Calcium-binding protein-2 (EhCaBP2) crystals were grown using MPD as a precipitant. EhCaBP2 also crystallized in complex with strontium (replacing calcium) at similar conditions. Preliminary data for EhCaBP2 crystals in complex with an IQ motif are also reported.
Calcium plays a pivotal role in the pathogenesis of amoebiasis, a major disease caused by Entamoeba histolytica. Two domains with four canonical EF-hand-containing calcium-binding proteins (CaBPs) have been identified from E. histolytica. Even though they have very high sequence similarity, these bind to different target proteins in a Ca2+-dependent manner, leading to different functional pathways. Calcium-binding protein-2 (EhCaBP2) crystals were grown using MPD as a precipitant. The crystals belong to space group P21, with unit-cell parameters a = 111.74, b = 68.83, c = 113.25 Å, β = 116.7°. EhCaBP2 also crystallized in complex with strontium (replacing calcium) at similar conditions. The crystals belong to space group P21, with unit-cell parameters a = 69.18, b = 112.03, c = 93.42 Å, β = 92.8°. Preliminary data for EhCaBP2 crystals in complex with an IQ motif are also reported. This complex was crystallized with MPD and ethanol as precipitating agents. These crystals belong to space group P21, with unit-cell parameters a = 60.5, b = 69.86, c = 86.5 Å, β = 97.9°.
calcium-binding proteins; Entamoeba histolytica; amoebiasis
Entamoeba histolytica, a protozoan parasite, is the causative agent of amoebiasis, and calcium signaling is thought to be involved in amoebic pathogenesis. EhCaBP1, a Ca2+ binding protein of E. histolytica, is essential for parasite growth. High resolution crystal structure of EhCaBP1 suggested an unusual arrangement of the EF-hand domains in the N-terminal part of the structure, while C-terminal part of the protein was not traced. The structure revealed a trimer with amino terminal domains of the three molecules interacting in a head-to-tail manner forming an assembled domain at the interface with EF1 and EF2 motifs of different molecules coming close to each other. In order to understand the specific roles of the two domains of EhCaBP1, the molecule was divided into two halves, and each half was separately expressed. The domains were characterized with respect to their structure, as well as specific functional features, such as ability to activate kinase and bind actin. The domains were also expressed in E. histolytica cells along with green fluorescent protein. The results suggest that the N-terminal domain retains some of the properties, such as localization in phagocytic cups and activation of kinase. Crystal structure of EhCaBP1 with Phenylalanine revealed that the assembled domains, which are similar to Calmodulin N-terminal domain, bind to Phenylalanine revealing the binding mode to the target proteins. The C-terminal domain did not show any of the activities tested. However, over-expression in amebic cells led to a dominant negative phenotype. The results suggest that the two domains of EhCaBP1 are functionally and structurally different from each other. Both the domains are required for structural stability and full range of functional diversity.
Phagocytosis is required for proliferation and pathogenesis of Entamoeba histolytica and erythrophagocytosis is considered to be a marker of invasive amoebiasis. Ca2+ has been found to play a central role in the process of phagocytosis. However, the molecular mechanisms and the signalling mediated by Ca2+ still remain largely unknown. Here we show that Calmodulin-like calcium binding protein EhCaBP3 of E. histolytica is directly involved in disease pathomechanism by its capacity to participate in cytoskeleton dynamics and scission machinery during erythrophagocytosis. Using imaging techniques EhCaBP3 was found in phagocytic cups and newly formed phagosomes along with actin and myosin IB. In vitro studies confirmed that EhCaBP3 directly binds actin, and affected both its polymerization and bundling activity. Moreover, it also binds myosin 1B in the presence of Ca2+. In cells where EhCaBP3 expression was down regulated by antisense RNA, the level of RBC uptake was reduced, myosin IB was found to be absent at the site of pseudopod cup closure and the time taken for phagocytosis increased, suggesting that EhCaBP3 along with myosin 1B mediate the closure of phagocytic cups. Experiments with EhCaBP3 mutant defective in Ca2+ -binding showed that Ca2+ binding is required for phagosome formation. Liposome binding assay revealed that EhCaBP3 recruitment and enrichment to membrane is independent of any cellular protein as it binds directly to phosphatidylserine. Taken together, our results suggest a novel pathway mediating phagocytosis in E. histolytica, and an unusual mechanism of modulation of cytoskeleton dynamics by two calcium binding proteins, EhCaBP1 and EhCaBP3 with mostly non-overlapping functions.
