The outer pore of the glutamate receptor channel has 2-fold rotational symmetry

The ligand binding domain of glutamate receptors (GluRs) has 2-fold rotational symmetry. The structure including the symmetry of the GluR ion channel remains undefined. Here we used substituted cysteines in the pore-lining M3 segment of the AMPAR GluR-A subunit and various cysteine-reactive agents to study the structure of the channel during gating. We find that cysteines substituted at A+6, located in the highly conserved SYTANLAAF motif, are grouped in pairs consistent with a 2-fold symmetry in the extracellular part of the pore. To account for this symmetry and crosslinking, we propose that the M3 segments in two neighboring GluR subunits are kinked within SYTANLAAF in opposite directions relative to the central axis of the pore. Our results extend the 2-fold rotational symmetry from the ligand binding domain to at minimum the extracellular part of the channel and suggest a model of gating movements in GluR pore-forming domains.     

A strain of Synechocystis sp. PCC 6803 without photosynthetic oxygen evolution and respiratory oxygen consumption: implications for the study of cyclic photosynthetic electron transport

Cyclic electron transport around photosystem (PS) I is believed to play a role in generation of ATP required for adaptation to stress in cyanobacteria and plants. However, elucidation of the pathway(s) of cyclic electron flow is difficult because of low rates of this electron flow relative to those of linear photosynthetic and respiratory electron transport. We have constructed a strain of Synechocystis sp. PCC 6803 that lacks both PSII and respiratory oxidases and that, consequently, neither evolves nor consumes oxygen. However, this strain is still capable of cyclic electron flow around PSI. The photoheterotrophic growth rate of this strain increased with light intensity up to an intensity of about 25 mumol photons m-2 s-1, supporting the notion that cyclic electron flow contributes to ATP generation in this strain. Indeed, the ATP-generating ability of PSI is demonstrated by the fact that the PSII-less oxidase-less strain is able to grow at much higher salt concentrations than a strain lacking PSI. A quinone electrode was used to measure the redox state of the plastoquinone pool in vivo in the various strains used in this study. In contrast to what is observed in chloroplasts, the plastoquinone pool was rather reduced in darkness and was oxidized in the light. This is in line with significant electron donation by respiratory pathways (NADPH dehydrogenase and particularly succinate dehydrogenase) in darkness. In the light, the pool becomes oxidized due to the presence of much more PSI than PSII. In the oxidase-less strains, the plastoquinone pool was very much reduced in darkness and was oxidized in the light by PSI. Photosystem II activity did not greatly alter the redox state of the plastoquinone pool. The results suggest that cyclic electron flow around PSI can contribute to generation of ATP, and a strain deficient in linear electron transport pathways provides an excellent model for further investigations of cyclic electron flow.     

Lectin-like proteins in model organisms: implications for evolution of carbohydrate-binding activity.

Classes of intracellular lectins that recognize core-type structures and mediate intracellular glycoprotein trafficking are present in vertebrates, model invertebrates such as Caenorhabditis elegans and Drosophila melanogaster, plants, and yeasts. Lectins that recognize more complex structures at the cell surface, such as C-type lectins and galectins, are also found in invertebrate organisms as well as vertebrates, but the functions of these proteins have evolved differently in different animal lineages.     

Membrane transport proteins: implications of sequence comparisons.

Analyses of the sequences and structures of many transport proteins that differ in substrate specificity, direction of transport and mechanism of transport suggest that they form a family of related proteins. Their sequence similarities imply a common mechanism of action. This hypothesis provides an objective basis for examining their mechanisms of action and relationships to other transporters.     

Convergence and divergence in the evolution of transport proteins

Different families of transport proteins catalyze transmembrane solute translocation, employing different mechanisms and energy sources. Several of these functionally dissimilar proteins nevertheless exhibit similar strutural units, consisting of six tightly packed α-helices which may comprise all or part of a transmembrane channel. It is now recognized that some of these families arose independently of each other by convergence, while others arose from common precursors by divergence. The former families apparently arose at different times in evolutionary history, in different groups of organisms, employing different routes.     

