Metal-binding Domain Research Articles

Molecular characterization and phylogenetic analysis of kaurene synthase protein in Stevia rebaudiana MS007

Stevia rebaudiana is a plant under the Asteraceae family and has been reported as a healthier alternative to sugar. Steviol glycosides (SGs) is the group of secondary metabolites responsible for the sweet taste. Among nine SGs synthesised by S. rebaudiana, stevioside and rebaudioside A are the sweetest. The biosynthetic pathway of SGs partly involves conversion of geranylgeranyl diphosphate (GGDP) into steviol, catalysed by ent- kaurene synthase (KS), ent-copalyl diphosphate synthase (CPPS), and kaurene oxidase (KO). This study focuses on in silico molecular characterization and phylogenetic analysis of KS from Malaysia’s S. rebaudiana MS007 variety (Stevia MS007). The transcriptomic dataset of S. rebaudiana accession MS007 was used in initial experiment toward analysing the KS. Through the blastx homology search using transcriptomic dataset query Cluster-31069.42907, the Stevia rebaudiana kaurene synthase (SrKS) sequence was identified with the highest similarity percentage identity (99.62%). The protein domain prediction using InterPro yields IPR005630 (terpene synthase metal-binding domain) at positions 490 to 755 and IPR001906 (terpene synthase-N-terminal-domain) at positions 258 to 477. Multiple sequence alignment was conducted using MUSCLE and MEGA-X as phylogenetic tree analysis tool for constructing the phylogenetic analysis tree. Based on the bootstrap value from the phylogenetic analysis, Cluster-31069.42907 represents relationships between the ancestors. Since both Helianthus annuus and S. rebaudiana are Asteraceae species, the bootstrap value for both species was 100%. In conclusion, this research contributes to a better understanding of Stevia MS007 KS via in silico analysis.

Chimeric MerR-Family Regulators and Logic Elements for the Design of Metal Sensitive Genetic Circuits in Bacillus subtilis.

Whole-cell biosensors are emerging as promising tools for monitoring environmental pollutants such as heavy metals. These sensors constitute a genetic circuit comprising a sensing module and an output module, such that a detectable signal is produced in the presence of the desired analyte. The MerR family of metal-responsive regulators offers great potential for the construction of metal sensing circuits, due to their high sensitivity, tight transcription control, and large diversity in metal-specificity. However, the sensing diversity is broadest in Gram-negative systems, while chassis organisms are often selected from Gram-positive species, particularly sporulating bacilli. This can be problematic, because Gram-negative biological parts, such as promoters, are frequently observed to be nonfunctional in Gram-positive hosts. Herein, we combined construction of synthetic genetic circuits and chimeric MerR regulators, supported by structure-guided design, to generate metal-sensitive biosensor modules that are functional in the biotechnological work-horse species Bacillus subtilis. These chimeras consist of a constant Gram-positive derived DNA-binding domain fused to variable metal binding domains of Gram-negative origins. To improve the specificity of the whole-cell biosensor, we developed a modular "AND gate" logic system based on the B.subtilis two-subunit σ-factor, SigO-RsoA, designed to maximize future use for synthetic biology applications in B.subtilis. This work provides insights into the use of modular regulators, such as the MerR family, in the design of synthetic circuits for the detection of heavy metals, with potentially wider applicability of the approach to other systems and genetic backgrounds.

Back to the future: asymmetrical DπA 2,2'-bipyridine ligands for homoleptic copper(i)-based dyes in dye-sensitised solar cells.

Metal complexes used as sensitisers in dye-sensitised solar cells (DSCs) are conventionally constructed using a push-pull strategy with electron-releasing and electron-withdrawing (anchoring) ligands. In a new paradigm we have designed new DπA ligands incorporating diarylaminophenyl donor substituents and phosphonic acid anchoring groups. These new ligands function as organic dyes. For two separate classes of DπA ligands with 2,2'-bipyridine metal-binding domains, the DSCs containing the copper(i) complexes [Cu(DπA)2]+ perform better than the push-pull analogues [Cu(DD)(AA)]+. Furthermore, we have shown for the first time that the complexes [Cu(DπA)2]+ perform better than the organic DπA dye in DSCs. The synthetic studies and the device performances are rationalised with the aid of density functional theory (DFT) and time-dependent DFT (TD-DFT) studies.

Electrochemical Analysis in Studying β-Amyloid Aggregation.

