NMR spectral mapping of Lipid A molecular patterns affected by interaction with the innate immune receptor CD14

Soluble CD14 (sCD14) is a serum glycoprotein that binds to the Lipid A moiety of lipopolysaccharides (LPS) with high affinity as part of the innate immune response to bacterial endotoxins. In order to investigate structural interactions of Lipid A with sCD14, we have prepared an isotopically labeled form of a fully active and chemically defined endotoxin, Kdo₂-Lipid A, which allowed us to carry out detailed NMR spectral mapping of this agonist ligand bound to sCD14 and identify for the first time structural regions that are strongly affected during complex formation with sCD14. These map to two adjacent areas comprising the lower portions of the sugar headgroup and upper half of the acyl chains I, III, and V, which are spatially proximal to the 1- and 4′-phosphate ends. Additionally, we have detected for the first time, presence of differential dynamic behavior for the affected resonances, suggesting a likely role for dynamics in the mechanism of Lipid A pattern recognition by sCD14.     

Structural studies on ligand–DNA systems: A robust approach in drug design

Molecular docking, molecular mechanics, molecular dynamics and relaxation matrix simulation protocols have been extensively used to generate the structural details of ligand-receptor complexes in order to understand the binding interactions between the two entities. Experimental methods like NMR spectroscopy and X-ray crystallography are known to provide structural information about ligand-receptor complexes. In addition, fluorescence spectroscopy, circular dichroism (CD) spectroscopy and molecular docking have also been utilized to decode the phenomenon of the ligand-DNA interactions, with good correlation between experimental and computational results. The DNA binding affinity was demonstrated by analysing fluorescence spectral data. Structural rigidity of DNA upon ligand binding was identified by CD spectroscopy. Docking is carried out using the DNA-Dock program which results in the binding affinity data along with structural information like interatomic distances and H-bonding, etc. The complete structural analyses of various drug-DNA complexes have afforded results that indicate a specific DNA binding pattern of these ligands. It also exhibited that certain structural features of ligands can make a ligand to be AT- or GC-specific. It was also demonstrated that changing specificity from AT base pairs to GC base pairs further improved the DNA topoisomerase inhibiting activity in certain ligands. Thus, a specific molecular recognition signature encrypted in the structure of ligand can be decoded and can be effectively employed in designing more potent antiviral and antitumour agents.     

Recognition and Detoxification of the Insecticide DDT by Drosophila melanogaster Glutathione S-Transferase D1

GSTD1 is one of several insect glutathione S-transferases capable of metabolizing the insecticide DDT. Here we use crystallography and NMR to elucidate the binding of DDT and glutathione to GSTD1. The crystal structure of Drosophila melanogaster GSTD1 has been determined to 1.1 Å resolution, which reveals that the enzyme adopts the canonical GST fold but with a partially occluded active site caused by the packing of a C-terminal helix against one wall of the binding site for substrates. This helix would need to unwind or be displaced to enable catalysis. When the C-terminal helix is removed from the model of the crystal structure, DDT can be computationally docked into the active site in an orientation favoring catalysis. Two-dimensional ¹H,¹⁵N heteronuclear single-quantum coherence NMR experiments of GSTD1 indicate that conformational changes occur upon glutathione and DDT binding and the residues that broaden upon DDT binding support the predicted binding site. We also show that the ancestral GSTD1 is likely to have possessed DDT dehydrochlorinase activity because both GSTD1 from D. melanogaster and its sibling species, Drosophila simulans, have this activity.     

The Solution Structure,Binding Properties,and Dynamics of the Bacterial Siderophore-binding Protein FepB

The periplasmic binding protein (PBP) FepB plays a key role in transporting the catecholate siderophore ferric enterobactin from the outer to the inner membrane in Gram-negative bacteria. The solution structures of the 34-kDa apo- and holo-FepB from Escherichia coli, solved by NMR, represent the first solution structures determined for the type III class of PBPs. Unlike type I and II PBPs, which undergo large “Venus flytrap” conformational changes upon ligand binding, both forms of FepB maintain similar overall folds; however, binding of the ligand is accompanied by significant loop movements. Reverse methyl cross-saturation experiments corroborated chemical shift perturbation results and uniquely defined the binding pocket for gallium enterobactin (GaEnt). NMR relaxation experiments indicated that a flexible loop (residues 225–250) adopted a more rigid and extended conformation upon ligand binding, which positioned residues for optimal interactions with the ligand and the cytoplasmic membrane ABC transporter (FepCD), respectively. In conclusion, this work highlights the pivotal role that structural dynamics plays in ligand binding and transporter interactions in type III PBPs.     

