scholarly journals The Structural Basis of the Binding of Various Aminopolycarboxylates by the Periplasmic EDTA-Binding Protein EppA from Chelativorans sp. BNC1

2020 ◽  
Vol 21 (11) ◽  
pp. 3940 ◽  
Author(s):  
Kevin M. Lewis ◽  
Chelsie L. Greene ◽  
Steven A. Sattler ◽  
Buhyun Youn ◽  
Luying Xun ◽  
...  
Keyword(s):  
Binding Protein ◽  
Metal Chelate ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Titration Calorimetry ◽  
Bacterial Cells ◽  
Chelate Complexes ◽  
Quantum Chemical Analysis ◽  
Ligand Selectivity

The widespread use of synthetic aminopolycarboxylates, such as ethylenediaminetetraacetate (EDTA), as chelating agents has led to their contamination in the environment as stable metal–chelate complexes. Microorganisms can transport free EDTA, but not metal–EDTA complexes, into cells for metabolism. An ABC-type transporter for free EDTA uptake in Chelativorans sp. BNC1 was investigated to understand the mechanism of the ligand selectivity. We solved the X-ray crystal structure of the periplasmic EDTA-binding protein (EppA) and analyzed its structure–function relations through isothermal titration calorimetry, site-directed mutagenesis, molecular docking, and quantum chemical analysis. EppA had high affinities for EDTA and other aminopolycarboxylates, which agrees with structural analysis, showing that its binding pocket could accommodate free aminopolycarboxylates. Further, key amino acid residues involved in the binding were identified. Our results suggest that EppA is a general binding protein for the uptake of free aminopolycarboxylates. This finding suggests that bacterial cells import free aminopolycarboxylates, explaining why stable metal–chelate complexes are resistant to degradation, as they are not transported into the cells for degradation.

scholarly journals Characterization of the High-Affinity Drug Ligand Binding Site of Mouse Recombinant TSPO

2019 ◽  
Vol 20 (6) ◽  
pp. 1444 ◽  
Author(s):  
Soria Iatmanen-Harbi ◽  
lucile Senicourt ◽  
Vassilios Papadopoulos ◽  
Olivier Lequin ◽  
Jean-Jacques Lacapere
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Conformational Changes ◽  
Protoporphyrin Ix ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Translocator Protein ◽  
Binding Affinities ◽  
Ligand Selectivity ◽  
High Affinity

The optimization of translocator protein (TSPO) ligands for Positron Emission Tomography as well as for the modulation of neurosteroids is a critical necessity for the development of TSPO-based diagnostics and therapeutics of neuropsychiatrics and neurodegenerative disorders. Structural hints on the interaction site and ligand binding mechanism are essential for the development of efficient TSPO ligands. Recently published atomic structures of recombinant mammalian and bacterial TSPO1, bound with either the high-affinity drug ligand PK 11195 or protoporphyrin IX, have revealed the membrane protein topology and the ligand binding pocket. The ligand is surrounded by amino acids from the five transmembrane helices as well as the cytosolic loops. However, the precise mechanism of ligand binding remains unknown. Previous biochemical studies had suggested that ligand selectivity and binding was governed by these loops. We performed site-directed mutagenesis to further test this hypothesis and measured the binding affinities. We show that aromatic residues (Y34 and F100) from the cytosolic loops contribute to PK 11195 access to its binding site. Limited proteolytic digestion, circular dichroism and solution two-dimensional (2-D) NMR using selective amino acid labelling provide information on the intramolecular flexibility and conformational changes in the TSPO structure upon PK 11195 binding. We also discuss the differences in the PK 11195 binding affinities and the primary structure between TSPO (TSPO1) and its paralogous gene product TSPO2.

scholarly journals Structural Basis of Ubiquitin Recognition by the Ubiquitin-associated (UBA) Domain of the Ubiquitin Ligase EDD

2007 ◽  
Vol 282 (49) ◽  
pp. 35787-35795 ◽  
Author(s):  
Guennadi Kozlov ◽  
Long Nguyen ◽  
Tong Lin ◽  
Gregory De Crescenzo ◽  
Morag Park ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Titration Calorimetry ◽  
Polyubiquitin Chains ◽  
C Terminus ◽  
Damage Repair ◽  
The Family ◽  
Uba Domain ◽  
Ordered Water

EDD (or HYD) is an E3 ubiquitin ligase in the family of HECT (homologous to E6-AP C terminus) ligases. EDD contains an N-terminal ubiquitin-associated (UBA) domain, which is present in a variety of proteins involved in ubiquitin-mediated processes. Here, we use isothermal titration calorimetry (ITC), NMR titrations, and pull-down assays to show that the EDD UBA domain binds ubiquitin. The 1.85Å crystal structure of the complex with ubiquitin reveals the structural basis of ubiquitin recognition by UBA helices α1 and α3. The structure shows a larger number of intermolecular hydrogen bonds than observed in previous UBA/ubiquitin complexes. Two of these involve ordered water molecules. The functional importance of residues at the UBA/ubiquitin interface was confirmed using site-directed mutagenesis. Surface plasmon resonance (SPR) measurements show that the EDD UBA domain does not have a strong preference for polyubiquitin chains over monoubiquitin. This suggests that EDD binds to monoubiquitinated proteins, which is consistent with its involvement in DNA damage repair pathways.