Entamoeba histolytica is one of the major causes of morbidity and mortality in developing countries. Phagocytosis plays an important role in both survival and virulence and has been used as a virulence marker. Inhibition of phagocytosis leads to a defect in cellular proliferation. Therefore, the molecules that participate in phagocytosis are good targets for developing new drugs. However, the molecular mechanism of the process is still largely unknown. Here, we demonstrate that Calmodulin-like calcium binding protein EhCaBP3 is involved in erythrophagocytosis. We show this by a number of different approaches including immunostaining of actin, myosin1B, EhCaBP1 and EhCaBP3 during uptake of RBC; over expression and down regulation of EhCaBP3, and over expression of calcium defective mutant of EhCaBP3. Our analysis suggests that EhCaBP3 can regulate actin dynamics. Along with actin and myosin 1B it can participate in both initiation and formation of phagosomes. The Ca2+-bound form of this protein is required only for progression from cups into early phagosomes but not for initiation. Our results demonstrate the complex role of Ca2+ binding proteins, EhCaBP1 and EhCaBP3 in regulation of phagocytosis in the protist parasite E. histolytica and the novel mechanisms of manipulating actin dynamics at multiple levels.
Calcium binding and signaling orchestrate a wide variety of essential cellular functions, many of which employ the EF-hand Ca2+ binding motif. The ion binding parameters of this motif are controlled, in part, by the structure of its Ca2+ binding loop, termed the EF-loop. The EF-loops of different proteins are carefully specialized, or fine-tuned, to yield optimized Ca2+ binding parameters for their unique cellular roles. The present study uses a structurally homologous Ca2+ binding loop, that of the Escherichia coli galactose binding protein, as a model for the EF-loop in studies examining the contribution of the third loop position to intramolecular tuning. 10 different side chains are compared at the third position of the model EF-loop with respect to their effects on protein stability, sugar binding, and metal binding equilibria and kinetics. Substitution of an acidic Asp side chain for the native Asn is found to generate a 6,000-fold increase in the ion selectivity for trivalent over divalent cations, providing strong support for the electrostatic repulsion model of divalent cation charge selectivity. Replacement of Asn by neutral side chains differing in size and shape each alter the ionic size selectivity in a similar manner, supporting a model in which large-ion size selectivity is controlled by complex interactions between multiple side chains rather than by the dimensions of a single coordinating side chain. Finally, the pattern of perturbations generated by side chain substitutions helps to explain the prevalence of Asn and Asp at the third position of natural EF-loops and provides further evidence supporting the unique kinetic tuning role of the gateway side chain at the ninth EF-loop position.
calcium signaling; calmodulin; troponin C; metal binding site; ion channels
In bacteria, P1-type ATPases are responsible for resistance to di- and monovalent toxic heavy metals by taking them out of the cell. These ATPases have a cytoplasmic N terminus comprising metal binding domains defined by a βαββαβ fold and a CXXC metal binding motif. To check how the structural properties of the metal binding site in the N terminus can influence the metal specificity of the ATPase, the first structure of a Cd(II)-ATPase N terminus was determined by NMR and its coordination sphere was investigated by X-ray absorption spectroscopy. A novel metal binding environment was found, comprising the two conserved Cys residues of the metal binding motif and a Glu in loop 5. A bioinformatic search identifies an ensemble of highly homologous sequences presumably with the same function. Another group of highly homologous sequences is found which can be referred to as zinc-detoxifying P1-type ATPases with the metal binding pattern DCXXC in the N terminus. Because no carboxylate groups participate in Cu(I) or Ag(I) binding sites, we suggest that the acidic residue plays a key role in the coordination properties of divalent cations, hence conferring a function to the N terminus in the metal specificity of the ATPase.