Structure-activity relationship studies of Bz amide-containing α-GalCer derivatives as natural killer T cell modulators

Abstract

CD1d is a non-polymorphic antigen-presenting glycoprotein that recognizes glycolipids as ligands. Ligands bind to the hydrophobic grooves of CD1d, and the resulting ligand-CD1d complexes activate natural killer T (NKT) cells by means of T cell receptor recognition, leading to the secretion of various cytokines. However, details of the ligand recognition mechanism of a large hydrophobic ligand binding pocket and the relationship between cytokine induction and ligand structure are unclear. We report the synthesis of α-GalCer derivatives containing a Bz amide group having various substituting groups in the ceramide moiety, and the analysis of the structure-activity relationships. The assays reveal that the Bz amide-containing CD1d ligands function as NKT cell modulators displaying Th2 cytokine biasing responses. Furthermore, molecular dynamics simulation studies suggest that the phenyl groups can interact with the aromatic amino acid residues in the lipid binding pocket of CD1d.     

Bioactive M(II) complexes of amino acid-based N<Subscript>3</Subscript>O donor mixed ligand: in vitro and in silico DNA binding studies

Three novel mixed ligand M(II) complexes, namely [CoL¹L²Cl₂] (1), [CuL¹L²Cl₂] (2), and [ZnL¹L²Cl₂] (3), were synthesized using 1,4-naphthoquinone, L-histidine, and 1,10-phenanthroline as ligands. The ligand framework and the corresponding structural changes on complexation were ascertained based on the results of elemental analysis, conductivity measurements, magnetic behavior, FT-IR, UV-visible, ¹H NMR, ¹³C NMR, ESR spectral studies, and ESI mass spectrometry. The biological action of the ligand (L) and complexes 1–3 such as DNA binding and cleaving ability were studied. Results suggest that the ligand and the complexes could interact with calf thymus-DNA (CT-DNA) via intercalation mode. Additionally, complex 2 displayed potential antioxidant activity in in vitro studies. Docking simulation was performed to position the ligand and the complexes into the active site of BDNA (IBNA) to determine the probable binding mode.     

Antagonism by ethanol of endotoxin-induced tissue factor activation in relation to the depressed endotoxin binding to monocyte-like U937 cells

Our previous study has reported that ethanol (ETOH) partially inhibited the endotoxin (LPS)-induced tissue factor (TF)-activation in monocytes including blood peripheral monocytes as well as cultured leukemic U937 and THP-1 cells. The present study shows a strong correlation (r=0·92; p<0·01) between TF-activation and depression in LPS binding blocked by ETOH in U937 cells. The antagonism by ETOH of LPS binding was not due to a direct extracellular blockade, since ETOH did not affect the affinity of fluorescein isothiocyanate (FITC)-LPS or -anti CD14 mAb on U937 cells. After U937 cells were treated with 2 per cent (v/v) ETOH for 3 h, LPS binding was however drastically inhibited as shown by immunostaining with FITC-LPS which was viewed on a confocal laser scanning microscope. The results imply that cellular events of the ETOH effect mediate this inhibition of LPS binding. Anti-CD14 mAb (UCHM-1) inhibited LPS binding in a dose-dependent fashion, revealing a competitive specific binding to the LPS receptor. The results suggest that CD14 plays an important role in the recognition of LPS. FITC-UCHM-1 binding was significantly reduced in the cells pretreated with 2 per cent (v/v) ETOH for 3 h, indicating that ETOH modulates the ability to express CD14. CD14 expression was upregulated by priming with LPS which was offset by ETOH. Acetaldehyde, a possible metabolite of ETOH, was tested with no effect on CD14 expression. Taken together, our results show that ETOH downregulates the recognition of LPS, and suggest that the inhibitory action is likely to be mediated by the depression in CD14 expression which was also accompanied by a significantly altered membrane fluidity. Thus, the antagonism by ETOH of the binding of LPS results in a depression in the LPS-induced TF-activation. © 1997 John Wiley & Sons, Ltd.     

