Biochemical Journal - BJ Gene

New insights into the structure of the reaction centre from Blastochloris viridis: evolution in the laboratory

2012-02-15

Newly determined crystal structures of the photosynthetic RC (reaction centre) from two substrains of the non-sulfur purple bacterium Blastochloris viridis strain DSM 133, together with analysis of their gene sequences, has revealed intraspecies evolutionary changes over a period of 14 years. Over 100 point mutations were identified between these two substrains in the four genes encoding the protein subunits of the RC, of which approximately one-fifth resulted in a total of 16 amino acid changes. The most interesting difference was in the M subunit where the change from a leucine residue to glycine in the carotenoid-binding pocket allowed NS5 (1,2-dihydroneurosporene) to adopt a more sterically favoured conformation, similar to the carotenoid conformation found in other related RCs. The results of the present study, together with a high rate of mutations in laboratory bacterial cultures described recently, suggest that bacteria evolve faster than has been generally recognized. The possibility that amino acid changes occur within protein sequences, without exhibiting any immediately observable phenotype, should be taken into account in studies that involve long-term continuous growth of pure bacterial cultures. The Blc. viridis RC is often studied with sophisticated biophysical techniques and changes such as those described here may well affect their outcome. In other words, there is a danger that laboratory-to-laboratory variation could well be due to different groups not realising that they are actually working with slightly different proteins. A way around this problem is suggested.

Identification of two independent nucleosome-binding domains in the transcriptional co-activator SPBP

2012-02-15

Transcriptional regulation requires co-ordinated action of transcription factors, co-activator complexes and general transcription factors to access specific loci in the dense chromatin structure. In the present study we demonstrate that the transcriptional co-regulator SPBP [stromelysin-1 PDGF (platelet-derived growth factor)-responsive element binding protein] contains two independent chromatin-binding domains, the SPBP-(1551–1666) region and the C-terminal extended PHD [ePHD/ADD (extended plant homeodomain/ATRX-DNMT3-DNMT3L)] domain. The region 1551–1666 is a novel core nucleosome-interaction domain located adjacent to the AT-hook motif in the DNA-binding domain. This novel nucleosome-binding region is critically important for proper localization of SPBP in the cell nucleus. The ePHD/ADD domain associates with nucleosomes in a histone tail-dependent manner, and has significant impact on the dynamic interaction between SPBP and chromatin. Furthermore, SPBP and its homologue RAI1 (retinoic-acid-inducible protein 1), are strongly enriched on chromatin in interphase HeLa cells, and both proteins display low nuclear mobility. RAI1 contains a region with homology to the novel nucleosome-binding region SPBP-(1551–1666) and an ePHD/ADD domain with ability to bind nucleosomes. These results indicate that the transcriptional co-regulator SPBP and its homologue RAI1 implicated in Smith–Magenis syndrome and Potocki–Lupski syndrome both belong to the expanding family of chromatin-binding proteins containing several domains involved in specific chromatin interactions.

Staphylococcus aureus DinG, a helicase that has evolved into a nuclease

2012-02-15

DinG (damage inducible gene G) is a bacterial superfamily 2 helicase with 5′→3′ polarity. DinG is related to the XPD (xeroderma pigmentosum complementation group D) helicase family, and they have in common an FeS (iron–sulfur)-binding domain that is essential for the helicase activity. In the bacilli and clostridia, the DinG helicase has become fused with an N-terminal domain that is predicted to be an exonuclease. In the present paper we show that the DinG protein from Staphylococcus aureus lacks an FeS domain and is not a DNA helicase, although it retains DNA-dependent ATP hydrolysis activity. Instead, the enzyme is an active 3′→5′ exonuclease acting on single-stranded DNA and RNA substrates. The nuclease activity can be modulated by mutation of the ATP-binding cleft of the helicase domain, and is inhibited by ATP or ADP, suggesting a modified role for the inactive helicase domain in the control of the nuclease activity. By degrading rather than displacing RNA or DNA strands, the S. aureus DinG nuclease may accomplish the same function as the canonical DinG helicase.