CadA; NMR; P1-type ATPase; cadmium detoxification; XAS
Transient protein-protein and protein-ligand interactions are fundamental components of biological activity. To understand biological activity, not only the structures of the involved proteins are important but also the energetics of the individual steps of a reaction. Here we use in vitro biophysical methods to deduce thermodynamic parameters of copper (Cu) transfer from the human copper chaperone Atox1 to the fourth metal-binding domain of the Wilson disease protein (WD4). Atox1 and WD4 have the same fold (ferredoxin-like fold) and Cu-binding site (two surface exposed cysteine residues) and thus it is not clear what drives metal transfer from one protein to the other. Cu transfer is a two-step reaction involving a metal-dependent ternary complex in which the metal is coordinated by cysteines from both proteins (i.e., Atox1-Cu-WD4). We employ size exclusion chromatography to estimate individual equilibrium constants for the two steps. This information together with calorimetric titration data are used to reveal enthalpic and entropic contributions of each step in the transfer process. Upon combining the equilibrium constants for both steps, a metal exchange factor (from Atox1 to WD4) of 10 is calculated, governed by a negative net enthalpy change of ∼10 kJ/mol. Thus, small variations in interaction energies, not always obvious upon comparing protein structures alone, may fuel vectorial metal transfer.
Analysis of metal-protein interaction distances, coordination numbers, B-factors (displacement parameters), and occupancies of metal binding sites in protein structures determined by X-ray crystallography and deposited in the PDB shows many unusual values and unexpected correlations. By measuring the frequency of each amino acid in metal ion binding sites, the positive or negative preferences of each residue for each type of cation were identified. Our approach may be used for fast identification of metal-binding structural motifs that cannot be identified on the basis of sequence similarity alone. The analysis compares data derived separately from high and medium resolution structures from the PDB with those from very high resolution small-molecule structures in the Cambridge Structural Database (CSD). For high resolution protein structures, the distribution of metal-protein or metal-water interaction distances agrees quite well with data from CSD, but the distribution is unrealistically wide for medium (2.0 – 2.5 Å) resolution data. Our analysis of cation B-factors versus average B-factors of atoms in the cation environment reveals substantial numbers of structures contain either an incorrect metal ion assignment or an unusual coordination pattern. Correlation between data resolution and completeness of the metal coordination spheres is also found.
Metalloprotein; protein structure; metal binding
To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T9TKE12 sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from Kd = 25±6 nM to Kd = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (Kd = 0.25±0.06 nM). FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the νas(P-O) and νs(P-O) IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in νas(UO2)2+ vibration (from 923 cm−1 to 908 cm−1) was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH.
Many essential physiological processes are regulated by the modulation of calcium concentration in the cell. The EF-hand proteins represent a superfamily of calcium-binding proteins involved in calcium signaling and homeostasis. Secretagogin is a hexa-EF-hand protein that is highly expressed in pancreatic islet of Langerhans and neuroendocrine cells and may play a role in the trafficking of secretory granules. We present the X-ray structure of Danio rerio secretagogin, which is 73% identical to human secretagogin, in calcium-free form at 2.1-Å resolution. Secretagogin consists of the three globular domains each of which contains a pair of EF-hand motifs. The domains are arranged into a V-shaped molecule with a distinct groove formed at the interface of the domains. Comparison of the secretagogin structure with the solution structure of calcium-loaded calbindin D28K revealed a striking difference in the spatial arrangement of their domains, which involves approximately a 180-degree rotation of the first globular domain with respect to the module formed by the remaining domains.
X-ray structure; calcium binding protein; EF-hand motif; structural genomics
The glycosylphosphatidylinositol (GPI) moiety is one of the ways by which many cell surface proteins, such as Gal/GalNAc lectin and proteophosphoglycans (PPGs) attach to the surface of Entamoeba histolytica, the agent of human amoebiasis. It is believed that these GPI-anchored molecules are involved in parasite adhesion to cells, mucus and the extracellular matrix. We identified an E. histolytica homolog of PIG-M, which is a mannosyltransferase required for synthesis of GPI. The sequence and structural analysis led to the conclusion that EhPIG-M1 is composed of one signal peptide and 11 transmembrane domains with two large intra luminal loops, one of which contains the DXD motif, involved in the enzymatic catalysis and conserved in most glycosyltransferases. Expressing a fragment of the EhPIG-M1 encoding gene in antisense orientation generated parasite lines diminished in EhPIG-M1 levels; these lines displayed reduced GPI production, were highly sensitive to complement and were dramatically inhibited for amoebic abscess formation. The data suggest a role for GPI surface anchored molecules in the survival of E. histolytica during pathogenesis.
