Making proteins green; biosynthesis of chlorophyll-binding proteins in cyanobacteria

Chlorophyll (Chl) is an essential component of the photosynthetic apparatus. Embedded into Chl-binding proteins, Chl molecules play a central role in light harvesting and charge separation within the photosystems. It is critical for the photosynthetic cell to not only ensure the synthesis of a sufficient amount of new Chl-binding proteins but also avoids any misbalance between apoprotein synthesis and the formation of potentially phototoxic Chl molecules. According to the available data, Chl-binding proteins are translated on membrane bound ribosomes and their integration into the membrane is provided by the SecYEG/Alb3 translocon machinery. It appears that the insertion of Chl molecules into growing polypeptide is a prerequisite for the correct folding and finishing of Chl-binding protein synthesis. Although the Chl biosynthetic pathway is fairly well-described on the level of enzymatic steps, a link between Chl biosynthesis and the synthesis of apoproteins remains elusive. In this review, I summarize the current knowledge about this issue putting emphasis on protein–protein interactions. I present a model of the Chl biosynthetic pathway organized into a multi-enzymatic complex and physically attached to the SecYEG/Alb3 translocon. Localization of this hypothetical large biosynthetic centre in the cyanobacterial cell is also discussed as well as regulatory mechanisms coordinating the rate of Chl and apoprotein synthesis.     

Di-iron—carboxylate proteins

Di-iron centers bridged by carboxylate residues and oxide/hydroxide groups have so far been seen in four classes of proteins involved in dioxygen chemistry or phosphoryl transfer reactions. The dinuclear iron centers in these proteins are coordinated by histidines and additional carboxylate ligands. Recent structural data on some of these enzymes, combined with spectroscopic and kinetic data, can now serve as a base for detailed mechanistic suggestions. The di-iron sites in the major class of hydroxylase-oxidase enzymes, which contains ribonucleotide reductase and methane monooxygenase, show significant flexibility in the geometry of their coordination of three or more carboxylate groups. This flexibility, combined with a relatively low coordination number, and a buried environment suitable for reactive oxygen chemistry, explains their efficient harnessing of the oxidation power of molecular oxygen.     

ELIPs – Light-induced stress proteins

Exposure of plants to light intensities higher than those required to saturate photosynthesis leads to a reduction in photosynthetic capacity. This effect is known as photoinhibition. Photoinhibition is followed by destruction of carotenoids, bleaching of chlorophylls and increased lipid peroxidation due to damage by oxygen-derivatives. The oxygen concentration in chloroplasts in the light is high because of oxygen production by photosystem II (PSII). This can result in the release of reactive intermediates of reduced dioxygen such as superoxide radicals, hydroxyl radicals, hydrogen peroxide or singlet oxygen. In order to maintain their normal function under light stress conditions, chloroplasts have developed multiple repair and protection systems. The induction of specific light stress proteins, the ELIPs (for early light-induced proteins) can be considered to be part of these protective responses. The accumulation of ELIPs under light stress conditions is correlated with the photoinactivation of PSII, degradation of the D_l-protein of PSII reaction centre and changes in the level of pigments. Futhermore, the accumulation of ELIPs in the thylakoids is strictly controlled by the pigment content, especially by chlorophylls. Isolation of ELIPs in a native form and analysis of pigments bound to these proteins revealed that ELIPs can bind chlorophyll a and lutein. These data indicate that ELIPs might represent unique chlorophyll-binding proteins which have a transient function(s) during light stress. A transient 'pigment-carrier' function is postulated for ELIPs.     