Solution structure of the cysteine‐rich domain in Fn14, a member of the tumor necrosis factor receptor superfamily

Fn14 is the smallest member of the tumor necrosis factor (TNF) receptor superfamily, and specifically binds to its ligand, TWEAK (TNF‐like weak inducer of apoptosis), which is a member of the TNF superfamily. The receptor‐ligand recognition between Fn14 and TWEAK induces a variety of cellular processes for tissue remodeling and is also involved in the pathogenesis of some human diseases, such as cancer, chronic autoimmune diseases, and acute ischaemic stroke. The extracellular ligand‐binding region of Fn14 is composed of 53 amino acid residues and forms a single, cysteine‐rich domain (CRD). In this study, we determined the solution structure of the Fn14 CRD (Glu28‐Ala70) by heteronuclear NMR, with a ¹³C‐/¹⁵N‐labeled sample. The tertiary structure of the CRD comprises a β‐sheet with two strands, followed by a 3₁₀ helix and a C‐terminal α‐helix, and is stabilized by three disulfide bonds connecting Cys36‐Cys49, Cys52‐Cys67, and Cys55‐Cys64. Comparison of the disulfide bond connectivities and the tertiary structures with those of other CRDs revealed that the Fn14 CRD is similar to the fourth CRD of TNF receptor 1 (A1‐C2 module type), but not to the CRD of B‐cell maturation antigen and the second CRD of transmembrane activator and CAML (calcium modulator and cyclophilin ligand) interactor (A1‐D2 module type). This is the first structural report about the A1‐C2 type CRD that could bind to the known target.     

CD44 Receptor Unfolding Enhances Binding by Freeing Basic Amino Acids to Contact Carbohydrate Ligand

The extracellular carbohydrate-binding domain of the Type I transmembrane receptor CD44 is known to undergo affinity switching, where change in conformation leads to enhanced binding of its carbohydrate ligand hyaluronan. Separate x-ray crystallographic and NMR experiments have led to competing explanations, with the former supporting minor conformational changes at the binding site and the latter a major order-to-disorder unfolding transition distant from the binding site. Here, all-atom explicit-solvent molecular dynamics studies employing adaptive biasing force sampling revealed a substantial favorable free-energy change associated with contact formation between the Arg⁴¹ side chain and hyaluronan at the binding site, independent of whether the distant site was ordered or disordered. Analogous computational experiments on Arg⁴¹Ala mutants showed loss of this favorable free-energy change, consistent with existing experimental data. More provocatively, the simulation data revealed the molecular mechanism by which the order-to-disorder transition enhances hyaluronan binding: in the disordered state, a number of basic residues gain sufficient conformational freedom—lacking in the ordered state—to spontaneously form side-chain contacts with hyaluronan. Mutation of these residues to Ala had been known to decrease binding affinity, but there had previously been no structural explanation, given their lack of proximity to the carbohydrate-binding site in existing structures of the complex.     

CD44 Receptor Unfolding Enhances Binding by Freeing Basic Amino Acids to Contact Carbohydrate Ligand

The extracellular carbohydrate-binding domain of the Type I transmembrane receptor CD44 is known to undergo affinity switching, where change in conformation leads to enhanced binding of its carbohydrate ligand hyaluronan. Separate x-ray crystallographic and NMR experiments have led to competing explanations, with the former supporting minor conformational changes at the binding site and the latter a major order-to-disorder unfolding transition distant from the binding site. Here, all-atom explicit-solvent molecular dynamics studies employing adaptive biasing force sampling revealed a substantial favorable free-energy change associated with contact formation between the Arg⁴¹ side chain and hyaluronan at the binding site, independent of whether the distant site was ordered or disordered. Analogous computational experiments on Arg⁴¹Ala mutants showed loss of this favorable free-energy change, consistent with existing experimental data. More provocatively, the simulation data revealed the molecular mechanism by which the order-to-disorder transition enhances hyaluronan binding: in the disordered state, a number of basic residues gain sufficient conformational freedom—lacking in the ordered state—to spontaneously form side-chain contacts with hyaluronan. Mutation of these residues to Ala had been known to decrease binding affinity, but there had previously been no structural explanation, given their lack of proximity to the carbohydrate-binding site in existing structures of the complex.     