The causative agent of the infectious disease, amoebiasis, is the parasite Entamoeba histolytica, which targets human intestine and liver. Once in the host, this parasite attaches to human cells and matrix components via factors at its surface such as the Gal/GalNAc lectin and proteophosphoglycans (PPGs). These factors are themselves anchored to the parasite surface by a glycosylphosphatidylinositol (GPI) moiety. To synthesise the GPI, a cascade of enzymes are necessary including the mannosyltransferase 1 (PIG-M1). A homolog of the PIG-M1 enzyme was shown to be present in E. histolytica (EhPIG-M1). To study the role of EhPIG-M1 in E. histolytica, parasites were constructed that had a reduced amount of mannosyltransferase. These parasites displayed a diminished production of GPI molecules and a lower amount of PPGs at the cell surface. Interestingly, the parasites were highly sensitive to the host blood complement and the formation of liver abscesses in hamsters was dramatically impaired. These results suggest that molecules anchored to the cell surface with the GPI moiety have a pivotal role in the survival of E. histolytica during pathogenesis.
The Entamoeba histolytica transcription factor Upstream Regulatory Element 3-Binding Protein (URE3-BP) is a calcium-responsive regulator of two E. histolytica virulence genes, hgl5 and fdx1. URE3-BP was previously identified by a yeast one-hybrid screen of E. histolytica proteins capable of binding to the sequence TATTCTATT (Upstream Regulatory Element 3 (URE3)) in the promoter regions of hgl5 and fdx1. In this work, precise definition of the consensus URE3 element was performed by electrophoretic mobility shift assays (EMSA) using base-substituted oligonucleotides, and the consensus motif validated using episomal reporter constructs. Transcriptome profiling of a strain induced to produce a dominant-positive URE3-BP was then used to identify additional genes regulated by URE3-BP. Fifty modulated transcripts were identified, and of these the EMSA defined motif T[atg]T[tc][cg]T[at][tgc][tg] was found in over half of the promoters (54% p<0.0001). Fifteen of the URE3-BP regulated genes were potential membrane proteins, suggesting that one function of URE3-BP is to remodel the surface of E. histolytica in response to a calcium signal. Induction of URE3-BP leads to an increase in tranwell migration, suggesting a possible role in the regulation of cellular motility.
Most infections with Entamoeba histolytica are asymptomatic. However, in a minority of cases, they develop into invasive and even life-threatening amebiasis. We suspect, based on prior studies of invasive amebae, that changes in amebic gene expression enable the transition from asymptomatic to invasive infection. Our long-term goal is to identify the genetic program required to cause amebic colitis. Here, we studied a transcription factor named URE3-BP that controls the expression of two virulence genes, the Galactose and Galactose N- acetyl- galactosamine inhibitable lectin (Gal/GalNAc lectin) and ferredoxin. We suspected that this factor might coordinate invasiveness by co-regulating additional virulence factors. The consensus DNA motif that is recognized by URE3-BP was identified by reporter gene assays and by electromobility shift assays. We then inducibly expressed a constitutively active form of the transcription factor, and measured the changes in total amebic gene expression mediated by overexpression of this dominant-positive version of URE3-BP. This analysis allowed for a further definition of the functional URE3 motif. Inducible expression of URE3-BP led to changes in the transcript levels of several novel amebic membrane proteins. In conclusion, this genome-wide analysis of a transcription factor and its cis-acting regulatory sequence in Entamoeba histolytica has identified new transcripts regulated by URE3-BP that may play a role in trophozoite motility within a coordinated virulence-specific gene regulatory network.
A right-handed, calcium-dependent β-roll structure found in secreted proteases and repeat-in-toxin proteins was used as a template for the design of minimal, soluble, monomeric polypeptides that would fold in the presence of Ca2+. Two polypeptides were synthesised to contain two and four metal-binding sites, respectively, and exploit stacked tryptophan pairs to stabilise the fold and report on the conformational state of the polypeptide.
Initial analysis of the two polypeptides in the presence of calcium suggested the polypeptides were disordered. The addition of lanthanum to these peptides caused aggregation. Upon further study by right angle light scattering and electron microscopy, the aggregates were identified as ordered protein filaments that required lanthanum to polymerize. These filaments could be disassembled by the addition of a chelating agent. A simple head-to-tail model is proposed for filament formation that explains the metal ion-dependency. The model is supported by the capping of one of the polypeptides with biotin, which disrupts filament formation and provides the ability to control the average length of the filaments.
Metal ion-dependent, reversible protein filament formation is demonstrated for two designed polypeptides. The polypeptides form filaments that are approximately 3 nm in diameter and several hundred nm in length. They are not amyloid-like in nature as demonstrated by their behaviour in the presence of congo red and thioflavin T. A capping strategy allows for the control of filament length and for potential applications including the "decoration" of a protein filament with various functional moieties.
