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.

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.

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.

Multicomponent Kinetic Analysis of Trace Metal Binding Sites on Iron Hydrous Oxide Colloids

1988 ◽  
Vol 23 (3) ◽  
pp. 379-387 ◽  
Author(s):  
Donald W. Gutzman ◽  
Cooper H. Langford
Keyword(s):  
Metal Binding ◽  
Binding Sites ◽  
Metal Ion ◽  
Base Hydrolysis ◽  
Diffusion Control ◽  
Hydrous Oxide ◽  
Ion Release ◽  
Metal Binding Sites ◽  
Metal Ion Release ◽  
Colloidal Species

Abstract The adsorption of copper(II) on a hydrous oxide colloid of iron formed by base hydrolysis in the presence of the adsorbate metal has been studied in two ways. Filtration-AAS of equilibrated solutions yielded data which fit well to a single variant adsorption isotherm even at high adsorbate metal concentration thus precluding the need for multivariant fitting. These same colloidal species were kinetically analyzed by reaction of copper with the chromophore reagent EHTTT (a = 13700 Mࢤ1 cmࢤ1 at 434 nm) which revealed the presence of three components, other than the very labile “free” species. Kinetic differentiation of thermodynamically similar sites may result from diffusion control of metal ion release.

Metal Ion Binding Properties of Perfluorosulfonate Membranes by Laser Excitation Spectroscopy

1988 ◽  
Vol 42 (2) ◽  
pp. 293-295 ◽  
Author(s):  
E. K. L. Wong ◽  
G. L. Richmond
Keyword(s):  
Metal Binding ◽  
Binding Sites ◽  
Metal Ion ◽  
Laser Excitation ◽  
Ion Binding ◽  
Metal Ion Binding ◽  
Binding Properties ◽  
Metal Binding Sites ◽  
Fluorescence Measurements ◽  
Experimental Parameters

The metal ion binding properties of the perfluorosulfonate membrane Nafion® have been investigated in this study. The experiments involve laser-induced fluorescence measurements of europium (III) ions which are bound to the membrane. By the exploitation of the hypersensitivity of the D → F transitions of europium (III) to the ligand binding environment, the properties of the metal binding sites have been analyzed as a function of various experimental parameters. The spectra and fluorescence lifetime measurements provide evidence for distinct metal binding sites within the polymer, each of which is sensitive to the conditions of the membrane preparation.

scholarly journals The structures of the CutA1 proteins fromThermus thermophilusandPyrococcus horikoshii: characterization of metal-binding sites and metal-induced assembly

2014 ◽  
Vol 70 (4) ◽  
pp. 404-413 ◽  
Author(s):  
Bagautdin Bagautdinov
Keyword(s):  
Metal Binding ◽  
Binding Sites ◽  
Metal Ion ◽  
Metal Tolerance ◽  
Structural Information ◽  
Acetylcholinesterase Activity ◽  
Thermophilic Bacterium ◽  
Copper Tolerance ◽  
Binding Modes ◽  
Metal Binding Sites

CutA1 (copper tolerance A1) is a widespread cytoplasmic protein found in archaea, bacteria, plants and animals, including humans. InEscherichia coliit is implicated in divalent metal tolerance, while the mammalian CutA1 homologue has been proposed to mediate brain enzyme acetylcholinesterase activity and copper homeostasis. The X-ray structures of CutA1 from the thermophilic bacteriumThermus thermophilus(TtCutA1) with and without bound Na+at 1.7 and 1.9 Å resolution, respectively, and from the hyperthermophilic archaeonPyrococcus horikoshii(PhCutA1) in complex with Na+at 1.8 Å resolution have been determined. Both are short and rigid proteins of about 12 kDa that form intertwined compact trimers in the crystal and solution. The main difference in the structures is a wide-type β-bulge on top of theTtCutA1 trimer. It affords a mechanism for lodging a single-residue insertion in the middle of β2while preserving the interprotomer main-chain hydrogen-bonding network. The liganded forms of the proteins provide new structural information about the metal-binding sites and CutA1 assembly. The Na+–TtCutA1 structure unveils a dodecameric assembly with metal ions in the trimer–trimer interfaces and the lateral clefts of the trimer. For Na+–PhCutA1, the metal ion associated with six waters in an octahedral geometry. The structures suggest that CutA1 may contribute to regulating intracellular metal homeostasis through various binding modes.

scholarly journals Check your metal - not every density blob is a water molecule

2014 ◽  
Vol 70 (a1) ◽  
pp. C1483-C1483
Author(s):  
Heping Zheng ◽  
Mahendra Chordia ◽  
David Cooper ◽  
Ivan Shabalin ◽  
Maksymilian Chruszcz ◽  
...  
Keyword(s):  
Coordination Chemistry ◽  
Metal Binding ◽  
Binding Sites ◽  
Metal Ion ◽  
Protein Structures ◽  
Data Bank ◽  
Careful Examination ◽  
Biological Macromolecules ◽  
Metal Binding Sites ◽  
Heterogeneous Chemical

Metals play vital roles in both the mechanism and architecture of biological macromolecules, and are the most frequently encountered ligands (i.e. non-solvent heterogeneous chemical atoms) in the determination of macromolecular crystal structures. However, metal coordinating environments in protein structures are not always easy to check in routine validation procedures, resulting in an abundance of misidentified and/or suboptimally modeled metal ions in the Protein Data Bank (PDB). We present a solution to identify these problems in three distinct yet related aspects: (1) coordination chemistry; (2) agreement of experimental B-factors and occupancy; and (3) the composition and motif of the metal binding environment. Due to additional strain introduced by macromolecular backbones, the patterns of coordination of metal binding sites in metal-containing macromolecules are more complex and diverse than those found in inorganic or organometallic chemistry. These complications make a comprehensive library of "permitted" coordination chemistry in protein structures less feasible, and the usage of global parameters such as the bond valence method more practical, in the determination and validation of metal binding environments. Although they are relatively infrequent, there are also cases where the experimental B-factor or occupancy of a metal ion suggests careful examination. We have developed a web-based tool called CheckMyMetal [1](http://csgid.org/csgid/metal_sites/) for the quick validation of metal binding sites. Moreover, the acquired knowledge of the composition and spatial arrangement (motif) of the coordinating atoms around the metal ion may also help in the modeling of metal binding sites in macromolecular structures. All of the studies described herein were performed using the NEIGHBORHOOD SQL database [2], which connects information about all modeled non-solvent heterogeneous chemical motifs in PDB structure by vectors describing all contacts to neighboring residues and atoms. NEIGHBORHOOD has broad applications for the validation and data mining of ligand binding environments in the PDB.

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.

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