β-amyloid (Aβ) is comprised of a group of peptides formed as a result of cleavage of the amyloid precursor protein by secretases. Aβ aggregation is considered as a central event in pathogenesis of Alzheimer's disease, the most common human neurodegenerative disorder. Molecular mechanisms of Aβ aggregation have intensively being investigated using synthetic Aβ peptides by methods based on monitoring of aggregates, including determination of their size and structure. In this review, an orthogonal approach to the study of Aβ aggregation is considered, which relies on electrochemical registration of the loss of peptide monomers. Electrochemical analysis of Aβ (by voltammetry and amperometric flow injection analysis) is based on registration of the oxidation signal of electroactive amino acid residues of the peptide on an electrode surface. The Aβ oxidation signal disappears, when the peptide is included in the aggregate. The advantages and disadvantages of electrochemical analysis for the study of spontaneous and metal-induced aggregation of Aβ, comparative analysis of various peptide isoforms, and study of the process of complexation of metal ions with the metal-binding domain of Aβ are discussed. It is concluded that the combined use of the electrochemical method and the methods based on detection of Aβ aggregates makes it possible to obtain more complete information about the mechanisms of peptide aggregation.

Di-Iron(II) [2+2] Helicates of Bis-(Dipyrazolylpyridine) Ligands: The Influence of the Ligand Linker Group on Spin State Properties.

Four bis[2-{pyrazol-1-yl}-6-{pyrazol-3-yl}pyridine] ligands have been synthesized, with butane-1,4-diyl (L1 ), pyrid-2,6-diyl (L2 ), benzene-1,2-dimethylenyl (L3 ) and propane-1,3-diyl (L4 ) linkers between the tridentate metal-binding domains. L1 and L2 form [Fe2 (μ-L)2 ]X4 (X- =BF4 - or ClO4 - ) helicate complexes when treated with the appropriate iron(II) precursor. Solvate crystals of [Fe2 (μ-L1 )2 ][BF4 ]4 exhibit three different helicate conformations, which differ in the torsions of their butanediyl linker groups. The solvates exhibit gradual thermal spin-crossover, with examples of stepwise switching and partial spin-crossover to a low-temperature mixed-spin form. Salts of [Fe2 (μ-L2 )2 ]4+ are high-spin, which reflects their highly twisted iron coordination geometry. The composition and dynamics of assembly structures formed by iron(II) with L1 -L3 vary with the ligand linker group, by mass spectrometry and 1 H NMR spectroscopy. Gas-phase DFT calculations imply the butanediyl linker conformation in [Fe2 (μ-L1 )2 ]4+ influences its spin state properties, but show anomalies attributed to intramolecular electrostatic repulsion between the iron atoms.

Expanded Ligands Based upon Iron(II) Coordination Compounds of Asymmetrical Bis(terpyridine) Domains.

The synthesis and characterization of two tritopic ligands containing a 2,2':6',2″-terpyridine (tpy) metal binding domain and either a 3,2':6',3″- or a 4,2':6',4″-tpy domain are detailed. The synthetic routes to these ligands involved the [Pd(dppf)Cl2]-catalyzed coupling of a boronic ester-functionalized 2,2':6',2″-tpy with bromo-derivatives of 3,2':6',3″-tpy or 4,2':6',4″-tpy. The 2,2':6',2″-tpy domains of the tritopic ligands preferentially bind Fe2+ in reactions with iron(II) salts leading to the formation of two homoleptic iron(II) complexes containing two peripheral 3,2':6',3″-tpy or 4,2':6',4″-tpy metal-binding sites, respectively. These iron(II) complexes are potentially tetratopic ligands and represent expanded versions of tetra(pyridin-4-yl)pyrazine.

The Modular Architecture of Metallothioneins Facilitates Domain Rearrangements and Contributes to Their Evolvability in Metal-Accumulating Mollusks.