Metal ion binding domains are found in proteins that mediate transport, buffering or detoxification of metal ions. The objective of the study is to
design and analyze metal binding motifs against the genes involved in phytoremediation. This is being done on the basis of certain pre-requisite
amino-acid residues known to bind metal ions/metal complexes in medicinal and aromatic plants (MAP's). Earlier work on MAP's have shown
that heavy metals accumulated by aromatic and medicinal plants do not appear in the essential oil and that some of these species are able to grow
in metal contaminated sites. A pattern search against the UniProtKB/Swiss-Prot and UniProtKB/TrEMBL databases yielded true positives in
each case showing the high specificity of the motifs designed for the ions of nickel, lead, molybdenum, manganese, cadmium, zinc, iron, cobalt
and xenobiotic compounds. Motifs were also studied against PDB structures. Results of the study suggested the presence of binding sites on the
surface of protein molecules involved. PDB structures of proteins were finally predicted for the binding sites functionality in their respective
phytoremediation usage. This was further validated through CASTp server to study its physico-chemical properties. Bioinformatics implications
would help in designing strategy for developing transgenic plants with increased metal binding capacity. These metal binding factors can be used
to restrict metal update by plants. This helps in reducing the possibility of metal movement into the food chain.
Phytoremediation; medicinal and aromatic plants (MAPs); putative metal binding sites
A de novo protein design strategy provides a powerful tool to elucidate how heavy metals interact with proteins. Cysteine derivatives of the TRI peptide family (Ac-G(LKALEEK)4G-NH2) have been shown to bind heavy metals in an unusual trigonal geometry. Our present objective was to design binding sites in α-helical scaffolds that are able to form higher coordination number complexes with Cd(II) and Hg(II). Herein, we evaluate the binding of Cd(II) and Hg(II) to double cysteine substituted TRI peptides lacking intervening leucines between sulfurs in the heptads. We compare a -Cysd-X-X-X-Cysa- binding motif found in TRIL12CL16C to the more common -Cysa-X-X-Cysd- sequence of native proteins found in TRIL9CL12C. Compared to TRI, these substitutions destabilize the helical aggregates, leading to mixtures of two and three stranded bundles. The three stranded coiled coils are stabilized by the addition of metals. TRIL9CL12C forms distorted tetrahedral complexes with both Cd(II) and Hg(II), as supported by UV-vis, CD, 113Cd NMR, 199Hg NMR and 111mCd PAC spectroscopy. Additionally, these signatures are very similar to those found for heavy metal substituted rubredoxin. These results suggest that in terms of Hg(II) binding, TRIL9CL12C can be considered as a good mimic of the metallochaperone HAH1, that has previously been shown to form protein dimers. TRIL12CL16C has limited ability to generate homoleptic tetrahedral complexes (Cd(SR)42−). These type of complexes were identified only for Hg(II). However, the spectroscopic signatures suggest a different geometry around the metal ion, demonstrating that effective metal sequestration into the hydrophobic interior of the bundle requires more than simply adding two sulfur residues in adjacent layers of the peptide core. Thus, proper design of metal binding sites must also consider the orientation of cysteine sidechains in a vs d positions of the heptads.
Wilson disease is an autosomal recessive disorder of copper metabolism. The Wilson disease protein is a putative copper-transporting P-type ATPase, ATP7B, whose malfunction results in the toxic accumulation of copper in the liver and brain, causing the hepatic and/or neurological symptoms accompanying this disease. The cytosolic N-terminal domain (approximately 70 kDa) of this ATPase comprises six heavy metal-associated domains, each of which contains the conserved metal-binding motif GMTCXXC. The N-terminal domain (Wilson disease copper-binding domain [WCBD]) has been expressed, purified, and characterized using various techniques. The WCBD binds six atoms of copper in the +1 oxidation state competitively, and with a greater affinity than all other metals. The copper atom is coordinated by two cysteines in a distorted linear geometry. Copper binds the WCBD in a cooperative manner and induces secondary and tertiary conformation changes. Zinc binding to the WCBD has also been characterized by circular dichroism spectroscopy and shown to produce conformational changes that are completely different from those induced by copper. The phosphorylation/nucleotide-binding domain of ATP7B has also been expressed and characterized and shown to be capable of binding ATP but lacking ATPase activity. A peptide corresponding to the sixth transmembrane domain of ATP7B has been constructed and shown to undergo secondary conformational changes upon binding a single atom of copper. Finally, a chimeric protein consisting of the WCBD and truncated ZntA, a zinc-transporting ATPase lacking the N-terminal domain, has been constructed and analyzed for metal ion selectivity. These results suggest that the core determines the metal ion specificity of P-type ATPases, and the N-terminal metal-binding domain may play a regulatory role.