Copper transport and its defect in Wilson disease: characterization of the copper-binding domain of Wilson disease ATPase

Copper is an essential trace element which forms an integral component of many enzymes. While trace amounts of copper are needed to sustain life, excess copper is extremely toxic. An attempt is made here to present the current understanding of the normal transport of copper in relation to the absorption, intracellular transport and toxicity. Wilson disease is a genetic disorder of copper transport resulting in the accumulation of copper in organs such as liver and brain which leads to progressive hepatic and neurological damage. The gene responsible for Wilson disease (ATP7B) is predicted to encode a putative copper-transporting P-type ATPase. An important feature of this ATPase is the presence of a large N-terminal domain that contains six repeats of a copper-binding motif which is thought to be responsible for binding this metal prior to its transport across the membrane. We have cloned, expressed and purified the N-terminal domain (approximately 70 kD) of Wilson disease ATPase. Metal-binding properties of the domain showed the protein to bind several metals besides copper; however, copper has a higher affinity for the domain. The copper is bound to the domain in Cu(I) form with a copper: protein ratio of 6.5:1. X-ray absorption studies strongly suggest Cu(I) atoms are ligated to cysteine residues. Circular dichroism spectral analyses suggest both secondary and tertiary structural changes upon copper binding to the domain. Copper-binding studies suggest some degree of cooperativity in binding of copper. These studies as well as detailed structural information of the copper-binding domain will be crucial in determining the specific role played by the copper-transporting ATPase in the homeostatic control of copper in the body and how the transport of copper is interrupted by mutations in the ATPase gene.     

NIFAS: visual analysis of domain evolution in proteins

MOTIVATION: Multi-domain proteins have evolved by insertions or deletions of distinct protein domains. Tracing the history of a certain domain combination can be important for functional annotation of multi-domain proteins, and for understanding the function of individual domains. In order to analyze the evolutionary history of the domains in modular proteins it is desirable to inspect a phylogenetic tree based on sequence divergence with the modular architecture of the sequences superimposed on the tree. RESULT: A Java applet, NIFAS, that integrates graphical domain schematics for each sequence in an evolutionary tree was developed. NIFAS retrieves domain information from the Pfam database and uses CLUSTAL W to calculate a tree for a given Pfam domain. The tree can be displayed with symbolic bootstrap values, and to allow the user to focus on a part of the tree, the layout can be altered by swapping nodes, changing the outgroup, and showing/collapsing subtrees. NIFAS is integrated with the Pfam database and is accessible over the internet (http://www.cgr.ki.se/Pfam). As an example, we use NIFAS to analyze the evolution of domains in Protein Kinases C.     

Intracellular transport of recombinant coronavirus spike proteins: implications for virus assembly.

Coronavirus spike protein genes were expressed in vitro by using the recombinant vaccinia virus expression system. Recombinant spike proteins were expressed at the cell surface and induced cell fusion in a host-cell-dependent fashion. The intracellular transport of recombinant spike proteins was studied. The half time of acquisition of resistance to endo-beta-N-acetylglucosaminidase H was approximately 3 h for the recombinant feline infectious peritonitis virus S protein. The S protein in feline infectious peritonitis virus-infected cells was found to have a half time of acquisition of resistance to endo-beta-N-acetylglucosaminidase H of approximately 1 h. This difference can be explained by the fact that coronavirus budding takes place at intracellular membranes and that the oligosaccharides of the spike protein are modified after budding. Apparently, spike protein incorporated into budded virions is transported faster through the Golgi apparatus than is spike protein alone. These findings provide new insights into the mechanism of coronavirus budding and are discussed in relation to current models of intracellular transport and sorting of proteins.     

On the frequency of arginine in proteins and its implications for molecular evolution

Evidence is presented against the concept that arginine appeared later in the evolution of life that the other common amino acids, as an ‘evolutionary intruder’. Alternative explanations for the relatively low frequency of arginine in proteins are considered, based on the proposition that there has been selection for such low frequency because of special properties of the amino acid.     

Role of the copper-binding domain in the copper transport function of ATP7B, the P-type ATPase defective in Wilson disease

We have analyzed the functional effect of site-directed mutations and deletions in the copper-binding domain of ATP7B (the copper transporting P-type ATPase defective in Wilson disease) using a yeast complementation assay. We have shown that the sixth copper-binding motif alone is sufficient, but not essential, for normal ATP7B function. The N-terminal two or three copper-binding motifs alone are not sufficient for ATP7B function. The first two or three N-terminal motifs of the copper-binding domain are not equivalent to, and cannot replace, the C-terminal motifs when placed in the same sequence position with respect to the transmembrane channel. From our data, we propose that the copper-binding motifs closest to the channel are required for the copper-transport function of ATP7B. We propose that cooperative copper binding to the copper-binding domain of ATP7B is not critical for copper transport function, but that cooperative copper binding involving the N-terminal two or three copper-binding motifs may be involved in initiating copper-dependent intracellular trafficking. Our data also suggest a functional difference between the copper-binding domains of ATP7A and ATP7B.     

