Bioinformatic Identification of the Most Ancient Copper Protein Architecture

Abstract

Since copper ions participate in many cellular processes and are implicated in pathogenesis of many diseases, copper proteins have important biological significance. Thus, it is of interest to explore their origins, especially to address the following question: which is the most ancient architecture of copper proteins? In this paper, through analyzing the architectural features of copper proteins, we find that the fold-domain relationship of these proteins follows a power law, which can be explained by preferential attachment principle and implicates that the architecture of the most ancient copper proteins belonged to Cupredoxin-like (b.6) fold. According to the chronology of protein folds, this architecture originated rather late, which can be understood in terms of the low abundance of reducing amino acids (e.g., His, Cys and/or Met) in the primordial world, because these amino acids are required by copper proteins to bind copper ions.     

Ragweed allergen Ra3: Relationship to some type 1 copper-binding proteins

Summary We have found ragweed allergen Ra3 to be related to the type 1 copper proteins; it is most closely related to stellacyanin and basic blue protein. The type 1 copper proteins form a diverse group of proteins, most of which are involved in electron transport. However, key amino acids believed to be involved in copper binding are absent from the allergen sequence; thus, the allergen is not likely to be functionally related to the type 1 copper proteins. We have grouped these proteins into one superfamily and we depict the relationships among them by an evolutionary tree. As indicated by this tree, an ancient gene duplication resulted in the divergence of plastocyanin from the line leading to basic blue protein, stellacyanin, and allergen Ra3.This paper is dedicated to the memory of Professor Margaret O. Dayhoff, whose contributions to the study of protein evolution made this investigation possible     

Organic cofactors participated more frequently than transition metals in redox reactions of primitive proteins

Protein redox reactions are one of the most basic and important biochemical actions. As amino acids are weak redox mediators, most protein redox functions are undertaken by protein cofactors, which include organic ligands and transition metal ions. Since both kinds of redox cofactors were available in the pre‐protein RNA world, it is challenging to explore which one was more involved in redox processes of primitive proteins? In this paper, using an examination of the redox cofactor usage of putative ancient proteins, we infer that organic ligands participated more frequently than transition metals in redox reactions of primitive proteins, at least as protein cofactors. This is further supported by the relative abundance of amino acids in the primordial world. Supplementary material for this article can be found on the BioEssays website. BioEssays 30:766–771, 2008. © 2008 Wiley Periodicals, Inc.     

Centrin isoforms in mammals. Relation to calmodulin

In mammals, three calmodulin (CaM) genes code for 100% identical proteins. In these species, four centrin (Cetn) genes have been reported to exist. They are examined in this paper. While the gene for Cetn 1 contains no introns and appears to be derived from Cetn 2 by retroposition, a gene product for Cetn 1 is expressed. Cetn 2, 3, and 4 represent bona fide genes. The major difference between the members of the CaM and the Cetn subfamilies is the presence (usually) in Cetn of an approximately 23 amino acids long (but occasionally much longer) protruding amino acid end. In all members of these two subgroups, four EF hand motifs (in this paper taken as loops containing 12 amino acids) are separated by 24, 25 and 24 amino acids (each a helix–loop–helix) positioned between motifs 1and 2, 2 and 3, and 3 and 4, respectively. This rule applies not only to CaM and Cetn in mammals but also to these two subfamilies in simpler eukaryotes such as Saccharomyces cerevisiae and Giardia lamblia. The various mRNA products can be identified most readily by their characteristic 3′ UTRs. While CaM is an ancient molecule that is expressed in all cells and is ubiquitous within these cells and interacts therein with almost 100 different proteins, many of which display the IQ or related binding motifs, the distribution and function of Cetn (an equally ancient molecule) is restricted mostly to basal bodies (e.g. in rods of the retina), axonemes, flagella, cilia and centrosomes. Are these two subclasses of calcium carriers (each molecule possessing four EF hands which possibly interact with different association constants)—if they are both present within a cell—randomly chosen for their service to the specific proteins with which they interact?     