Protein domains are independent structural and functional modules that can rearrange to create new proteins. While the evolution of multidomain proteins through the shuffling of different preexisting domains has been well documented, the evolution of domain repeat proteins and the origin of new domains are less understood. Metallothioneins (MTs) provide a good case study considering that they consist of metal-binding domain repeats, some of them with a likely de novo origin. In mollusks, for instance, most MTs are bidomain proteins that arose by lineage-specific rearrangements between six putative domains: α, β1, β2, β3, γ and δ. Some domains have been characterized in bivalves and gastropods, but nothing is known about the MTs and their domains of other Mollusca classes. To fill this gap, we investigated the metal-binding features of NpoMT1 of Nautilus pompilius (Cephalopoda class) and FcaMT1 of Falcidens caudatus (Caudofoveata class). Interestingly, whereas NpoMT1 consists of α and β1 domains and has a prototypical Cd2+ preference, FcaMT1 has a singular preference for Zn2+ ions and a distinct domain composition, including a new Caudofoveata-specific δ domain. Overall, our results suggest that the modular architecture of MTs has contributed to MT evolution during mollusk diversification, and exemplify how modularity increases MT evolvability.

Mechanistic insights into the nickel-dependent allosteric response of the Helicobacter pylori NikR transcription factor

In Helicobacter pylori, the nickel-responsive NikR transcription factor plays a key role in regulating intracellular nickel concentrations, which is an essential process for survival of this pathogen in the acidic human stomach. Nickel binding to H.pylori NikR (HpNikR) allosterically activates DNA binding to target promoters encoding genes involved in nickel homeostasis and acid adaptation, to either activate or repress their transcription. We previously showed that HpNikR adopts an equilibrium between an open conformation and DNA-binding competent cis and trans states. Nickel binding slows down conformational exchange between these states and shifts the equilibrium toward the binding-competent states. The protein then becomes stabilized in a cis conformation upon binding the ureA promoter. Here, we investigate how nickel binding creates this response and how it is transmitted to the DNA-binding domains. Through mutagenesis, DNA-binding studies, and computational methods, the allosteric response to nickel was found to be propagated from the nickel-binding sites to the DNA-binding domains via the β-sheets of the metal-binding domain and a network of residues at the inter-domain interface. Our computational results suggest that nickel binding increases protein rigidity to slow down the conformational exchange. A thymine base in the ureA promoter sequence, known to be critical for high affinity DNA binding by HpNikR, was also found to be important for the allosteric response, while a modified version of this promoter further highlighted the importance of the DNA sequence in modulating the response. Collectively, our results provide insights into regulation of a key protein for H.pylori survival.

Genome-wide analysis elucidates the roles of GhHMA genes in different abiotic stresses and fiber development in upland cotton

The heavy metal-binding domain is involved in heavy metal transporting and plays a significant role in plant detoxification. However, the functions of HMAs are less well known in cotton. In this study, a total of 143 GhHMAs (heavy metal-binding domain) were detected by genome-wide identification in G. hirsutum L. All the GhHMAs were classified into four groups via phylogenetic analysis. The exon/intron structure and protein motifs indicated that each branch of the GhHMA genes was highly conserved. 212 paralogous GhHMA gene pairs were identified, and the segmental duplications were the main role to the expansion of GhHMAs. The Ka/Ks values suggested that the GhHMA gene family has undergone purifying selection during the long-term evolutionary process. GhHMA3 and GhHMA75 were located in the plasma membrane, while GhHMA26, GhHMA117 and GhHMA121 were located in the nucleus, respectively. Transcriptomic data and qRT-PCR showed that GhHMA26 exhibited different expression patterns in each tissue and during fiber development or under different abiotic stresses. Overexpressing GhHMA26 significantly promoted the elongation of leaf trichomes and also improved the tolerance to salt stress. Therefore, GhHMA26 may positively regulate fiber elongation and abiotic stress. Yeast two-hybrid assays indicated that GhHMA26 and GhHMA75 participated in multiple biological functions. Our results suggest some genes in the GhHMAs might be associated with fiber development and the abiotic stress response, which could promote further research involving functional analysis of GhHMA genes in cotton.

Activity and Synergy of Cu-ATCUN Antimicrobial Peptides.

Antibiotic resistance demands innovative strategies and therapies. The pairs of antimicrobial peptides tested in this work show broad-spectrum synergy and are capable of interacting with diverse bacterial membranes. In most cases, the ATCUN motif enhanced the activity of peptides tested in combination. Our studies also show CP10A to be a multifaceted peptide, displaying both cell membrane and intracellular activity and acting as a chameleon, improving the activity of other peptides as needed. The results of the synergy experiments demonstrate the importance of varied modes of action and how these changes can affect the ability to combat pathogens, while also illustrating the value of the metal-binding domain in enhancing the activity of antimicrobial peptides in combination.

Molecular characteristics of isoprene synthase and its control effects on isoprene emissions from tropical trees.