Structures of the substrate-binding protein provide insights into the multiple compatible solute binding specificities of the Bacillus subtilis ABC transporter OpuC

2011 ◽  
Vol 436 (2) ◽  
pp. 283-289 ◽  
Author(s):  
Yang Du ◽  
Wei-Wei Shi ◽  
Yong-Xing He ◽  
Yi-Hu Yang ◽  
Cong-Zhao Zhou ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Abc Transporter ◽  
Binding Protein ◽  
Substrate Binding ◽  
Compatible Solutes ◽  
Binding Pocket ◽  
Compatible Solute ◽  
Structural Basis ◽  
Binding Property ◽  
Substrate Binding Protein

The compatible solute ABC (ATP-binding cassette) transporters are indispensable for acquiring a variety of compatible solutes under osmotic stress in Bacillus subtilis. The substrate-binding protein OpuCC (Opu is osmoprotectant uptake) of the ABC transporter OpuC can recognize a broad spectrum of compatible solutes, compared with its 70% sequence-identical paralogue OpuBC that can solely bind choline. To explore the structural basis of this difference of substrate specificity, we determined crystal structures of OpuCC in the apo-form and in complex with carnitine, glycine betaine, choline and ectoine respectively. OpuCC is composed of two α/β/α globular sandwich domains linked by two hinge regions, with a substrate-binding pocket located at the interdomain cleft. Upon substrate binding, the two domains shift towards each other to trap the substrate. Comparative structural analysis revealed a plastic pocket that fits various compatible solutes, which attributes the multiple-substrate binding property to OpuCC. This plasticity is a gain-of-function via a single-residue mutation of Thr94 in OpuCC compared with Asp96 in OpuBC.

scholarly journals Transferrin-Binding Protein B of Neisseria meningitidis: Sequence-Based Identification of the Transferrin-Binding Site Confirmed by Site-Directed Mutagenesis

2004 ◽  
Vol 186 (3) ◽  
pp. 850-857 ◽  
Author(s):  
Geneviève Renauld-Mongénie ◽  
Laurence Lins ◽  
Tino Krell ◽  
Laure Laffly ◽  
Michèle Mignon ◽  
...  
Keyword(s):  
Neisseria Meningitidis ◽  
Binding Protein ◽  
Prediction Method ◽  
Binding Activity ◽  
Site Directed Mutagenesis ◽  
Titration Calorimetry ◽  
Directed Mutagenesis ◽  
Binding Domains ◽  
Homologous Strain ◽  
Protein B

ABSTRACT A sequence-based prediction method was employed to identify three ligand-binding domains in transferrin-binding protein B (TbpB) of Neisseria meningitidis strain B16B6. Site-directed mutagenesis of residues located in these domains has led to the identification of two domains, amino acids 53 to 57 and 240 to 245, which are involved in binding to human transferrin (htf). These two domains are conserved in an alignment of different TbpB sequences from N. meningitidis and Neisseria gonorrhoeae, indicating a general functional role of the domains. Western blot analysis and BIAcore and isothermal titration calorimetry experiments demonstrated that site-directed mutations in both binding domains led to a decrease or abolition of htf binding. Analysis of mutated proteins by circular dichroism did not provide any evidence for structural alterations due to the amino acid replacements. The TbpB mutant R243N was devoid of any htf-binding activity, and antibodies elicited by the mutant showed strong bactericidal activity against the homologous strain, as well as against several heterologous tbpB isotype I strains.

scholarly journals Structural basis of terephthalate recognition by solute binding protein TphC

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Trishnamoni Gautom ◽  
Dharmendra Dheeman ◽  
Colin Levy ◽  
Thomas Butterfield ◽  
Guadalupe Alvarez Gonzalez ◽  
...  
Keyword(s):  
Genetic Resource ◽  
Binding Protein ◽  
Structural Basis ◽  
Bacterial Cells ◽  
Ligand Specificity ◽  
Biological Degradation ◽  
Genomic Context ◽  
Context Analysis ◽  
Structural Characterisation ◽  
Genomic Context Analysis

