unchanged Km. Although E866A had a kcat of 0.235 s−1, the Kmfor cyclic adenosine monophosphate (cAMP) was increased 11-fold and the Ki for cyclic guanosine monophosphate (cGMP) was 27-fold higher than PDE3A▵1. The Ki of H836A for cGMP was 177-fold higher than that of PDE3A▵1. The Km for E971A was 5-fold higher than PDE3A▵1. These results suggest that the cAMP and cGMP binding sites are overlapping, but not identical, involving both common and different amino acids. Mutants E825A, H836A, and E866A showed low activity in a metal ion-free assay; however, their enzymatic activities were increased 4- to 10-fold in buffers containing Mn2+, Mg2+, or Co2+. This observation indicates that conserved amino acids in the second metal-binding motif might not be involved in binding divalent cations but may serve other functions such as substrate or inhibitor binding in PDE3A.

scholarly journals Exploring the structure and function of Thermotoga maritima CorA reveals the mechanism of gating and ion selectivity in Co2+/Mg2+ transport

2013 ◽  
Vol 451 (3) ◽  
pp. 365-374 ◽  
Author(s):  
Nurhuda Nordin ◽  
Albert Guskov ◽  
Terri Phua ◽  
Newsha Sahaf ◽  
Yu Xia ◽  
...  
Keyword(s):  
Metal Binding ◽  
Binding Sites ◽  
Metal Ion ◽  
Thermotoga Maritima ◽  
Ion Selectivity ◽  
Metal Binding Sites ◽  
Gating Mechanism ◽  
Structural Details ◽  
And Function ◽  
Conserved Amino Acids

The CorA family of divalent cation transporters utilizes Mg2+ and Co2+ as primary substrates. The molecular mechanism of its function, including ion selectivity and gating, has not been fully characterized. Recently we reported a new structure of a CorA homologue from Methanocaldococcus jannaschii, which provided novel structural details that offered the conception of a unique gating mechanism involving conversion of an open hydrophilic gate into a closed hydrophobic one. In the present study we report functional evidence for this novel gating mechanism in the Thermotoga maritima CorA together with an improved crystal structure of this CorA to 2.7 Å (1 Å=0.1 nm) resolution. The latter reveals the organization of the selectivity filter to be similar to that of M. jannaschii CorA and also the previously unknown organization of the second signature motif of the CorA family. The proposed gating is achieved by a helical rotation upon the binding of a metal ion substrate to the regulatory binding sites. Additionally, our data suggest that the preference of this CorA for Co2+ over Mg2+ is controlled by the presence of threonine side chains in the channel. Finally, the roles of the intracellular metal-binding sites have been assigned to increased thermostability and regulation of the gating. These mechanisms most likely apply to the entire CorA family as they are regulated by the highly conserved amino acids.

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.

scholarly journals Metal ion recognition of the metal-binding motif in hammerhead ribozymes

2003 ◽  
Vol 43 (supplement) ◽  
pp. S28
Author(s):  
Y. Tanaka ◽  
Y. Kasai ◽  
C. Kojima ◽  
K. Yamasaki ◽  
H. Morita ◽  
...  
Keyword(s):  
Metal Binding ◽  
Metal Ion ◽  
Binding Motif ◽  
Hammerhead Ribozymes ◽  
Ion Recognition ◽  
Metal Binding Motif

scholarly journals Investigation of the nature of the two metal-binding sites in 5-aminolaevulinic acid dehydratase from Escherichia coli

1994 ◽  
Vol 300 (2) ◽  
pp. 373-381 ◽  
Author(s):  
P Spencer ◽  
P M Jordan
Keyword(s):  
Escherichia Coli ◽  
Metal Ions ◽  
Metal Binding ◽  
Binding Sites ◽  
Metal Ion ◽  
Protein Fluorescence ◽  
Metal Binding Sites ◽  
Aminolaevulinic Acid ◽  
Acid Dehydratase ◽  
Aminolaevulinic Acid Dehydratase

Two distinct metal-binding sites, termed alpha and beta, have been characterized in 5-aminolaevulinic acid dehydratase from Escherichia coli. The alpha-site binds a Zn2+ ion that is essential for catalytic activity. This site can also utilize other metal ions able to function as a Lewis acid in the reaction mechanism, such as Mg2+ or Co2+. The beta-site is exclusively a transition-metal-ion-binding site thought to be involved in protein conformation, although a metal bound at this site only appears to be essential for activity if Mg2+ is to be bound at the alpha-site. The alpha- and beta-sites may be distinguished from one another by their different abilities to bind divalent-metal ions at different pH values. The occupancy of the beta-site with Zn2+ results in a decrease of protein fluorescence at pH 6. Occupancy of the alpha- and beta-sites with Co2+ results in u.v.-visible spectral changes. Spectroscopic studies with Co2+ have tentatively identified three cysteine residues at the beta-site and one at the alpha-site. Reaction with N-ethyl[14C]maleimide preferentially labels cysteine-130 at the alpha-site when Co2+ occupies the beta-site.

scholarly journals Computational approaches for de novo design and redesign of metal-binding sites on proteins

2017 ◽  
Vol 37 (2) ◽  
Author(s):  
Gunseli Bayram Akcapinar ◽  
Osman Ugur Sezerman
Keyword(s):  
Metal Ions ◽  
Metal Binding ◽  
Binding Sites ◽  
Metal Ion ◽  
De Novo ◽  
De Novo Design ◽  
Ion Binding ◽  
Chemical Transformations ◽  
Metal Ion Binding ◽  
Metal Binding Sites