Plant hemoglobins: a molecular fossil record for the evolution of oxygen transport

The evolution of oxygen transport hemoglobins occurred on at least two independent occasions. The earliest event led to myoglobin and red blood cell hemoglobin in animals. In plants, oxygen transport "leghemoglobins" evolved much more recently. In both events, pentacoordinate heme sites capable of inert oxygen transfer evolved from hexacoordinate hemoglobins that have unrelated functions. High sequence homology between hexacoordinate and pentacoordinate hemoglobins in plants has poised them for potential structural analysis leading to a molecular understanding of this important evolutionary event. However, the lack of a plant hexacoordinate hemoglobin structure in the exogenously ligand-bound form has prevented such comparison. Here we report the crystal structure of the cyanide-bound hexacoordinate hemoglobin from barley. This presents the first opportunity to examine conformational changes in plant hexacoordinate hemoglobins upon exogenous ligand binding, and reveals structural mechanisms for stabilizing the high-energy pentacoordinate heme conformation critical to the evolution of reversible oxygen binding hemoglobins.     

Molecular evolution of PAS domain-containing proteins of filamentous cyanobacteria through domain shuffling and domain duplication.

When the entire genome of a filamentous heterocyst-forming N2-fixing cyanobacterium, Anabaena sp. PCC 7120 (Anabaena) was determined in 2001, a large number of PAS domains were detected in signal-transducing proteins. The draft genome sequence is also available for the cyanobacterium, Nostoc punctiforme strain ATCC 29133 (Nostoc), that is closely related to Anabaena. In this study, we extracted all PAS domains from the Nostoc genome sequence and analyzed them together with those of Anabaena. Clustering analysis of all the PAS domains gave many specific pairings, indicative of evolutionary conservations. Ortholog analysis of PAS-containing proteins showed composite multidomain architecture in some cases of conserved domains and domains of disagreement between the two species. Further inspection of the domains of disagreement allowed us to trace them back in evolution. Thus, multidomain proteins could have been generated by duplication or shuffling in these cyanobacteria. The conserved PAS domains in the orthologous proteins were analyzed by structural fitting to the known PAS domains. We detected several subclasses with unique sequence features, which will be the target of experimental analysis.     

An inactivated nuclease-like domain in RecC with novel function: implications for evolution

Background  

The PD-(D/E)xK superfamily, containing a wide variety of other exo- and endonucleases, is a notable example of general function conservation in the face of extreme sequence and structural variation. Almost all members employ a small number of shared conserved residues to bind catalytically essential metal ions and thereby effect DNA cleavage. The crystal structure of the RecBCD prokaryotic DNA repair machinery shows that RecB contains such a nuclease domain at its C-terminus. The RecC C-terminal region was reported as having a novel fold.     

Copper-induced production of copper-binding supernatant proteins by the marine bacterium Vibrio alginolyticus.

Growth of the marine bacterium Vibrio alginolyticus is temporarily inhibited by micromolar levels of copper. During the copper-induced lag phase, supernatant compounds which complex and detoxify copper are produced. In this study two copper-inducible supernatant proteins having molecular masses of ca. 21 and 19 kilodaltons (CuBP1 and CuBP2) were identified; these proteins were, respectively, 25 and 46 times amplified in supernatants of copper-challenged cultures compared with controls. Experiments in which chloramphenicol was added to cultures indicated that there was de novo synthesis of these proteins in response to copper. When supernatants were separated by gel permeation chromatography, CuBP1 and CuBP2 coeluted with a copper-induced peak in copper-binding activity. CuBP1 and CuBP2 from whole supernatants were concentrated and partially purified by using a copper-charged immobilized metal ion affinity chromatography column, confirming the affinity of these proteins for copper. A comparison of cell pellets and supernatants demonstrated that CuBP1 was more concentrated in supernatants than in cells. Our data are consistent with a model for a novel mechanism of copper detoxification in which excretion of copper-binding protein is induced by copper.     

Inhibition of Photosystem II by formate. Possible evidence for a direct role of bicarbonate in photosynthetic oxygen evolution

In broken chloroplasts the presence of 100 mM sodium formate at pH 8.2 will specifically lengthen the Photosystem II relaxation times of the reactions S′₂ → S₃ and S′₃ → S₀. Rates of reactions S′₀ → S₁ and S′₁ → S₂ remain unaffected. Evidence is presented which indicates the discrimination among S-states by formate cannot be attributed to a block imposed on the reducing side of Photosystem II. The results are interpreted in context of the known interaction of formate and CO₂ which is bound to the Photosystem II reaction center complex. It is proposed that those S-state transitions which show extended relaxation times in the presence of formate must result in the momentary release and rebinding of CO₂. Furthermore since formate is acting on the oxygen-evolving side of Photosystem II, it would seem that CO₂ is released in reactions that occur there. A chemical model of oxygen evolution is presented. It is based on the hypothesis that hydrated CO₂ is the immediate source of photosynthetically evolved oxygen and explains why, under certain conditions formate slows only the S-state transitions S′₂ → S₃ and S′₃ → S₀.     