The isoprene emission rate from plants is simulated by a function of light intensity and leaf temperature, and the G-93 formula is the most extensively applied algorithm for this purpose. Isoprene is biosynthesized by the enzyme isoprene synthase (IspS), and instantly emitted from the leaf. Enzyme kinetics of IspS and substrate availability are important factors involved in the short-term leaf-level control of isoprene emissions. It is thus assumed that the parameters of G-93 may correlate with the kinetics of IspSs, however, at present there is no data available on the relationship between these two parameters. In this investigation, six IspS genes from tropical trees were cloned, their properties characterized, and the relationship between the enzyme kinetics of IspSs and the parameters of G-93 examined. There was a negative correlation between the enzyme kinetics of IspS Km and parameter CT1 of G93, which is used to define the temperature dependency of isoprene emissions. However, performance constant of IspS (kcat/Km) only showed slight positive correlation with CT1.suggesting that the enzyme kinetics of IspS has limited significance in controlling the temperature response of isoprene emissions. The molecular structure of IspS was further elucidated using a molecular dynamics simulation with a focus on the active site in the 6 α-helices bundle. The simulation of the enzyme-substrate complex of IspS from B. variegata predicted a new metal binding domain in helix F (E383) and catalytic motif FXRDRLXE in the A-C loop that could involve the deprotonation of dimethylallyl diphosphate (DMADP) to form a carbocation. Notably, after the binding of a metal ion and DMADP, the active-site closure mechanism was found to involve conformational alterations in the helix H-α1 and transition from a loose to tight enclosure of the 6 α-helices bundles to tune the active pocket size. The characteristics identified for the IspSs from tropical trees could help to explain regional isoprene emissions in tropical areas.

Disrupting Cu trafficking as a potential therapy for cancer.

Copper ions play a crucial role in various cellular biological processes. However, these copper ions can also lead to toxicity when their concentration is not controlled by a sophisticated copper-trafficking system. Copper dys-homeostasis has been linked to a variety of diseases, including neurodegeneration and cancer. Therefore, manipulating Cu-trafficking to trigger selective cancer cell death may be a viable strategy with therapeutic benefit. By exploiting combined in silico and experimental strategies, we identified small peptides able to bind Atox1 and metal-binding domains 3-4 of ATP7B proteins. We found that these peptides reduced the proliferation of cancer cells owing to increased cellular copper ions concentration. These outcomes support the idea of harming copper trafficking as an opportunity for devising novel anti-cancer therapies.

Copper binding leads to increased dynamics in the regulatory N-terminal domain of full-length human copper transporter ATP7B.

ATP7B is a human copper-transporting P1B-type ATPase that is involved in copper homeostasis and resistance to platinum drugs in cancer cells. ATP7B consists of a copper-transporting core and a regulatory N-terminal tail that contains six metal-binding domains (MBD1-6) connected by linker regions. The MBDs can bind copper, which changes the dynamics of the regulatory domain and activates the protein, but the underlying mechanism remains unknown. To identify possible copper-specific structural dynamics involved in transport regulation, we constructed a model of ATP7B spanning the N-terminal tail and core catalytic domains and performed molecular dynamics (MD) simulations with (holo) and without (apo) copper ions bound to the MBDs. In the holo protein, MBD2, MBD3 and MBD5 showed enhanced mobilities, which resulted in a more extended N-terminal regulatory region. The observed separation of MBD2 and MBD3 from the core protein supports a mechanism where copper binding activates the ATP7B protein by reducing interactions among MBD1-3 and between MBD1-3 and the core protein. We also observed an increased interaction between MBD5 and the core protein that brought the copper-binding site of MBD5 closer to the high-affinity internal copper-binding site in the core protein. The simulation results assign specific, mechanistic roles to the metal-binding domains involved in ATP7B regulation that are testable in experimental settings.