Wheat PR‐1 proteins are targeted by necrotrophic pathogen effector proteins

Recent studies have identified that proteinaceous effectors secreted by Parastagonospora nodorum are required to cause disease on wheat. These effectors interact in a gene‐for‐gene manner with host‐dominant susceptibilty loci, resulting in disease. However, whilst the requirement of these effectors for infection is clear, their mechanisms of action remain poorly understood. A yeast‐two‐hybrid library approach was used to search for wheat proteins that interacted with the necrotrophic effector SnTox3. Using this strategy we indentified an interaction between SnTox3 and the wheat pathogenicity‐related protein TaPR‐1‐1, and confirmed it by in‐planta co‐immunprecipitation. PR‐1 proteins represent a large family (23 in wheat) of proteins that are upregulated early in the defence response; however, their function remains ellusive. Interestingly, the P. nodorum effector SnToxA has recently been shown to interact specifically with TaPR‐1‐5. Our analysis of the SnTox3–TaPR‐1 interaction demonstrated that SnTox3 can interact with a broader range of TaPR‐1 proteins. Based on these data we utilised homology modeling to predict, and validate, regions on TaPR‐1 proteins that are likely to be involved in the SnTox3 interaction. Precipitating from this work, we identified that a PR‐1‐derived defence signalling peptide from the C‐terminus of TaPR‐1‐1, known as CAPE1, enhanced the infection of wheat by P. nodorum in an SnTox3‐dependent manner, but played no role in ToxA‐mediated disease. Collectively, our data suggest that P. nodorum has evolved unique effectors that target a common host‐protein involved in host defence, albeit with different mechanisms and potentially outcomes.     

Helix‐sheet packing in proteins

The three‐dimensional structure of a protein is organized around the packing of its secondary structure elements. Although much is known about the packing geometry observed between α‐helices and between β‐sheets, there has been little progress on characterizing helix–sheet interactions. We present an analysis of the conformation of αβ₂ motifs in proteins, corresponding to all occurrences of helices in contact with two strands that are hydrogen bonded. The geometry of the αβ₂ motif is characterized by the azimuthal angle θ between the helix axis and an average vector representing the two strands, the elevation angle ψ between the helix axis and the plane containing the two strands, and the distance D between the helix and the strands. We observe that the helix tends to align to the two strands, with a preference for an antiparallel orientation if the two strands are parallel; this preference is diminished for other topologies of the β‐sheet. Side‐chain packing at the interface between the helix and the strands is mostly hydrophobic, with a preference for aliphatic amino acids in the strand and aromatic amino acids in the helix. From the knowledge of the geometry and amino acid propensities of αβ₂ motifs in proteins, we have derived different statistical potentials that are shown to be efficient in picking native‐like conformations among a set of non‐native conformations in well‐known decoy datasets. The information on the geometry of αβ₂ motifs as well as the related statistical potentials have applications in the field of protein structure prediction. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Structure‐based barcoding of proteins

A reduced representation in the format of a barcode has been developed to provide an overview of the topological nature of a given protein structure from 3D coordinate file. The molecular structure of a protein coordinate file from Protein Data Bank is first expressed in terms of an alpha‐numero code and further converted to a barcode image. The barcode representation can be used to compare and contrast different proteins based on their structure. The utility of this method has been exemplified by comparing structural barcodes of proteins that belong to same fold family, and across different folds. In addition to this, we have attempted to provide an illustration to (i) the structural changes often seen in a given protein molecule upon interaction with ligands and (ii) Modifications in overall topology of a given protein during evolution. The program is fully downloadable from the website http://www.iitg.ac.in/probar/ .     

Carotenoid-binding proteins; accessories to carotenoid function

Understanding of the widespread biological importance of carotenoids is increasing. Accompanying this is the developing recognition that the interaction of carotenoids with other molecules, such as proteins, is also essential. Here the significance of carotenoid-protein interactions with respect to biological function is reviewed for three well characterised carotenoprotein complexes; crustacyanin, the orange carotenoid protein and glutathione-S-transferase P1. In addition a preliminary report is made on the recent partial purification of an echinenone-binding protein extracted from a New Zealand sea urchin, Evechinus chloroticus.     

Water‐mediated interactions destabilize proteins

Proteins are folded to avoid exposure of the nonpolar groups to water because water‐mediated interactions between nonpolar groups are a promising factor in the thermodynamic stabilities of proteins—which is a well‐accepted view as one of the unique effects of hydrophobic interactions. This article poses a critical question for this classical view by conducting an accurate solvation free‐energy calculation for a thermodynamic cycle of a protein folding using a liquid‐state density functional theory. Here, the solvation‐free energy for a leucine zipper formation was examined in the coiled‐coil protein GCN4‐p1, a typical model for hydrophobic interactions, which demonstrated that water‐mediated interactions were unfavorable for the association of nonpolar groups in the native state, while the dispersion forces between them were, instead, responsible for the association. Furthermore, the present analysis well predicted the isolated helical state stabilized by pressure, which was previously observed in an experiment. We reviewed the problems in the classical concept and semiempirical presumption that the energetic cost of the hydration of nonpolar groups is a driving force of folding.     