This summary examines some of the known and hypothesized roles of metallothionein and related proteins in mediating the metal metabolism and toxicity from a chemical perspective. It attempts to examine in kinetic terms how such molecules may exert homeostatic control over the intracellular bioavailability of metal ions to essential enzymatic or other molecular systems. The concept of ongoing competition between metallothionein and related proteins with other intracellular metal-binding sites for various metals is also examined in relation to the thermodynamic stability of these proteins. Comparisons between mammalian metallothionein and analogous nonmammalian proteins demonstrate both similarities and great differences in types of metal-binding sites, metal-binding constants, amino acid composition, and secondary structures such that apparent diversity of these low molecular weight metal-binding molecules in nature appears to be growing ever wider. The potential value of these data rests both in delineating new hypotheses for metallothionein evolution and in suggesting new model systems for discovering the normal function of metallothionein and related proteins in cells.
The crystal structure of the aspartyl-tRNA synthetase from the eukaryotic parasite Entamoeba histolytica has been determined at 2.8 Å resolution. Relative to homologous sequences, the E. histolytica protein contains a 43-residue insertion between the N-terminal anticodon binding domain and the C-terminal catalytic domain. The present structure reveals that this insertion extends an arm of the hinge region that has previously been shown to mediate interaction of aspartyl-tRNA synthetase with the cognate tRNA D-stem. Modeling indicates that this Entamoeba-specific insertion is likely to increase the interaction surface with the cognate tRNAAsp. In doing so it may substitute functionally for an RNA-binding motif located in N-terminal extensions found in AspRS sequences from lower eukaryotes but absent in Entamoeba. The E. histolytica AspRS structure shows a well-ordered N-terminus that contributes to the AspRS dimer interface.
Terpenoid synthases are ubiquitous enzymes that catalyze the formation of structurally and stereochemically diverse isoprenoid natural products. Many isoprenoid coupling enzymes and terpenoid cyclases from bacteria, fungi, protists, plants, and animals share the class I terpenoid synthase fold. Despite generally low amino acid sequence identity among these examples, class I terpenoid synthases contain conserved metal binding motifs that coordinate to a trinuclear metal cluster. This cluster not only serves to bind and orient the flexible isoprenoid substrate in the precatalytic Michaelis complex, but it also triggers the departure of the diphosphate leaving group to generate a carbocation that initiates catalysis. Additional conserved hydrogen bond donors assist the metal cluster in this function. Crystal structure analysis reveals that the constellation of three metal ions required for terpenoid synthase catalysis is generally identical among all class I terpenoid synthases of known structure.
enzyme catalysis; inorganic pyrophosphate; geranyl diphosphate; farnesyl diphosphate; Mg2+
Calmodulin (CaM) is a 16.8 kDa calcium binding protein involved in calcium-signal transduction. It is the canonical member of the EF-hand family of proteins, which are characterized by a helix-loop-helix calcium-binding motif. CaM is comprised of N- and C-terminal globular domains (N-CaM and C-CaM), and within each domain there are two EF-hand motifs. Upon binding calcium, CaM undergoes a significant, global conformational change involving reorientation of the four helix bundles in each of its two domains. This conformational change upon ion binding is a key component of the signal transduction and regulatory roles of CaM, yet the precise nature of this transition is still unclear. Here, we present a 1.3Å structure of zinc-bound N-terminal calmodulin (N-CaM) solved by single wavelength anomalous diffraction (SAD) phasing of a selenomethionyl-N-CaM. Our zinc-bound N-CaM structure differs from previously reported CaM structures and resembles calcium-free apo-CaM despite the zinc binding to both EF-hand motifs. Structural comparison with calcium-free apo-CaM, calcium-loaded CaM, and a crosslinked calcium-loaded CaM suggests that our zinc-bound N-CaM reveals an intermediate step in the initiation of metal ion binding at the first EF-hand motif. Our data also suggests that metal ion coordination by two key residues in the first metal-binding site represents an initial step in the conformational transition induced by metal binding. This is followed by reordering of the N-terminal region of the helix exiting from this first binding loop. This conformational switch should be incorporated into models of either step-wise conformational transition or flexible, dynamic energetic state-sampling based transition.