ZraP is a periplasmic molecular chaperone and a repressor of the zinc-responsive two-component regulator ZraSR

2012-02-15

The bacterial envelope is the interface with the surrounding environment and is consequently subjected to a barrage of noxious agents including a range of compounds with antimicrobial activity. The ESR (envelope stress response) pathways of enteric bacteria are critical for maintenance of the envelope against these antimicrobial agents. In the present study, we demonstrate that the periplasmic protein ZraP contributes to envelope homoeostasis and assign both chaperone and regulatory function to ZraP from Salmonella Typhimurium. The ZraP chaperone mechanism is catalytic and independent of ATP; the chaperone activity is dependent on the presence of zinc, which is shown to be responsible for the stabilization of an oligomeric ZraP complex. Furthermore, ZraP can act to repress the two-component regulatory system ZraSR, which itself is responsive to zinc concentrations. Through structural homology, ZraP is a member of the bacterial CpxP family of periplasmic proteins, which also consists of CpxP and Spy. We demonstrate environmental co-expression of the CpxP family and identify an important role for these proteins in Salmonella's defence against the cationic antimicrobial peptide polymyxin B.

Insights into the regulation of eukaryotic elongation factor 2 kinase and the interplay between its domains

2012-02-15

eEF2K (eukaryotic elongation factor 2 kinase) is a Ca²⁺/CaM (calmodulin)-dependent protein kinase which regulates the translation elongation machinery. eEF2K belongs to the small group of so-called ‘α-kinases’ which are distinct from the main eukaryotic protein kinase superfamily. In addition to the α-kinase catalytic domain, other domains have been identified in eEF2K: a CaM-binding region, N-terminal to the kinase domain; a C-terminal region containing several predicted α-helices (resembling SEL1 domains); and a probably rather unstructured ‘linker’ region connecting them. In the present paper, we demonstrate: (i) that several highly conserved residues, implicated in binding ATP or metal ions, are critical for eEF2K activity; (ii) that Ca²⁺/CaM enhance the ability of eEF2K to bind to ATP, providing the first insight into the allosteric control of eEF2K; (iii) that the CaM-binding/α-kinase domain of eEF2K itself possesses autokinase activity, but is unable to phosphorylate substrates in trans; (iv) that phosphorylation of these substrates requires the SEL1-like domains of eEF2K; and (v) that highly conserved residues in the C-terminal tip of eEF2K are essential for the phosphorylation of eEF2, but not a peptide substrate. On the basis of these findings, we propose a model for the functional organization and control of eEF2K.

Induction of group IVC phospholipase A2 in allergic asthma: transcriptional regulation by TNFα in bronchoepithelial cells

2012-02-15

Airway inflammation in allergen-induced asthma is associated with eicosanoid release. These bioactive lipids exhibit anti- and pro-inflammatory activities with relevance to pulmonary pathophysiology. We hypothesized that sensitization/challenge using an extract from the ubiquitous fungus Aspergillus fumigatus in a mouse model of allergic asthma would result in altered phospholipase gene expression, thus modulating the downstream eicosanoid pathway. We observed the most significant induction in the group IVC PLA₂ (phospholipase A₂) [also known as cPLA₂γ (cytosolic PLA₂γ) or PLA2G4C]. Our results infer that A. fumigatus extract can induce cPLA₂γ levels directly in eosinophils, whereas induction in lung epithelial cells is most likely to be a consequence of TNFα (tumour necrosis factor α) secretion by A. fumigatus-activated macrophages. The mechanism of TNFα-dependent induction of cPLA₂γ gene expression was elucidated through a combination of promoter deletions, ChIP (chromatin immunoprecipitation) and overexpression studies in human bronchoepithelial cells, leading to the identification of functionally relevant CRE (cAMP-response element), NF-κB (nuclear factor κB) and E-box promoter elements. ChIP analysis demonstrated that RNA polymerase II, ATF-2 (activating transcription factor 2)–c-Jun, p65–p65 and USF (upstream stimulating factor) 1–USF2 complexes are recruited to the cPLA₂γ enhancer/promoter in response to TNFα, with overexpression and dominant-negative studies implying a strong level of co-operation and interplay between these factors. Overall, our results link cytokine-mediated alterations in cPLA₂γ gene expression with allergic asthma and outline a complex regulatory mechanism.