Heterogeneous interactome between Litopenaeus vannamei plasma proteins and Vibrio parahaemolyticus outer membrane proteins

A great loss has been suffered by microbial infectious diseases under intensive shrimp farming in recent years. In this background, the understanding of shrimp innate immunity becomes an importantly scientific issue, but little is known about the heterogeneous protein–protein interaction between pathogenic cells and hosts, which is a key step for the invading microbes to infect internet organs through bloodstream. In the present study, bacterial outer membrane (OM) protein array and pull-down approaches are used to isolate both Vibrio parahaemolyticus OM proteins that bind to shrimp serum proteins and the shrimp serum proteins that interact with bacterial cells, respectively. Three interacting shrimp serum proteins, hemocyanin, β-1,3-glucan binding protein and LV_HP_RA36F08r and thirty interacting OM proteins were determined. They form 63 heterogeneous protein–protein interactions. Nine out of the 30 OM proteins were randomly demonstrated to be up-regulated or down-regulated when bacterial cells were cultured with shrimp sera, indicating the biological significance of the network. The interesting findings uncover the complexity of struggle between host immunity and bacterial infection. Compared with our previous report on heterogeneous interactome between fish grill and bacterial OM proteins, the present study further extends the investigation from lower vertebrates to invertebrates and develops a bacterial OM protein array to identify the OM proteins bound with shrimp serum proteins, which elevates the frequencies of the bound OM proteins. Our results highlight the way to determine and understand the heterogeneous interaction between hosts and microbes.     

β-Lactam resistance in Streptococcus pneumoniae: penicillin-binding proteins and non-penicillin-binding proteins

The beta-lactams are by far the most widely used and efficacious of all antibiotics. Over the past few decades, however, widespread resistance has evolved among most common pathogens. Streptococcus pneumoniae has become a paradigm for understanding the evolution of resistance mechanisms, the simplest of which, by far, is the production of beta-lactamases. As these enzymes are frequently plasmid encoded, resistance can readily be transmitted between bacteria. Despite the fact that pneumococci are naturally transformable organisms, no beta-lactamase-producing strain has yet been described. A much more complex resistance mechanism has evolved in S. pneumoniae that is mediated by a sophisticated restructuring of the targets of the beta-lactams, the penicillin-binding proteins (PBPs); however, this may not be the whole story. Recently, a third level of resistance mechanisms has been identified in laboratory mutants, wherein non-PBP genes are mutated and resistance development is accompanied by deficiency in genetic transformation. Two such non-PBP genes have been described: a putative glycosyltransferase, CpoA, and a histidine protein kinase, CiaH. We propose that these non-PBP genes are involved in the biosynthesis of cell wall components at a step prior to the biosynthetic functions of PBPs, and that the mutations selected during beta-lactam treatment counteract the effects caused by the inhibition of penicillin-binding proteins.     

‘Holy’ proteins I: ribonuclease inhibitor

The X-ray structure of the ribonuclease inhibitor from porcine pancreas shows a remarkable non-globular fold. It possesses a large central hole that forms part of the RNase A binding site.     

The TolQ–TolR proteins energize TolA and share homologies with the flagellar motor proteins MotA–MotB

The Tol-Pal system of Escherichia coli is required for the maintenance of outer membrane stability. Recently, proton motive force (pmf) has been found to be necessary for the co-precipitation of the outer membrane lipoprotein Pal with the inner membrane TolA protein, indicating that the Tol-Pal system forms a transmembrane link in which TolA is energized. In this study, we show that both TolQ and TolR proteins are essential for the TolA-Pal interaction. A point mutation within the third transmembrane (TM) segment of TolQ was found to affect the TolA-Pal interaction strongly, whereas suppressor mutations within the TM segment of TolR restored this interaction. Modifying the Asp residue within the TM region of TolR indicated that an acidic residue was important for the pmf-dependent interaction of TolA with Pal and outer membrane stabilization. Analysis of sequence alignments of TolQ and TolR homologues from numerous Gram-negative bacterial genomes, together with analyses of the different tolQ-tolR mutants, revealed that the TM domains of TolQ and TolR present structural and functional homologies not only to ExbB and ExbD of the TonB system but also with MotA and MotB of the flagellar motor. The function of these three systems, as ion potential-driven molecular motors, is discussed