Calmodulin; EF-hand motif; metal ion binding; metal-ion induced conformational switch; X-ray crystallography; SAD phasing
The rubella virus (RUB) nonstructural protein (NS) open reading frame (ORF) encodes a polypeptide precursor that is proteolytically self cleaved into two replicase components involved in viral RNA replication. A putative EF-hand Ca2+-binding motif that was conserved across different genotypes of RUB was predicted within the nonstructural protease that cleaves the precursor by using bioinformatics tools. To probe the metal-binding properties of this motif, we used an established grafting approach and engineered the 12-residue Ca2+-coordinating loop into a non-Ca2+-binding scaffold protein, CD2. The grafted EF-loop bound to Ca2+ and its trivalent analogs Tb3+ and La3+ with Kds of 214, 47, and 14 μM, respectively. Mutations (D1210A and D1217A) of two of the potential Ca2+-coordinating ligands in the EF-loop led to the elimination of Tb3+ binding. Inductive coupled plasma mass spectrometry was used to confirm the presence of Ca2+ ([Ca2+]/[protein] = 0.7 ± 0.2) in an NS protease minimal metal-binding domain, RUBCa, that spans the EF-hand motif. Conformational studies on RUBCa revealed that Ca2+ binding induced local conformational changes and increased thermal stability (ΔTm = 4.1°C). The infectivity of an RUB infectious cDNA clone containing the mutations D1210A/D1217A was decreased by ∼20-fold in comparison to the wild-type (wt) clone, and these mutations rapidly reverted to the wt sequence. The NS protease containing these mutations was less efficient at precursor cleavage than the wt NS protease at 35°C, and the mutant NS protease was temperature sensitive at 39°C, confirming that the Ca2+-binding loop played a structural role in the NS protease and was specifically required for optimal stability under physiological conditions.
113Cd-NMR studies have been used to elucidate the structure of the metal-binding sites in mammalian and invertebrate ( Scylla serrata) metallothioneins (MTs). Chemical shift data have shown that all Cd ions are tetrahedrally coordinated to four cysteine thiolate ligands with single cysteinyl sulfurs bridging adjacent metals. Homonuclear decoupling experiments have shown that the 7 g-atoms of metal bound per mole of mammalian protein are located in a three- and a four-metal cluster while the 6 g-atoms of metal in the invertebrate MT are located in two three-metal clusters. The different metal binding affinities of the two mammalian clusters have been determined by 113Cd-NMR. The three-metal cluster prefers Cu greater than Zn greater than Cd whereas exactly the reverse order applies in the four-metal cluster. Proteolytic cleavage of the protein produced a 32-residue fragment which contained the four-metal cluster and demonstrated the presence of two separate domains in the protein. 500 MHz 1H-NMR has been employed to elucidate the arrangement of these metal clusters in the tertiary structure of the protein. The 1H resonances were assigned from their scalar and dipolar connectivities obtained from extensive one and two-dimensional NMR experiments. A specific application of 2D correlation spectroscopy ( COSY ) to the assignment of the 1H resonances in crab MT-1 is discussed. A molecular model, representing the three-dimensional solution structure of this protein, has been constructed based on an analysis of all these data. Detailed structural features of this model are discussed, with particular emphasis on their relationship to the function and evolution of the protein.
Metallothionein is a ubiquitous protein with a wide range of proposed physiological roles, including
the transport, storage and detoxification of essential and nonessential trace metals. The amino
acid sequence of isoform 2a of rabbit liver metallothionein, the isoform used in our spectroscopic
studies, includes 20 cysteinyl groups out of 62 amino acids. Metallothioneins in general represent
an impressive chelating agent for a wide range of metals. Structural studies carried out by a
number of research groups (using 1H and 113Cd NMR, X-ray crystallography, more recently EXAFS,
as well as optical spectroscopy) have established that there are three structural motifs for metal
binding to mammalian metallothioneins. These three structures are defined by metal to protein
stoichiometric ratios, which we believe specifically determine the coordination geometry adopted
by the metal in the metal binding site at that metal to protein molar ratio. Tetrahedral geometry is
associated with the thiolate coordination of the metals in the M7-MT species, for M = Zn(II), Cd(II),
and possibly also Hg(II), trigonal coordination is proposed in the M11-12-MT species, for M = Ag(I),
Cu(I), and possibly also Hg(II), and digonal coordination is proposed for the metal in the M17-18-MT
species for M = Hg(II), and Ag(I). The M7-MT species has been completely characterized for M =
Cd(II) and Zn(II). 113Cd NMR spectroscopic and x-ray crystallographic data show that mammalian
Cd7-MT and Zn7-MT have a two domain structure, with metal-thiolate clusters of the form M4(Scys)11 (the α domain) and M3(Scys)9 (the β domain). A similar two domain structure involving Cu6(Scys)11 (α) and Cu6(Scys)9 (β) copper-thiolate clusters has been proposed for the Cu12-MT species.