Expression, purification and copper-binding studies of the first metal-binding domain of Menkes protein.

The cDNA, coding for the first metal-binding domain (MBD1) of Menkes protein, was cloned into the T7-system based vector, pCA. The T7 lysozyme-encoding plasmid, pLysS, is shown to be crucial for expression, suggesting that the protein is toxic to the cells. Adding copper to the growth medium did not affect the plasmid stability. MBD1 is purified in two steps with a typical yield of 12 mg.L-1. Menkes protein, a P-type ATPase, contains a sequence GMXCXSC that is repeated six times, at the N-terminus. The paired cysteine residues are involved in metal binding. MBD1 has only two cysteine residues, which can exist as free thiol groups (reduced), as a disulphide bond (oxidized) or bound to a metal ion [e.g. Cu(I)-MBD1]. These three MBD1 forms have been investigated using CD. No major spectral change was seen between the different MBD1 forms, indicating that the folding is not changed upon metal binding. A copper-bound MBD1 was also studied by EPR, and the lack of an EPR signal suggests that the oxidation state of copper bound to MBD1 is Cu(I). Cu(I) binding studies were performed by equilibrium dialysis and revealed a stoichiometry of 1 : 1 and an apparent Kd = 46 microM. Oxidized MBD1, however, is not able to bind copper. Different copper complexes were investigated for their ability to reconstitute apo-MBD1. Given the same total copper concentration CuCl43- was superior to Cu(I)-thiourea (structural analogue of metallothionein) and Cu(I)-glutathione (used at fivefold higher copper concentration) although the latter two were able to partially reconstitute apo-MBD1. Cu(II) was not able to reconstitute apo-MBD1, presumably due to Cu(II)-induced oxidation of the thiol groups. Based on our results, glutathione and/or metallothionein are likely candidates for the in vivo incorporation of copper to Menkes protein.     

A second symmetry mismatch at the portal vertex of bacteriophage T7: 8-fold symmetry in the procapsid core

Like other bacteriophages, T7 has a singular vertex that is the site of a symmetry mismatch involving the portal/connector protein, a 12-fold ring at the vertex site which is also a 5-fold axis for the icosahedral capsid. In the mature virion, a 6-fold-symmetric tail extends outwards from the connector. T7 also has a cylindrical "core" that assembles on the inner surface of the connector during procapsid formation, is retained in the mature virion, and is required for infectivity. We have investigated the core structure by cryo-electron microscopy and image analysis of procapsids and find that it observes 8-fold symmetry. Stoichiometry data indicate that its major constituent is an octamer of gp15.     

Possible implications in the use of exogenous phospholipids.

The development of new techniques has shed more light on the physical state of phospholipids within the living cell. This has helped in the development of new concepts of the structural inter-relationship of phospholipids with proteins and other natural constituents and also of their functional role (e.g. enzyme modulators). On the other hand, understanding of phospholipid behaviour within the biological environment has encouraged research into the possibility of assigning to these molecules the role of carriers for exogenous or endogenous biologically active agents. Possible applications of this carrier potential in biology and medicine are discussed.     

The role of facilitated diffusion in oxygen transport by cell-free hemoglobins: implications for the design of hemoglobin-based oxygen carriers

We compared rates of oxygen transport in an in vitro capillary system using red blood cells (RBCs) and cell-free hemoglobins. The axial PO(2) drop down the capillary was calculated using finite-element analysis. RBCs, unmodified hemoglobin (HbA(0)), cross-linked hemoglobin (alpha alpha-Hb) and hemoglobin conjugated to polyethylene-glycol (PEG-Hb) were evaluated. According to their fractional saturation curves, PEG-Hb showed the least desaturation down the capillary, which most closely matched the RBCs; HbA(0) and alpha alpha-Hb showed much greater desaturation. A lumped diffusion parameter, K*, was calculated based on the Fick diffusion equation with a term for facilitated diffusion. The overall rates of oxygen transfer are consistent with hemoglobin diffusion rates according to the Stokes-Einstein Law and with previously measured blood pressure responses in rats. This study provides a conceptual framework for the design of a 'blood substitute' based on mimicking O(2) transport by RBCs to prevent autoregulatory changes in blood flow and pressure.