Amino Acid Metabolic Origin as an Evolutionary Influence on Protein Sequence in Yeast

The metabolic cycle of Saccharomyces cerevisiae consists of alternating oxidative (respiration) and reductive (glycolysis) energy-yielding reactions. The intracellular concentrations of amino acid precursors generated by these reactions oscillate accordingly, attaining maximal concentration during the middle of their respective yeast metabolic cycle phases. Typically, the amino acids themselves are most abundant at the end of their precursor’s phase. We show that this metabolic cycling has likely biased the amino acid composition of proteins across the S. cerevisiae genome. In particular, we observed that the metabolic source of amino acids is the single most important source of variation in the amino acid compositions of functionally related proteins and that this signal appears only in (facultative) organisms using both oxidative and reductive metabolism. Periodically expressed proteins are enriched for amino acids generated in the preceding phase of the metabolic cycle. Proteins expressed during the oxidative phase contain more glycolysis-derived amino acids, whereas proteins expressed during the reductive phase contain more respiration-derived amino acids. Rare amino acids (e.g., tryptophan) are greatly overrepresented or underrepresented, relative to the proteomic average, in periodically expressed proteins, whereas common amino acids vary by a few percent. Genome-wide, we infer that 20,000 to 60,000 residues have been modified by this previously unappreciated pressure. This trend is strongest in ancient proteins, suggesting that oscillating endogenous amino acid availability exerted genome-wide selective pressure on protein sequences across evolutionary time. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Benjamin L. de Bivort and Ethan O. Perlstein have contributed equally to this work.     

Copper sensing based on the far-red fluorescent protein, HcRed, from Heteractis crispa

In this article, we report for the first time on the copper (Cu(2+)) binding characteristics of the far-red fluorescent protein, HcRed, and its application in the development of a reagentless sensing system for copper. The far-red emission of HcRed (lambda(max) = 645 nm) where background cellular fluorescence is low should prove to be advantageous in the development of the sensing system. In the studies performed in our laboratory, we found that the fluorescence of HcRed is quenched in the presence of copper ions (Cu(2+)). The results obtained through UV-visible and circular dichroism spectra generated in the presence and absence of copper, as well as Stern-Volmer plots at different temperatures, indicate static quenching of HcRed fluorescence in the presence of copper, possibly through the formation of a copper-protein complex. On the basis of this observation, we developed a reagentless sensing system for the detection of copper(II) based on HcRed as the biosensing element. A detection limit for Cu(2+) in the nanomolar range was obtained. HcRed was found to bind copper ions selectively when compared with other divalent ions. A dissociation constant of 3.6muM was observed for copper binding. Histidine and cysteine residues are commonly involved in copper binding within proteins; therefore, to investigate the role of these amino acids present in HcRed, we chemically modified Cys and His residues using iodoacetamide and diethyl pyrocarbonate, respectively. The effect of copper addition on the fluorescence of the chemically modified HcRed was investigated. The His modification of HcRed substantially affected copper ion binding, pointing to histidine as the possible amino acid residue involved in the binding of copper ions in HcRed. A purification strategy for HcRed was also developed based on a copper immobilized affinity column without the addition of any affinity tag on the protein. The HcRed-based copper sensing system can potentially be employed to perform intracellular copper detection by genetically encoding the biosensing element or can be employed in environmental sensing.     

Induction of stress proteins in chicken embryo cells by low-level zinc contamination in amino acid-free media

It has been reported that chicken embryo cells deprived of exogenous amino acids for 4 hours synthesize stress (heat-shock) proteins. Herein, we show that amino acid deprivation is not sufficient to cause induction of stress proteins. Zinc contaminating a component of commercial cell culture medium used to prepare amino acid-free medium was an inducer in our cultures. In the absence of exogenous amino acids, the concentration of zinc ions needed for half-maximal induction of stress proteins was an order of magnitude lower than the dose required for cells in complete medium. Histidine and cystine, which have high affinities for zinc ions, were the amino acids most effective in blocking the induction of stress proteins by zinc. Problems posed by heavy metal ions in culture media and biologic fluids for searches for in vivo inducers of the cellular stress (heat shock) response are discussed.     