Role of the conserved acidic residue Asp21 in the structure of phosphatidylinositol 3-kinase Src homology 3 domain: circular dichroism and nuclear magnetic resonance studies

To elucidate a role of the Src homology 3 (SH3)-conserved acidic residue Asp21 of the phosphatidylinositol 3-kinase (PI3K) SH3 domain, structural changes induced by the D21N mutation (Asp21 --> Asn) were examined by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. In the previous study, we demonstrated that environmental alterations occurred at the side chains of Trp55 and some Tyr residues from the comparison of the near-UV CD spectra of the PI3K SH3 domain with or without a D21N mutation [Okishio, N., et al. (2000) Biopolymers 57, 208-217]. In this work, the affected Tyr residues were identified as Tyr14 and Tyr73 by the CD analysis of a series of mutants, in which every single Tyr residue was replaced by a Phe residue with or without a D21N mutation. The (1)H and (15)N resonance assignments of the PI3K SH3 domain and its D21N mutant revealed that significant chemical shift changes occurred to the aromatic side-chain protons of Trp55 and Tyr14 upon the D21N mutation. All these aromatic residues are implicated in ligand recognition. In addition, the NMR analysis showed that the backbone conformations of Lys15-Asp23, Gly54-Trp55, Asn57-Gly58, and Gly67-Pro70 were affected by the D21N mutation. Furthermore, the (15)N[(1)H] nuclear Overhauser effect values of Tyr14, Glu19, and Glu20 were remarkably changed by the mutation. These results show that the D21N mutation causes structural deformation of more than half of the ligand binding cleft of the domain and provide evidence that Asp21 plays an important role in forming a well-ordered ligand binding cleft in cooperation with the RT loop (Lys15-Glu20).     

Crystal structures of the Apo and Holo form of rat catechol-O-methyltransferase

Catechol-O-methyltransferase (COMT, EC 2.1.1.6) is a monomeric enzyme that catalyzes the transfer of a methyl group from S-adenosyl-l-methionine (AdoMet) to the phenolic oxygen of substituted catechols. Although the inhibitor recognition pattern and AdoMet site have already been studied crystallographically, structural information on the catalytic cycle of COMT has not yet been obtained. In this study, comparison of the co-factor and inhibitor-bound structures revealed that the Apo form of COMT shows a conformational change and there was no cleft corresponding to the AdoMet-binding site; the overall structure was partially open form and the substrate recognition site was not clearly defined. The Holo form of COMT was similar to the quaternary structure except for the β6–β7 and α2–α3 ligand recognition loops. These conformational changes provide a deeper insight into the structural events occurring in reactions catalyzed by AdoMet.     

Alpha-bungarotoxin and the competing antibody WF6 interact with different amino acids within the same cholinergic subsite

In the nicotinic acetylcholine receptors (AChRs), the sequence segment surrounding two invariant vicinal cysteinyl residues at positions 192 and 193 of the alpha subunit contains important structural component(s) of the binding site for acetylcholine and high molecular weight cholinergic antagonists, like snake alpha-neurotoxins. At least a second sequence region contributes to the formation of the cholinergic site. Studying the binding of alpha-bungarotoxin and three different monoclonal antibodies, able to compete with alpha-neurotoxins and cholinergic ligands, to a panel of synthetic peptides as representative structural elements of the AChR from Torpedo, we recently identified the sequence segments alpha 181-200 and alpha 55-74 as contributing to form the cholinergic site (Conti-Tronconi et al., 1990). As a first attempt to elucidate the structural requirements for ligand binding to the subsite formed by the sequence alpha 181-200, we have now studied the binding of alpha-bungarotoxin and of antibody WF6 to the synthetic peptide alpha 181-200, and to a panel of peptide analogues differing from the parental sequence alpha 181-200 by substitution of a single amino acid residue. CD spectral analysis of the synthetic peptide analogues indicated that they all have comparable structures in solution, and they can therefore be used to analyze the influence of single amino acid residues on ligand binding. Distinct clusters of amino acid residues, discontinuously positioned along the sequence 181-200, seem to serve as attachment points for the two ligands studied, and the residues necessary for binding of alpha-bungarotoxin are different from those crucial for binding of antibody WF6. In particular, residues at positions 188-190 (VYY) and 192-194 (CCP) were necessary for binding of alpha-bungarotoxin, while residues W187, T191, and Y198 and the three residues at positions 193-195 (CPD) were necessary for binding of WF6. Comparison of the CD spectra of the toxin/peptide complexes, and those obtained for the same peptides and alpha-bungarotoxin in solution, indicates that structural changes of the ligand(s) occur upon binding, with a net increase of the beta-structure component. The cholinergic binding site is therefore a complex surface area, formed by discontinuous clusters of amino acid residues from different sequence regions. Such complex structural arrangement is similar to the "discontinuous epitopes" observed by X-ray diffraction studies of antibody/antigen complexes [reviewed in Davies et al. (1988)]. Within this relatively large structure, cholinergic ligands bind with multiple points of attachment, and ligand-specific patterns of the attachment points exist.(ABSTRACT TRUNCATED AT 400 WORDS)     