Metal ions play pivotal roles in protein structure, function and stability. The functional and structural diversity of proteins in nature expanded with the incorporation of metal ions or clusters in proteins. Approximately one-third of these proteins in the databases contain metal ions. Many biological and chemical processes in nature involve metal ion-binding proteins, aka metalloproteins. Many cellular reactions that underpin life require metalloproteins. Most of the remarkable, complex chemical transformations are catalysed by metalloenzymes. Realization of the importance of metal-binding sites in a variety of cellular events led to the advancement of various computational methods for their prediction and characterization. Furthermore, as structural and functional knowledgebase about metalloproteins is expanding with advances in computational and experimental fields, the focus of the research is now shifting towards de novo design and redesign of metalloproteins to extend nature’s own diversity beyond its limits. In this review, we will focus on the computational toolbox for prediction of metal ion-binding sites, de novo metalloprotein design and redesign. We will also give examples of tailor-made artificial metalloproteins designed with the computational toolbox.

scholarly journals Specificity and affinity of binding of phosphate-containing compounds to CheY protein

1992 ◽  
Vol 287 (2) ◽  
pp. 533-543 ◽  
Author(s):  
L Kar ◽  
P Z De Croos ◽  
S J Roman ◽  
P Matsumura ◽  
M E Johnson
Keyword(s):  
Metal Binding ◽  
Binding Sites ◽  
Metal Ion ◽  
Bacterial Chemotaxis ◽  
Phosphate Binding ◽  
Metal Binding Sites ◽  
Low Concentrations ◽  
Bivalent Metal Ions ◽  
Proton Resonances ◽  
Inorganic Phosphates

1H- and 31P-n.m.r. have been used to study the interaction of the bacterial chemotaxis protein, CheY, with ATP and a variety of other phosphates in the presence and absence of bivalent metal ions. In the metal-bound conformation, CheY will bind nucleotide phosphates and phosphates in general, while in the metal-free conformation CheY loses its affinity for phosphates. In the presence of low concentrations of nitroxide-spin-labelled ATP (SL-ATP), specific proton resonances of metal-bound CheY are suppressed, indicating that ATP binds to a specific site on this metal-bound form of the protein. These studies also show that the same resonances are affected by the binding of SL-ATP and Mn2+, indicating that the phosphate- and metal-binding sites are close to each other and to Asp-57 (the site of phosphorylation in CheY). 1H- and 31P-n.m.r. studies using ATP, GTP, TTP, UTP, ADP, AMP and inorganic phosphates show that the binding is not specific for adenine, and does not involve the base directly, but is mediated primarily by the phosphate groups. Experiments with a phosphorylation mutant (Asp-13-->Asn) suggest that the observed phosphate binding and activation of CheY by phosphorylation may be related. Our results indicate that the conformational change and charge interactions brought about by the binding of a metal ion at the active site are required for CheY to interact with a phosphate. These studies also demonstrate the utility of spin-label-induced relaxation in conjunction with two-dimensional-n.m.r. measurements for exploring ligand-binding sites.

Probing Metal Ion Complexation of Ligands with Multiple Metal Binding Sites: The Case of Spiropyrans

2016 ◽  
Vol 22 (39) ◽  
pp. 13976-13984 ◽  
Author(s):  
Michele Baldrighi ◽  
Giulia Locatelli ◽  
John Desper ◽  
Christer B. Aakeröy ◽  
Silvia Giordani
Keyword(s):  
Metal Binding ◽  
Binding Sites ◽  
Metal Ion ◽  
Metal Ion Complexation ◽  
Metal Binding Sites ◽  
Ion Complexation

scholarly journals Unravelling the Structure of the Tetrahedral Metal-Binding Site in METP3 through an Experimental and Computational Approach

Molecules ◽  
2021 ◽  
Vol 26 (17) ◽  
pp. 5221
Author(s):  
Salvatore La Gatta ◽  
Linda Leone ◽  
Ornella Maglio ◽  
Maria De Fenza ◽  
Flavia Nastri ◽  
...  
Keyword(s):  
Metal Ions ◽  
Binding Site ◽  
Metal Binding ◽  
Binding Sites ◽  
Metal Ion ◽  
Structural Determinants ◽  
Binding Properties ◽  
Tetrahedral Geometry ◽  
Design Synthesis ◽  
Metal Binding Sites

Understanding the structural determinants for metal ion coordination in metalloproteins is a fundamental issue for designing metal binding sites with predetermined geometry and activity. In order to achieve this, we report in this paper the design, synthesis and metal binding properties of METP3, a homodimer made up of a small peptide, which self assembles in the presence of tetrahedrally coordinating metal ions. METP3 was obtained through a redesign approach, starting from the previously developed METP molecule. The undecapeptide sequence of METP, which dimerizes to house a Cys4 tetrahedral binding site, was redesigned in order to accommodate a Cys2His2 site. The binding properties of METP3 were determined toward different metal ions. Successful assembly of METP3 with Co(II), Zn(II) and Cd(II), in the expected 2:1 stoichiometry and tetrahedral geometry was proven by UV-visible spectroscopy. CD measurements on both the free and metal-bound forms revealed that the metal coordination drives the peptide chain to fold into a turned conformation. Finally, NMR data of the Zn(II)-METP3 complex, together with a retrostructural analysis of the Cys-X-X-His motif in metalloproteins, allowed us to define the model structure. All the results establish the suitability of the short METP sequence for accommodating tetrahedral metal binding sites, regardless of the first coordination ligands.

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