Structure of the Mg-Chelatase Cofactor GUN4 Reveals a Novel Hand-Shaped Fold for Porphyrin Binding

In plants, the accumulation of the chlorophyll precursor Mg-protoporphyrin IX (Mg-Proto) in the plastid regulates the expression of a number of nuclear genes with functions related to photosynthesis. Analysis of the plastid-to-nucleus signaling activity of Mg-Proto in Arabidopsis thaliana led to the discovery of GUN4, a novel porphyrin-binding protein that also dramatically enhances the activity of Mg-chelatase, the enzyme that synthesizes Mg-Proto. GUN4 may also play a role in both photoprotection and the cellular shuttling of tetrapyrroles. Here we report a 1.78-Å resolution crystal structure of Synechocystis GUN4, in which the porphyrin-binding domain adopts a unique three dimensional fold with a “cupped hand” shape. Biophysical and biochemical analyses revealed the specific site of interaction between GUN4 and Mg-Proto and the energetic determinants for the GUN4 • Mg-Proto interaction. Our data support a novel protective function for GUN4 in tetrapyrrole trafficking. The combined structural and energetic analyses presented herein form the physical-chemical basis for understanding GUN4 biological activity, including its role in the stimulation of Mg-chelatase activity, as well as in Mg-Proto retrograde signaling

Plasmodium vivax Tryptophan-Rich Antigen PvTRAg33.5 Contains Alpha Helical Structure and Multidomain Architecture

Tryptophan-rich proteins from several malarial parasites have been identified where they play an important role in host-parasite interaction. Structural characterization of these proteins is needed to develop them as therapeutic targets. Here, we describe a novel Plasmodium vivax tryptophan-rich protein named PvTRAg33.5. It is expressed by blood stage(s) of the parasite and its gene contains two exons. The exon 1 encodes for a 23 amino acids long putative signal peptide which is likely to be cleaved off whereas the exon 2 encodes for the mature protein of 252 amino acids. The mature protein contains B-cell epitopes which were recognized by the human immune system during P.vivax infection. The PvTRAg33.5 contains 24 (9.5%) tryptophan residues and six motifs whose patterns were similar among tryptophan-rich proteins. The modeled structure of the PvTRAg33.5 consists of a multidomain architecture which is stabilized by the presence of large number of tryptophan residues. The recombinant PvTRAg33.5 showed predominantly α helical structure and alpha helix to beta sheet transition at pH below 4.5. Protein acquires an irreversible non-native state at temperature more than 50°C at neutral pH. Its secondary and tertiary structures remain stable in the presence of 35% alcohol but these structures are destabilized at higher alcohol concentrations due to the disturbance of hydrophobic interactions between tryptophanyl residues. These structural changes in the protein might occur during its translocation to interact with other proteins at its final destination for biological function such as erythrocyte invasion

The Photosynthetic Apparatus and Its Regulation in the Aerobic Gammaproteobacterium Congregibacter litoralis gen. nov., sp. nov

BACKGROUND: There is accumulating evidence that in some marine environments aerobic bacteriochlorophyll a-producing bacteria represent a significant part of the microbial population. The interaction of photosynthesis and carbon metabolism in these interesting bacteria is still largely unknown and requires further investigation in order to estimate their contribution to the marine carbon cycle. METHODOLOGY/PRINCIPAL FINDINGS: Here, we analyzed the structure, composition and regulation of the photosynthetic apparatus in the obligately aerobic marine gammaproteobacterium KT71(T). Photoheterotrophically grown cells were characterized by a poorly developed lamellar intracytoplasmic membrane system, a type 1 light-harvesting antenna complex and a photosynthetic reaction center associated with a tetraheme cytochrome c. The only photosynthetic pigments produced were bacteriochlorophyll a and spirilloxanthin. Under semiaerobic conditions KT71(T) cells expressing a photosynthetic apparatus showed a light-dependent increase of growth yield in the range of 1.3-2.5 fold. The expression level of the photosynthetic apparatus depended largely on the utilized substrate, the intermediary carbon metabolism and oxygen tension. In addition, pigment synthesis was strongly influenced by light, with blue light exerting the most significant effect, implicating that proteins containing a BLUF domain may be involved in regulation of the photosynthetic apparatus. Several phenotypic traits in KT71(T) could be identified that correlated with the assumed redox state of growing cells and thus could be used to monitor the cellular redox state under various incubation conditions. CONCLUSIONS/SIGNIFICANCE: In a hypothetical model that explains the regulation of the photosynthetic apparatus in strain KT71(T) we propose that the expression of photosynthesis genes depends on the cellular redox state and is maximal under conditions that allow a balanced membrane redox state. So far, bacteria capable of an obligately aerobic, photosynthetic metabolism constitute a unique phenotype within the class Gammaproteobacteria, so that it is justified to propose a new genus and species, Congregibacter litoralis gen. nov, sp. nov., represented by the type strain KT71(T) ( = DSM 17192(T) = NBRC 104960(T))

Application of a Small EF Hand Affinity Tag for Expression, Purification and Biophysical Studies of G Protein-Coupled Membrane Receptors

Heptahelical G protein-coupled receptors (GPCR) comprise a large family of integral membrane proteins involved in a wide array of cell signaling pathways. For high resolution structural studies of these receptors, multi-milligram quantities of pure and structurally unperturbed proteins are required. Purification of recombinant GPCRs typically involves their solubilization into detergent micelles followed by chromatographic purification. Because of relatively low expression levels of these recombinant receptors, it is challenging to design an efficient strategy for selective and efficient purification with high yield. Here, we describe a recently introduced purification system employing a high affinity molecular switch based on fragment complementation, with a calcium dependent capture and EDTA mediated chelation elution. This technique was successfully applied to the purification of the recombinant cannabinoid receptor CB2, a promising target for the development of drugs for inflammation, immunological disorders and pain. It is feasible that similar strategies can be successfully employed for expression and purification of other membrane protein targets.Science Foundation IrelandIntramural Research Program of the NIAA

TspO as a Modulator of the Repressor/Antirepressor (PpsR/AppA) Regulatory System in Rhodobacter sphaeroides 2.4.1

The TspO outer membrane protein of Rhodobacter sphaeroides has been shown to be involved in controlling the transcription of a number of genes which encode enzymes involved in photopigment biosynthesis and the puc operon. The display of regulated genes appears identical to those genes encompassing the PpsR/AppA repressor/antirepressor regulon, although the effect of TspO is modest relative to that of PpsR/AppA. To directly address the hypothesis that TspO is effective through the PpsR/AppA system, we constructed mutant strains with mutations in both tspO and appA. In all cases, the phenotypes examined resembled those of the appA lesion by itself, leading us to conclude that TspO works through or modulates the PpsR/AppA system and acts upstream of the site of action of these regulatory proteins. In earlier publications, we had suggested that TspO is involved in the efflux of a certain intermediate(s) of the porphyrin biosynthesis pathway and that transcriptional regulation of target gene expression could be explained by the accumulation of a coactivator of AppA function. Although the data reported here do not precisely identify this coactivator, they lend support to this hypothesis. We discuss the importance of this form of gene control as the result of the recent extension of the TspO system to Sinorhizobium meliloti, as described by Davey and de Bruijn (M. E. Davey and F. J. de Bruijn, Appl. Environ. Microbiol. 66:5353–5359, 2000). It is therefore possible that this system constitutes a more widely, although not universally, demonstrated form of gene regulation