Copper-, silver- and gold-containing metallothioneins luminesce in the 500-600 nm region from
excited triplet, metal-based states that are populated by absorption into the 260-300 nm region of
the metal-thiolate charge transfer states. The luminescence spectrum provides a very sensitive
probe of the metal-thiolate cluster structures that form when Ag(I), Au(I), and Cu(I) are added to
metallothionein. CD spectroscopy has been used in our laboratory to probe the formation of
species that exhibit well-defined three-dimensional structures. Saturation of the optical signals
during titrations of MT with Cu(I) or Ag(I) clearly show formation of unique metal-thiolate structures
at specific metal:protein ratios. However, we have proposed that these M=7, 12 and 18 structures
form within a continuum of stoichiometries. Compounds prepared at these specific molar ratios
have been examined by X-ray Absorption Spectroscopy (XAS) and bond lengths have been
determined for the metal-thiolate clusters through the EXAFS technique. The stoichiometric ratio
data from the optical experiments and the bond lengths from the XAS experiments are used to
propose structures for the metal-thiolate binding site with reference to known inorganic
Ferritin, an iron homeostasis protein, has important functions in transition and storage of toxic metal ions. In this study, the full-length cDNA of ferritin was isolated from Dendrorhynchus zhejiangensis by cDNA library and RACE approaches. The higher similarity and conserved motifs for ferritin were also identified in worm counterparts, indicating that it belonged to a new member of ferritin family. The temporal expression of worm ferritin in haemocytes was analyzed by RT-PCR, and revealed the ferritin could be induced by Cd2+, Pb2+ and Fe2+. The heavy metal binding activity of recombinant ferritin was further elucidated by atomic force microscopy (AFM). It was observed that the ferritin protein could form a chain of beads with different size against three metals exposure, and the largest one with 35∼40 nm in height was identified in the Cd2+ challenge group. Our results indicated that worm ferritin was a promising candidate for heavy metals detoxification.
Crystal structures of human recombinant calmodulin were determined in the presence of lead and barium ions.
Calmodulin is a calcium sensor that is also capable of binding and being activated by other metal ions. Of specific interest in this respect is lead, which is known to be neurotoxic and to have a very high affinity towards calmodulin. Crystal structures of human calmodulin complexed with lead and barium ions have been solved. The results will help in understanding the activation mechanisms of calmodulin by different heavy metals and will provide a detailed view of a putative target for lead neurotoxicity in humans.
calmodulin; lead; barium; neurotoxicity; heavy-metal binding
All prokaryotes encode a panel of metal sensor or metalloregulatory proteins that govern the expression of genes that allows an organism to quickly adapt to toxicity or deprivation of both biologically essential transition metal ions, e.g., Zn, Cu, Fe, and heavy metal pollutants. As such, metal sensor proteins can be considered arbiters of intracellular transition metal bioavailability and thus potentially control the metallation state of the metalloproteins in the cell. Metal sensor proteins are specialized allosteric proteins that regulate transcription as a result direct binding of one or two cognate metal ions, to the exclusion of all others. In most cases, the binding of the cognate metal ion induces a structural change in a protein oligomer that either activates or inhibits operator DNA binding. A quantitative measure of the degree to which a particular metal drives metalloregulation of operator DNA-binding is the allosteric coupling free energy, ΔGc. In this review, we summarize recent work directed toward understanding metal occupancy and metal selectivity of these allosteric switches in selected families of metal sensor proteins and examine the structural origins of ΔGc in the functional context a thermodynamic “set-point” model of intracellular metal homeostasis.
metalloregulation; metal sensor protein; transition metal ions; allosteric coupling free energy; protein-DNA interactions; linkage