Coordination Properties of the Zinc Domains of BigR4 and SmtB Proteins in Nickel Systems─Designation of Key Donors

The increasing number of antibiotic-resistant pathogens has become one of the foremost health problems of modern times. One of the most lethal and multidrug-resistant bacteria is Mycobacterium tuberculosis (Mtb), which causes tuberculosis (TB). TB continues to engulf health systems due to the significant development of bacterial multidrug-resistant strains. Mammalian immune system response to mycobacterial infection includes, but is not limited to, increasing the concentration of zinc(II) and other divalent metal ions in phagosome vesicles up to toxic levels. Metal ions are necessary for the survival and virulence of bacteria but can be highly toxic to organisms if their concentrations are not strictly controlled. Therefore, understanding the mechanisms of how bacteria use metal ions to maintain their optimum concentrations and survive under lethal environmental conditions is essential. The mycobacterial SmtB protein, one of the metal-dependent transcription regulators of the ArsR/SmtB family, dissociates from DNA in the presence of high concentrations of metals, activating the expression of metal efflux proteins. In this work, we explore the properties of α5 metal-binding domains of SmtB/BigR4 proteins (the latter being the SmtB homolog from nonpathogenic Mycobacterium smegmatis), and two mutants of BigR4 as ligands for nickel(II) ions. The study focuses on the specificity of metal–ligand interactions and describes the effect of mutations on the coordination properties of the studied systems. The results of this research reveal that the Ni(II)-BigR4 α5 species are more stable than the Ni(II)-SmtB α5 complexes. His mutations, exchanging one of the histidines for alanine, cause a decrease in the stability of Ni(II) complexes. Surprisingly, the lack of His102 resulted also in increased involvement of acidic amino acids in the coordination. The results of this study may help to understand the role of critical mycobacterial virulence factor—SmtB in metal homeostasis. Although SmtB prefers Zn(II) binding, it may also bind metal ions that prefer other coordination modes, for example, Ni(II). We characterized the properties of such complexes in order to understand the nature of mycobacterial SmtB when acting as a ligand for metal ions, given that nickel and zinc ArsR family proteins possess analogous metal-binding motifs. This may provide an introduction to the design of a new antimicrobial strategy against the pathogenic bacterium M. tuberculosis.

Bioinformatic prediction of putative metallothioneins in non-ciliate protists.

Intracellular ligands that bind heavy metals (HMs) and thereby minimize their detrimental effects to cellular metabolism are attracting great interest for a number of applications including bioremediation and development of HM-biosensors. Metallothioneins (MTs) are short, cysteine-rich, genetically encoded proteins involved in intracellular metal-binding and play a key role in detoxification of HMs. We searched approximately 700 genomes and transcriptomes of non-ciliate protists for novel putative MTs by similarity and structural analyses and found 21 unique proteins playing a potential role as MTs. Most putative MTs derive from heterokonts and dinoflagellates and share common features such as (i) a putative metal-binding domain in proximity of the N-terminus, (ii) two putative MT-specific domains near the C-terminus and (iii) one to three CTCGXXCXCGXXCXCXXC patterns. Although the biological function of these proteins has not been experimentally proven, knowledge of their genetic sequences adds useful information on proteins that are potentially involved in HM-binding and can contribute to the design of future biomolecular assays on HM-microbe interactions and MT-based biosensors.

Cu(I) Binding to Designed Proteins Reveals a Putative Copper Binding Site of the Human Line1 Retrotransposon Protein ORF1p.

Long interspersed nuclear elements-1 (L1) are autonomous retrotransposons that encode two proteins in different open reading frames (ORF1 and ORF2). The ORF1p, which may be an RNA binding and chaperone protein, contains a three-stranded coiled coil (3SCC) domain that facilitates the formation of the biologically active homotrimer. This 3SCC domain is composed of seven amino acid (heptad) repeats as found in native and designed peptides and a stammer that modifies the helical structure. Cysteine residues occur at three hydrophobic positions (2 a and 1 d sites) within this domain. We recently showed that the cysteine layers in ORF1p and model de novo designed peptides bind the toxic metalloid lead(II) with high affinities, a feature that had not been previously recognized. However, there is little understanding of how essential metal ions might interact with this metal binding domain. We have, therefore, investigated the copper(I) binding properties of analogous de novo designed 3SCCs that contain cysteine layers within the hydrophobic core. The results from UV-visible and X-ray absorption spectroscopy show that these designed peptides bind Cu(I) with high affinity in a pH-dependent manner. At pH 9, monomeric trigonal planar Cu(I)S3 centers are formed with 1 equiv of metal, while dinuclear centers form with a second equivalent of metal. At physiologic pH conditions, the dinuclear center forms cooperatively. These data suggest that ORF1p is capable of binding two copper ions to its tris(cysteine) layers. This has major implications for ORF1p coiled coil domain stability and dynamics, ultimately potentially impacting the resulting biological activity.

Structure of the Wilson disease copper transporter ATP7B.