AbstractBiological degradation of Polyethylene terephthalate (PET) plastic and assimilation of the corresponding monomers ethylene glycol and terephthalate (TPA) into central metabolism offers an attractive route for bio-based molecular recycling and bioremediation applications. A key step is the cellular uptake of the non-permeable TPA into bacterial cells which has been shown to be dependent upon the presence of the key tphC gene. However, little is known from a biochemical and structural perspective about the encoded solute binding protein, TphC. Here, we report the biochemical and structural characterisation of TphC in both open and TPA-bound closed conformations. This analysis demonstrates the narrow ligand specificity of TphC towards aromatic para-substituted dicarboxylates, such as TPA and closely related analogues. Further phylogenetic and genomic context analysis of the tph genes reveals homologous operons as a genetic resource for future biotechnological and metabolic engineering efforts towards circular plastic bio-economy solutions.

scholarly journals Determinants of nucleoside specificity of a macrolide phosphotransferase

2014 ◽  
Vol 70 (a1) ◽  
pp. C705-C705
Author(s):  
Desiree Fong ◽  
Jonathan Blanchet ◽  
Albert Berghuis
Keyword(s):  
Ternary Complex ◽  
Binding Pocket ◽  
Structural Basis ◽  
Titration Calorimetry ◽  
Nucleoside Triphosphate ◽  
Type I ◽  
Purine Nucleotides ◽  
Phosphate Donor ◽  
Aminoglycoside Phosphotransferases ◽  
Complex Forms

2'-macrolide phosphotransferase type I [MPH(2')-I] is an antibiotic kinase that renders many macrolides, such as erythromycin, inactive by catalyzing the transfer of a phosphate group from a nucleoside triphosphate to the hydroxyl at the 2'-position of the antibiotic. MPH(2')-I is functionally and structurally analogous to the aminoglycoside kinases (APHs). However, it is distinct from most APHs in that it utilizes GTP exclusively as its phosphate donor. We will present the crystal structure of MPH(2')-I in its apo and ternary complex forms with guanosine nucleotide and different macrolide substrates. We will compare its nucleoside-binding pocket to that of the 2''-aminoglycoside phosphotransferases [APH(2'')], a subclass of aminoglycoside kinases that are capable of utilizing GTP as a phosphate donor. To further decipher the structural basis of the nucleoside specificity of MPH(2')-I, mutations of amino acid resides in the nucleoside-binding pocket have been carried out and their effects on the binding affinity of purine nucleotides were examined by isothermal titration calorimetry. Our preliminary results show that the "gatekeeper" residue plays a role in governing the nucleoside selectivity.

scholarly journals Molecular Determinants of the Cofactor Specificity of Ribitol Dehydrogenase, a Short-Chain Dehydrogenase/Reductase

2012 ◽  
Vol 78 (9) ◽  
pp. 3079-3086 ◽  
Author(s):  
Hee-Jung Moon ◽  
Manish Kumar Tiwari ◽  
Ranjitha Singh ◽  
Yun Chan Kang ◽  
Jung-Kul Lee
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Titration Calorimetry ◽  
Content Type ◽  
Cofactor Specificity ◽  
Charged Amino Acids ◽  
Molecular Determinants ◽  
Ribitol Dehydrogenase

ABSTRACTRibitol dehydrogenase fromZymomonas mobilis(ZmRDH) catalyzes the conversion of ribitol tod-ribulose and concomitantly reduces NAD(P)+to NAD(P)H. A systematic approach involving an initial sequence alignment-based residue screening, followed by a homology model-based screening and site-directed mutagenesis of the screened residues, was used to study the molecular determinants of the cofactor specificity of ZmRDH. A homologous conserved amino acid, Ser156, in the substrate-binding pocket of the wild-type ZmRDH was identified as an important residue affecting the cofactor specificity of ZmRDH. Further insights into the function of the Ser156 residue were obtained by substituting it with other hydrophobic nonpolar or polar amino acids. Substituting Ser156 with the negatively charged amino acids (Asp and Glu) altered the cofactor specificity of ZmRDH toward NAD+(S156D, [kcat/Km,NAD]/[kcat/Km,NADP] = 10.9, whereKm,NADis theKmfor NAD+andKm,NADPis theKmfor NADP+). In contrast, the mutants containing positively charged amino acids (His, Lys, or Arg) at position 156 showed a higher efficiency with NADP+as the cofactor (S156H, [kcat/Km,NAD]/[kcat/Km,NADP] = 0.11). These data, in addition to those of molecular dynamics and isothermal titration calorimetry studies, suggest that the cofactor specificity of ZmRDH can be modulated by manipulating the amino acid residue at position 156.