Unmasking of CD22 Co-receptor on Germinal Center B-cells Occurs by Alternative Mechanisms in Mouse and Man

CD22 is an inhibitory B-cell co-receptor whose function is modulated by sialic acid (Sia)-bearing glycan ligands. Glycan remodeling in the germinal center (GC) alters CD22 ligands, with as yet no ascribed biological consequence. Here, we show in both mice and humans that loss of high affinity ligands on GC B-cells unmasks the binding site of CD22 relative to naive and memory B-cells, promoting recognition of trans ligands. The conserved modulation of CD22 ligands on GC B-cells is striking because high affinity glycan ligands of CD22 are species-specific. In both species, the high affinity ligand is based on the sequence Siaα2–6Galβ1–4GlcNAc, which terminates N-glycans. The human ligand has N-acetylneuraminic acid (Neu5Ac) as the sialic acid, and the high affinity ligand on naive B-cells contains 6-O-sulfate on the GlcNAc. On human GC B-cells, this sulfate modification is lost, giving rise to lower affinity CD22 ligands. Ligands of CD22 on naive murine B-cells do not contain the 6-O-sulfate modification. Instead, the high affinity ligand for mouse CD22 has N-glycolylneuraminic acid (Neu5Gc) as the sialic acid, which is replaced on GC B-cells with Neu5Ac. Human naive and memory B-cells express sulfated glycans as high affinity CD22 ligands, which are lost on GC B-cells. In mice, Neu5Gc-containing glycans serve as high affinity CD22 ligands that are replaced by Neu5Ac-containing glycans on GC B-cells. Our results demonstrate that loss of high affinity CD22 ligands on GC B-cells occurs in both mice and humans through alternative mechanisms, unmasking CD22 relative to naive and memory B-cells.     

Ca2+-dependent Structural Changes in the B-cell Receptor CD23 Increase Its Affinity for Human Immunoglobulin E

Immunoglobulin E (IgE) antibodies play a fundamental role in allergic disease and are a target for therapeutic intervention. IgE functions principally through two receptors, FcϵRI and CD23 (FcϵRII). Minute amounts of allergen trigger mast cell or basophil degranulation by cross-linking IgE-bound FcϵRI, leading to an inflammatory response. The interaction between IgE and CD23 on B-cells regulates IgE synthesis. CD23 is unique among Ig receptors in that it belongs to the C-type (calcium-dependent) lectin-like superfamily. Although the interaction of CD23 with IgE is carbohydrate-independent, calcium has been reported to increase the affinity for IgE, but the structural basis for this activity has previously been unknown. We have determined the crystal structures of the human lectin-like head domain of CD23 in its Ca²⁺-free and Ca²⁺-bound forms, as well as the crystal structure of the Ca²⁺-bound head domain of CD23 in complex with a subfragment of IgE-Fc consisting of the dimer of Cϵ3 and Cϵ4 domains (Fcϵ3-4). Together with site-directed mutagenesis, the crystal structures of four Ca²⁺ ligand mutants, isothermal titration calorimetry, surface plasmon resonance, and stopped-flow analysis, we demonstrate that Ca²⁺ binds at the principal and evolutionarily conserved binding site in CD23. Ca²⁺ binding drives Pro-250, at the base of an IgE-binding loop (loop 4), from the trans to the cis configuration with a concomitant conformational change and ordering of residues in the loop. These Ca²⁺-induced structural changes in CD23 lead to additional interactions with IgE, a more entropically favorable interaction, and a 30-fold increase in affinity of a single head domain of CD23 for IgE. Taken together, these results suggest that binding of Ca²⁺ brings an extra degree of modulation to CD23 function.     