ATP7A and ATP7B, two homologous copper-transporting P1B-type ATPases, play crucial roles in cellular copper homeostasis, and mutations cause Menkes and Wilson diseases, respectively. ATP7A/B contains a P-type ATPase core consisting of a membrane transport domain and three cytoplasmic domains, the A, P, and N domains, and a unique amino terminus comprising six consecutive metal-binding domains. Here, we present a cryo–electron microscopy structure of frog ATP7B in a copper-free state. Interacting with both the A and P domains, the metal-binding domains are poised to exert copper-dependent regulation of ATP hydrolysis coupled to transmembrane copper transport. A ring of negatively charged residues lines the cytoplasmic copper entrance that is presumably gated by a conserved basic residue sitting at the center. Within the membrane, a network of copper-coordinating ligands delineates a stepwise copper transport pathway. This work provides the first glimpse into the structure and function of ATP7 proteins and facilitates understanding of disease mechanisms and development of rational therapies.

Metal ion scavenging activity of elastin-like peptide analogues containing a cadmium ion binding sequence

The development of simple and safe methods for recovering environmental pollutants, such as heavy metals, is needed for sustainable environmental management. Short elastin-like peptide (ELP) analogues conjugated with metal chelating agents are considered to be useful as metal sequestering agents as they are readily produced, environment friendly, and the metal binding domain can be selected based on any target metal of interest. Due to the temperature dependent self-assembly of ELP, the peptide-based sequestering agents can be transformed from the solution state into the particles that chelate metal ions, which can then be collected as precipitates. In this study, we developed a peptide-based sequestering agent, AADAAC-(FPGVG)4, by introducing the metal-binding sequence AADAAC on the N-terminus of a short ELP, (FPGVG)4. In turbidity measurements, AADAAC-(FPGVG)4 revealed strong self-assembling ability in the presence of metal ions such as Cd2+ and Zn2+. The results from colorimetric analysis indicated that AADAAC-(FPGVG)4 could capture Cd2+ and Zn2+. Furthermore, AADAAC-(FPGVG)4 that bound to metal ions could be readily recycled by treatment with acidic solution without compromising its metal binding affinity. The present study indicates that the fusion of the metal-binding sequence and ELP is a useful and powerful strategy to develop cost-effective heavy metal scavenging agents with low environmental impacts.

Copper-dependent ATP7B N-terminal domain dynamics in two reaction-cycle intermediates

Membrane proteins control the flow of ions across biological membranes by selective import or export and are of vital importance for all living cells. Disturbances in ion transport can cause severe diseases such as Wilson’s disease, which is caused by mutations in the human copper-transporting P-type ATPase ATP7B. ATP7B activity is regulated by a dynamic N-terminal domain consisting of six metal binding domains (MBDs) that move as rigid bodies and interact transiently with each other and the core transporter in a copper-dependent manner.

Multitopic 3,2':6',3''-terpyridine ligands as 4-connecting nodes in two-dimensional 4,4-networks.

The tetratopic 1,4-bis(2-phenylethoxy)-2,5-bis(3,2':6',3''-terpyridin-4'-yl)benzene (1) and 1,4-bis(3-phenylpropoxy)-2,5-bis(3,2':6',3''-terpyridin-4'-yl)benzene (2) ligands have been prepared and fully characterised. Combination of ligand 1 or 2 and [M(hfacac)2]·xH2O (M = Cu, x = 1; M = Zn, x = 2) under conditions of crystal growth by layering led to the formation of [Cu2(hfacac)4(1)] n ·3.6n(1,2-Cl2C6H4)·2nCHCl3, [Zn2(hfacac)4(1)] n ·nMeC6H5·1.8nCHCl3, [Cu2(hfacac)4(2)] n ·nMeC6H5·2nH2O, [Cu2(hfacac)4(2)] n ·2.8nC6H5Cl and [Cu2(hfacac)4(2)] n ·2n(1,2-Cl2C6H4)·0.4nCHCl3·0.5nH2O. For each compound, single-crystal X-ray analysis revealed the assembly of a planar (4,4)-net in which the tetratopic ligands 1 or 2 define the nodes. The metal centres link two different bis(3,2':6',3''-tpy) ligands via the outer pyridine rings; whereas copper(ii) has N-donors in a trans-arrangement, zinc(ii) has them in cis. This difference between the copper(ii) and zinc(ii) coordination polymers modifies the architecture of the assembly without changing the underlying (4,4)-network.