Critical residues for ligand binding in blade 2 of the propeller domain of the integrin αIIb subunit

2004 ◽  
Vol 91 (01) ◽  
pp. 111-118 ◽  
Author(s):  
Tatsushiro Tamura ◽  
Jun Yamanouchi ◽  
Shigeru Fujita ◽  
Takaaki Hato
Keyword(s):  
Crystal Structure ◽  
Ligand Binding ◽  
Binding Site ◽  
Thrombus Formation ◽  
Direct Interaction ◽  
Binding Pocket ◽  
Crystal Structure Analysis ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Critical Residues

SummaryLigand binding to integrin αIIbβ3 is a key event of thrombus formation. The propeller domain of the αIIb subunit has been implicated in ligand binding. Recently, the ligand binding site of the αV propeller was determined by crystal structure analysis. However, the structural basis of ligand recognition by the αIIb propeller remains to be determined. In this study, we conducted site-directed mutagenesis of all residues located in the loops extending above blades 2 and 4 of the αIIb propeller, which are spatially close to, but distinct from, the loops that contain the binding site for an RGD ligand in the crystal structure of the αV propeller. Replacement by alanine of Q111, H112 or N114 in the loop within the blade 2 (the W2:2-3 loop in the propeller model) abolished binding of a ligand-mimetic antibody and fibrinogen to αIIbβ3 induced by different types of integrin activation including activation of αIIbβ3 by β3 cytoplasmic mutation. CHO cells stably expressing recombinant αIIbβ3 bearing Q111A, H112A or N114A mutation did not exhibit αIIbβ3mediated adhesion to fibrinogen. According to the crystal structure of αVβ3, the αV residue corresponding to αIIbN114 is exposed on the integrin surface and close to the RGD binding site. These results suggest that the Q111, H112 and N114 residues in the loop within blade 2 of the αIIb propeller are critical for ligand binding, possibly because of direct interaction with ligands or modulation of the RGD binding pocket.

Probing the structural basis of oxygen binding in a cofactor-independent dioxygenase

2017 ◽  
Vol 73 (7) ◽  
pp. 573-580 ◽  
Author(s):  
Kunhua Li ◽  
Elisha N. Fielding ◽  
Heather L. Condurso ◽  
Steven D. Bruner
Keyword(s):  
Catalytic Mechanism ◽  
Binding Pocket ◽  
Binding Mode ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
General Mechanism ◽  
Oxygen Binding ◽  
Migration Pathway ◽  
And Migration ◽  
Key Residues

The enzyme DpgC is included in the small family of cofactor-independent dioxygenases. The chemistry of DpgC is uncommon as the protein binds and utilizes dioxygen without the aid of a metal or organic cofactor. Previous structural and biochemical studies identified the substrate-binding mode and the components of the active site that are important in the catalytic mechanism. In addition, the results delineated a putative binding pocket and migration pathway for the co-substrate dioxygen. Here, structural biology is utilized, along with site-directed mutagenesis, to probe the assigned dioxygen-binding pocket. The key residues implicated in dioxygen trafficking were studied to probe the process of binding, activation and chemistry. The results support the proposed chemistry and provide insight into the general mechanism of dioxygen binding and activation.

scholarly journals Structural Basis for the Inhibition of the Autophosphorylation Activity of HK853 by Luteolin

Molecules ◽  
2019 ◽  
Vol 24 (5) ◽  
pp. 933 ◽  
Author(s):  
Yuan Zhou ◽  
Liqun Huang ◽  
Shixia Ji ◽  
Shi Hou ◽  
Liang Luo ◽  
...  
Keyword(s):  
Adenosine Diphosphate ◽  
Binding Pocket ◽  
Structural Basis ◽  
Inhibition Mechanism ◽  
Titration Calorimetry ◽  
Two Component System ◽  
Histidine Kinases ◽  
Variable Environments ◽  
Π Stacking Interaction ◽  
Novel Target

The two-component system (TCS) is a significant signal transduction system for bacteria to adapt to complicated and variable environments, and thus has recently been regarded as a novel target for developing antibacterial agents. The natural product luteolin (Lut) can inhibit the autophosphorylation activity of the typical histidine kinase (HK) HK853 from Thermotoga maritime, but the inhibition mechanism is not known. Herein, we report on the binding mechanism of a typical flavone with HK853 by using solution NMR spectroscopy, isothermal titration calorimetry (ITC), and molecular docking. We show that luteolin inhibits the activity of HK853 by occupying the binding pocket of adenosine diphosphate (ADP) through hydrogen bonds and π-π stacking interaction structurally. Our results reveal a detailed mechanism for the inhibition of flavones and observe the conformational and dynamics changes of HK. These results should provide a feasible approach for antibacterial agent design from the view of the histidine kinases.

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