Relevance of lysine snorkeling in the outer transmembrane domain of small viral potassium ion channels

Transmembrane domains (TMDs) are often flanked by Lys or Arg because they keep their aliphatic parts in the bilayer and their charged groups in the polar interface. Here we examine the relevance of this so-called "snorkeling" of a cationic amino acid, which is conserved in the outer TMD of small viral K(+) channels. Experimentally, snorkeling activity is not mandatory for Kcv(PBCV-1) because K29 can be replaced by most of the natural amino acids without any corruption of function. Two similar channels, Kcv(ATCV-1) and Kcv(MT325), lack a cytosolic N-terminus, and neutralization of their equivalent cationic amino acids inhibits their function. To understand the variable importance of the cationic amino acids, we reanalyzed molecular dynamics simulations of Kcv(PBCV-1) and N-terminally truncated mutants; the truncated mutants mimic Kcv(ATCV-1) and Kcv(MT325). Structures were analyzed with respect to membrane positioning in relation to the orientation of K29. The results indicate that the architecture of the protein (including the selectivity filter) is only weakly dependent on TMD length and protonation of K29. The penetration depth of Lys in a given protonation state is independent of the TMD architecture, which leads to a distortion of shorter proteins. The data imply that snorkeling can be important for K(+) channels; however, its significance depends on the architecture of the entire TMD. The observation that the most severe N-terminal truncation causes the outer TMD to move toward the cytosolic side suggests that snorkeling becomes more relevant if TMDs are not stabilized in the membrane by other domains.     

Human apo-lipoprotein B from normal plasma contains oxidised peptides

Oxidation of low density lipoprotein (LDL) may be atherogenic, but radical-initiated oxidation of its apoprotein B-100 (apoB) has been little studied. Transition metal ions iron and copper are candidates for mediating radical oxidation of LDL in vivo. Therefore, we studied the copper-ion-induced oxidation of apoB in human LDL. Using HPLC methods developed in our recent work, we studied the destruction of native and the generation of six oxidised amino acids; we also assessed the release of peptides from the LDL particle by FPLC. We observed time-dependent losses of apoB histidine, lysine and glycine. Long-lived reactive species, the reductant DOPA, and the oxidant hydroperoxides of valine and leucine (measured as hydroxides after reduction), were generated. Their relative abundance (mol/mol of parent amino acid) was DOPA>o- and m-tyrosine>dityrosine, valine-hydroxides, leucine hydroxides. Low molecular weight fragments were also released from the LDL in a time-dependent manner, contained hydroperoxides sensitive to GSH peroxidase, and generated radicals on reaction with iron–EDTA. The fragments contained peptides active in the quinone redox cycling procedure, comprising 0.25% of the supplied LDL amino acids. Characteristic peptides were present in each FPLC fraction containing the fragments, as judged by further HPLC fractionation. Some fragments were present in the unoxidised LDL preparations, and when these were largely removed by FPLC, copper oxidation could still generate fragments, suggesting that those present in the starting material might indicate prior oxidation. Concordantly, we found that fresh plasma LDL apoB contained 3% of total plasma protein-bound oxidised amino acids, and with the same relative abundance. We conclude that plasma proteins including apoB are subject to physiological oxidation, similar to that inflicted by copper ions; the latter may contribute to intimal LDL oxidation, which could be the source of oxidised plasma apoB.     

Spasmin and a Putative Spasmin Binding Protein(s) Isolated from Solubilized Spasmonemes1

ABSTRACT The amino acid composition and hydrophobicity scale (hydropathy) of calcium-binding proteins contained in the contractile spasmoneme of Carchesium polypinum was compared with other calcium-binding proteins from eukaryotes. Spasmins which may hind at most 4 calcium ions simultaneously and initiate spasmoneme contraction cooperatively belong to a super family of proteins including; centrin/caltractin and calmodulin. Based on chemical modification of tryptophan and methionine, these residues are involved in contraction but the spasmin proteins contain little or none of these amino acids. Based on this evidence, it is suggested that another, non-calcium binding protein(s) is involved in spasmoneme contraction.     