Circular Permutation Provides an Evolutionary Link between Two Families of Calcium-dependent Carbohydrate Binding Modules

The microbial deconstruction of the plant cell wall is a critical biological process, which also provides important substrates for environmentally sustainable industries. Enzymes that hydrolyze the plant cell wall generally contain non-catalytic carbohydrate binding modules (CBMs) that contribute to plant cell wall degradation. Here we report the biochemical properties and crystal structure of a family of CBMs (CBM60) that are located in xylanases. Uniquely, the proteins display broad ligand specificity, targeting xylans, galactans, and cellulose. Some of the CBM60s display enhanced affinity for their ligands through avidity effects mediated by protein dimerization. The crystal structure of vCBM60, displays a β-sandwich with the ligand binding site comprising a broad cleft formed by the loops connecting the two β-sheets. Ligand recognition at site 1 is, exclusively, through hydrophobic interactions, whereas binding at site 2 is conferred by polar interactions between a protein-bound calcium and the O2 and O3 of the sugar. The observation, that ligand recognition at site 2 requires only a β-linked sugar that contains equatorial hydroxyls at C2 and C3, explains the broad ligand specificity displayed by vCBM60. The ligand-binding apparatus of vCBM60 displays remarkable structural conservation with a family 36 CBM (CBM36); however, the residues that contribute to carbohydrate recognition are derived from different regions of the two proteins. Three-dimensional structure-based sequence alignments reveal that CBM36 and CBM60 are related by circular permutation. The biological and evolutionary significance of the mechanism of ligand recognition displayed by family 60 CBMs is discussed.     

Assembly of chloroplast signal recognition particle involves structural rearrangement in cpSRP43

Signal recognition particle in chloroplasts (cpSRP) exhibits the unusual ability to bind and target full-length proteins to the thylakoid membrane. Unlike cytosolic SRPs in prokaryotes and eukaryotes, cpSRP lacks an RNA moiety and functions as a heterodimer composed of a conserved 54-kDa guanosine triphosphatase (cpSRP54) and a unique 43-kDa subunit (cpSRP43). Assembly of the cpSRP heterodimer is a prerequisite for post-translational targeting activities and takes place through interactions between chromatin modifier domain 2 (CD2) of cpSRP43 and a unique 10-amino-acid region in cpSRP54 (cpSRP54_pep). We have used multidimensional NMR spectroscopy and other biophysical methods to examine the assembly and structure of the cpSRP43-cpSRP54 interface. Our data show that CD2 of cpSRP43 binds to cpSRP54_pep in a 1:1 stoichiometry with an apparent K_d of ∼ 1.06 μM. Steady-state fluorescence and far-UV circular dichroism data suggest that the CD2-cpSRP54_pep interaction causes significant conformational changes in both CD2 and the peptide. Comparison of the three-dimensional solution structures of CD2 alone and in complex with cpSRP54_pep shows that significant structural changes are induced in CD2 in order to establish a binding interface contributed mostly by residues in the N-terminal segment of CD2 (Phe5-Val10) and an arginine doublet (Arg536 and Arg537) in the cpSRP54 peptide. Taken together, our results provide new insights into the mechanism of cpSRP assembly and the structural forces that stabilize the functionally critical cpSRP43-cpSRP54 interaction.     

The X-ray structure of the zinc transporter ZnuA from Salmonella enterica discloses a unique triad of zinc-coordinating histidines

ZnuA is the soluble component of the high-affinity ZnuABC zinc transporter belonging to the cluster 9 group of ATP-binding cassette-type periplasmic Zn- and Mn-binding proteins. In Gram-negative bacteria, the ZnuABC system is essential for zinc uptake and homeostasis and is an important determinant of bacterial resistance to the host defense mechanisms. The cluster 9 members share a two (α/β)₄ domain architecture with a long α-helix connecting the two domains. In the Zn-specific proteins, the so-called α3c and the α4 helices are separated by an insert of variable length, rich in histidine and negatively charged residues. This distinctive His-rich loop is proposed to play a role in the management of zinc also due to its location at the entrance of the metal binding site located at the domain interface. The known Synechocystis 6803 and Escherichia coli ZnuA structures show the same metal coordination involving three conserved histidines and a glutamic acid or a water molecule as fourth ligand. The structures of Salmonella enterica ZnuA, with a partially or fully occupied zinc binding site, and of a deletion mutant missing a large part of the His-rich loop revealed unexpected differences in the metal-coordinating ligands, as histidine 140 from the mobile (at the C-terminal) part of the loop substitutes the conserved histidine 60. This unforeseen coordination is rendered possible by the “open conformation” of the two domains. The possible structural determinants of these peculiarities and their functional relevance are discussed.