The catalytic effect of L- and D-histidine on alanine and lysine peptide formation

A starting phase of chemical evolution on our ancient Earth around 4 billion years ago was the formation of amino acids and their combination to peptides and proteins. The salt-induced peptide formation (SIPF) reaction has been shown to be appropriate for this condensation reaction under moderate and plausible primitive Earth conditions, forming short peptides from amino acids in aqueous solution containing sodium chloride and Cu(II) ions. In this paper we report results about the formation of dialanine and dilysine from their monomers in this reaction. The catalytic influence of l- and d-histidine dramatically increases dialanine yields when starting from lower alanine concentrations, but also dilysine formation is markedly boosted by these catalysts. Attention is paid to measurable preferences for one enantiomeric form of alanine and lysine in the SIPF reaction. Alanine, especially, shows stereospecific behaviour, mostly in favour of the l-form.     

Is salivary histatin 5 a metallopeptide?

Histatins are small histidine-rich salivary polypeptides which exhibit antimicrobial activity against Candida albicans. This antimicrobial activity has been ascribed in part to a high content of basic amino acids. However, unlike most other antimicrobial proteins histatins have a high content of histidine, tyrosine and acidic amino acids known to participate in metal ion coordination. This study was conducted to test whether histatin 5 could bind zinc and copper which are metals present in salivary secretions and whole saliva. Physical binding parameters and spectral properties of zinc- and copper-histatin complexes were investigated in order to obtain direct evidence of these interactions. A spectrophotometric competition assay using the metallochromic indicator murexide showed that histatin 5 dissociates metal indicator complexes containing zinc or copper ions. Absorption spectra of histatin 5 at increasing copper chloride concentrations resulted in higher absorbance in the 230-280 nm wavelength range and this spectral change was saturated at a peptide:metal molar ratio of approx. 1:1. A corresponding band was observed in the visible range of the spectrum with a maximum and molar extinction coefficient corresponding to that of copper binding to an ATCUN motif. Quantitative assessment of zinc and copper binding to histatin 5 using isothermal titration calorimetry revealed at least one high affinity site for each metal, with binding constants of 1.2x10(5) and 2.6x10(7) M(-1), respectively. These results indicate that histatin 5 exhibits metallopeptide-like properties. The precise biological significance of this has not yet been established but histatins may contribute significantly to salivary metal binding capacity.     

Efforts towards the design of 'teflon' proteins: in vivo translation with trifluorinated leucine and methionine analogues

In vivo incorporation of monofluorinated noncanonical amino acids into recombinant proteins has been well-established for decades. Proteins fluorinated in this way proved to be useful tools for many practical applications. In contrast, trifluorinated amino acids have been incorporated in only a few peptides and relatively small proteins by using expression systems in living cells. A novel class of proteins with a fluorous core can be envisaged only if full replacement of the core-building hydrophobic and aliphatic amino acids such as leucine or methionine with the related analogues trifluoromethionine and trifluoroleucine would be feasible. However, our systematic efforts to introduce these amino acids in larger proteins (over 10 Da) that contain different structural motifs clearly show that only partial substitutions are possible. The reasons are high toxicity of these substances and difficulties to accommodate them into the compact cores of natural proteins without adverse effects on their structural integrity. Therefore, engineering of such three dimensional 'Teflon'-like structures would require, besides an expansion of the amino acid repertoire of the genetic code, a de novo protein design as well.     

Acquisition of dietary copper: a role for anion transporters in intestinal apical copper uptake

Copper is an essential micronutrient in humans and is required for a wide range of physiological processes, including neurotransmitter biosynthesis, oxidative metabolism, protection against reactive oxygen species, and angiogenesis. The first step in the acquisition of dietary copper is absorption from the intestinal lumen. The major human high-affinity copper uptake protein, human copper transporter hCTR1, was recently shown to be at the basolateral or blood side of both intestinal and renal epithelial cell lines and thus does not play a direct role in this initial step. We sought to functionally identify the major transport pathways available for the absorption of dietary copper across the apical intestinal membrane using Caco2 cells, a well-established model for human enterocytes. The initial rate of apical copper uptake into confluent monolayers of Caco2 cells is greatly elevated if amino acids and serum proteins are removed from the growth media. Uptake from buffered saline solutions at neutral pH (but not at lower pH) is inhibited by either d- or l-histidine, unaltered by the removal of sodium ions, and inhibited by ～90% when chloride ions are replaced by gluconate or sulfate. Chloride-dependent copper uptake occurs with Cu(II) or Cu(I), although Cu(I) uptake is not inhibited by histidine, nor by silver ions. A well-characterized inhibitor of anion exchange systems, DIDS, inhibited apical copper uptake by 60-70%, while the addition of Mn(II) or Fe(II), competitive substrates for the divalent metal transporter DMT1, had no effect on copper uptake. We propose that anion exchangers play an unexpected role in copper absorption, utilizing copper-chloride complexes as pseudo-substrates. This pathway is also observed in mouse embryonic fibroblasts, human embryonic kidney cells, and Cos-7 cells. The special environment of low pH, low concentration of protein, and protonation of amino acids in the early intestinal lumen make this pathway especially important in dietary copper acquisition.     

Viscosity B-coefficients and standard partial molar volumes of amino acids, and their roles in interpreting the protein (enzyme) stabilization

This review systematically surveys the viscosity B-coefficients and standard partial molar volumes of amino acids at various temperatures as these data are quite important for interpreting the hydration and other properties of peptides and proteins. The effect of organic solutes and various ions on the viscometric and volumetric properties of amino acids has also been discussed in terms of their kosmotropic ('structure-making') effects on the hydration of amino acids. The comparison of these effects on the amino acid hydration enables us to have a better understanding of the influence of organic solute and salt on the protein stabilization. In addition, the viscometric and volumetric behaviors of amino acid ions (cations and anions) are also summarized because these ions have recently been incorporated as part of novel ionic liquids, which have wide applications in biocatalysis and protein stabilization.     

Oxidative modification of human ceruloplasmin by methylglyoxal: an in vitro study

Methylglyoxal (MG) is an endogenous physiological metabolite which is present in increased concentrations in diabetics. MG reacts with the amino acids of proteins to form advanced glycation end products. In this in vitro study, we investigated the effect of MG on the structure and function of ceruloplasmin (CP) a serum oxidase carrier of copper ions in the human. When CP was incubated with MG, the protein showed increased electrophoretic mobility which represented the aggregates at a high concentration of MG (100 mM). MG-mediated CP aggregation led to the loss of enzymatic activity and the release of copper ions from the protein. Radical scavengers and copper ion chelators significantly prevented CP aggregation. CP is an important protein that circulates in plasma as a major copper transport protein. It is suggested that oxidative damage of CP by MG may induce perturbations of the copper transport system and subsequently lead to harmful intracellular condition. The proposed mechanism, in part, may provide an explanation for the deterioration of organs in the diabetic patient.     

Cohesion Group Approach for Evolutionary Analysis of Aspartokinase,an Enzyme That Feeds a Branched Network of Many Biochemical Pathways

Summary: Aspartokinase (Ask) exists within a variable network that supports the synthesis of 9 amino acids and a number of other important metabolites. Lysine, isoleucine, aromatic amino acids, and dipicolinate may arise from the ASK network or from alternative pathways. Ask proteins were subjected to cohesion group analysis, a methodology that sorts a given protein assemblage into groups in which evolutionary continuity is assured. Two subhomology divisions, ASK_α and ASK_β, have been recognized. The ASK_α subhomology division is the most ancient, being widely distributed throughout the Archaea and Eukarya and in some Bacteria. Within an indel region of about 75 amino acids near the N terminus, ASK_β sequences differ from ASK_α sequences by the possession of a proposed ancient deletion. ASK_β sequences are present in most Bacteria and usually exhibit an in-frame internal translational start site that can generate a small Ask subunit that is identical to the C-terminal portion of the larger subunit of a heterodimeric unit. Particularly novel are ask genes embedded in gene contexts that imply specialization for ectoine (osmotic agent) or aromatic amino acids. The cohesion group approach is well suited for the easy recognition of relatively recent lateral gene transfer (LGT) events, and many examples of these are described. Given the current density of genome representation for Proteobacteria, it is possible to reconstruct more ancient landmark LGT events. Thus, a plausible scenario in which the three well-studied and iconic Ask homologs of Escherichia coli are not within the vertical genealogy of Gammaproteobacteria, but rather originated via LGT from a Bacteroidetes donor, is supported.     

Minireview: Recent progress in hemocyanin research

This review summarizes recent highlights of our joint work on the structure, evolution, and function of a family of highly complex proteins, the hemocyanins. They are blue-pigmented oxygen carriers, occurring freely dissolved in the hemolymph of many arthropods and molluscs. They are copper type-3 proteins and bind one dioxygen molecule between two copper atoms in a side-on coordination. They possess between 6 and 160 oxygen-binding sites, and some of them display the highest molecular cooperativity observed in nature. The functional properties of hemocyanins can be convincingly described by either the Monod-Wyman-Changeux (MWC) model or its hierarchical extension, the Nested MWC model; the latter takes into account the structural hierarchies in the oligomeric architecture. Recently, we applied these models to interpret the influence of allosteric effectors in detailed terms. Effectors shift the allosteric equilibria but have no influence on the oxygen affinities characterizing the various conformational states. We have shown that hemocyanins from species living at different environmental temperatures have a cooperativity optimum at the typical temperature of their natural habitat. Besides being oxygen carriers, some hemocyanins function as a phenoloxidase (tyrosinase/catecholoxidase) which, however, requires activation. Chelicerates such as spiders and scorpions lack a specific phenoloxidase, and in these animals activated hemocyanin might catalyse melanin synthesis in vivo. We propose a similar activation mechanism for arthropod hemocyanins, molluscan hemocyanins and tyrosinases: amino acid(s) that sterically block the access of phenolic compounds to the active site have to be removed. The catalysis mechanism itself can now be explained on the basis of the recently published crystal structure of a tyrosinase. In a series of recent publications, we presented the complete gene and primary structure of various hemocyanins from different molluscan classes. From these data, we deduced that the molluscan hemocyanin molecule evolved ca. 740 million years ago, prior to the separation of the extant molluscan classes. Our recent advances in the 3D cryo-electron microscopy of hemocyanins also allow considerable insight into the oligomeric architecture of these proteins of high molecular mass. In the case of molluscan hemocyanin, the structure of the wall and collar of the basic decamers is now rapidly becoming known in greater detail. In the case of arthropod hemocyanin, a 10-? structure and molecular model of the Limulus 8 × 6mer shows the amino acids at the various interfaces between the eight hexamers, and reveals histidine-rich residue clusters that might be involved in transferring the conformational signals establishing cooperative oxygen binding.     

Sodium ion-dependent amino acid transport in membrane vesicles of Bacillus stearothermophilus.

Amino acid transport in membrane vesicles of Bacillus stearothermophilus was studied. A relatively high concentration of sodium ions is needed for uptake of L-alanine (Kt = 1.0 mM) and L-leucine (Kt = 0.4 mM). In contrast, the Na(+)-H(+)-L-glutamate transport system has a high affinity for sodium ions (Kt less than 5.5 microM). Lithium ions, but no other cations tested, can replace sodium ions in neutral amino acid transport. The stimulatory effect of monensin on the steady-state accumulation level of these amino acids and the absence of transport in the presence of nonactin indicate that these amino acids are translocated by a Na+ symport mechanism. This is confirmed by the observation that an artificial delta psi and delta mu Na+/F but not a delta pH can act as a driving force for uptake. The transport system for L-alanine is rather specific. L-Serine, but not L-glycine or other amino acids tested, was found to be a competitive inhibitor of L-alanine uptake. On the other hand, the transport carrier for L-leucine also translocates the amino acids L-isoleucine and L-valine. The initial rates of L-glutamate and L-alanine uptake are strongly dependent on the medium pH. The uptake rates of both amino acids are highest at low external pH (5.5 to 6.0) and decline with increasing pH. The pH allosterically affects the L-glutamate and L-alanine transport systems. The maximal rate of L-glutamate uptake (Vmax) is independent of the external pH between pH 5.5 and 8.5, whereas the affinity constant (Kt) increases with increasing pH. A specific transport system for the basic amino acids L-lysine and L-arginine in the membrane vesicles has also been observed. Transport of these amino acids occurs most likely by a uniport mechanism.