Nanomechanical and thermophoretic analyses of the nucleotide-dependent interactions between the AAA+ subunits of magnesium chelatase

In chlorophyll biosynthesis, the magnesium chelatase enzyme complex catalyzes the insertion of a Mg2+ ion into protoporphyrin IX. Prior to this event, two of the three subunits, the AAA+ proteins ChlI and ChlD, form a ChlID− MgATP complex. We used microscale thermophoresis to directly determine dissociation constants for the I-D subunits from Synechocystis, and to show that the formation of a ChlID− MgADP complex, mediated by the arginine finger and the sensor II domain on ChlD, is necessary for the assembly of the catalytically active ChlHID−MgATP complex. The N-terminal AAA+ domain of ChlD is essential for complex formation, but some stability is preserved in the absence of the C-terminal integrin domain of ChlD, particularly if the intervening polyproline linker region is retained. Single molecule force spectroscopy (SMFS) was used to determine the factors that stabilize formation of the ChlID−MgADP complex at the single molecule level; ChlD was attached to an atomic force microscope (AFM) probe in two different orientations, and the ChlI subunits were tethered to a silica surface; the probability of subunits interacting more than doubled in the presence of MgADP, and we show that the N-terminal AAA+ domain of ChlD mediates this process, in agreement with the microscale thermophoresis data. Analysis of the unbinding data revealed a most probable interaction force of around 109 pN for formation of single ChlID−MgADP complexes. These experiments provide a quantitative basis for understanding the assembly and function of the Mg chelatase complex

PucC and LhaA direct efficient assembly of the light-harvesting complexes in <i>Rhodobacter sphaeroides</i>

The mature architecture of the photosynthetic membrane of the purple phototroph Rhodobacter sphaeroides has been characterised to a level where an atomic-level membrane model is available, but the roles of the putative assembly proteins LhaA and PucC in establishing this architecture are unknown. Here we investigate the assembly of light-harvesting LH2 and reaction centre-light-harvesting1-PufX (RC-LH1-PufX) photosystem complexes using spectroscopy, pull-downs, native gel electrophoresis, quantitative mass spectrometry and fluorescence lifetime microscopy to characterise a series of lhaA and pucC mutants. LhaA and PucC are important for specific assembly of LH1 or LH2 complexes, respectively, but they are not essential; the few LH1 subunits found in ΔlhaA mutants assemble to form normal RC-LH1-PufX core complexes showing that, once initiated, LH1 assembly round the RC is cooperative and proceeds to completion. LhaA and PucC form oligomers at sites of initiation of membrane invagination; LhaA associates with RCs, bacteriochlorophyll synthase (BchG), the protein translocase subunit YajC and the YidC membrane protein insertase. These associations within membrane nanodomains likely maximise interactions between pigments newly arriving from BchG and nascent proteins within the SecYEG-SecDF-YajC-YidC assembly machinery, thereby co-ordinating pigment delivery, the co-translational insertion of LH polypeptides and their folding and assembly to form photosynthetic complexes. LhaA and PucC form oligomers at the sites where invagination of the cytoplasmic membranes is initiated, and they play important roles in photosystem assembly in the purple phototrophic bacterium Rhodobacter sphaeroides. Establishing the architecture of the photosynthetic membrane involves interplay between LhaA, reaction centre complexes, bacteriochlorophyll synthase, the protein translocase subunit YajC, and the YidC membrane protein insertase. These associations likely coordinate the delivery of pigments and the membrane insertion, folding and assembly of photosystem polypeptides

Porphyrin Binding to Gun4 protein, Facilitated by a Flexible Loop, Controls Metabolite Flow through the Chlorophyll Biosynthetic Pathway

In oxygenic phototrophs, chlorophylls, hemes and bilins are synthesized by a common branched pathway. Given the phototoxic nature of tetrapyrroles, this pathway must be tightly regulated and an important regulatory role is attributed to Mgchelatase enzyme at the branching between the heme and chlorophyll pathway. Gun4 is a porphyrin-binding protein known to stimulate in vitro the Mg-chelatase activity but how the Gun4-porphyrin complex acts in the cell was unknown. To address this issue we first performed simulations to determine the porphyrin-docking mechanism to the cyanobacterial Gun4 structure. After correcting crystallographic loop contacts, we determined the binding site for Mgprotoporphyrin IX. It revealed that the orientation of 6/7 loop is critical for the binding and the magnesium ion held within the porphyrin is coordinated by Asn211 residue. We also identified the basis for stronger binding in the Gun4-1 variant and for weaker binding in the W192A mutant. The W192A-Gun4 was further characterized in Mg-chelatase assay showing that tight porphyrin-binding in Gun4 facilitates its interaction with the Mg-chelatase ChlH subunit. Finally, we introduced the W192A mutation into Synechocystis 6803 cells and show that the Gun4-porphyrin complex is important for the accumulation of ChlH and for channeling metabolites into the chlorophyll biosynthetic pathway.This work was supported by project P501/12/G055 of the Czech Science Foundation, and by the National Programme of Sustainability I (LO1416) and by ERC 2009-Adg25027-PELE (to V.G). J.K. was supported by project Algain (EE2.3.30.0059). N.B.P.A., P.A.D., A.A.B. and C.N.H. thank the Biotechnology and Biological Sciences Research Council (BBSRC) U.K. for funding, under award numbers BB/G021546/1 and BB/M000265/1. CNH was also supported by an Advanced Award 338895 from the European Research Council.Peer ReviewedPostprint (author's final draft

Structural and functional consequences of removing the N-terminal domain from the magnesium chelatase ChlH subunit of Thermosynechococcus elongatus

Magnesium chelatase (MgCH) initiates chlorophyll biosynthesis by catalysing the ATP-dependent insertion of Mg2+ into protoporphyrin. This large enzyme complex comprises ChlH, I and D subunits, with I and D involved in ATP hydrolysis, and H the protein that handles the substrate and product. The 148 kDa ChlH subunit has a globular N-terminal domain attached by a narrow linker to a hollow cage-like structure. Following deletion of this ~18 kDa domain from the Thermosynechoccus elongatus ChlH, we used single particle reconstruction to show that the apo- and porphyrin-bound forms of the mutant subunit consist of a hollow globular protein with three connected lobes; superposition of the mutant and native ChlH structures shows that, despite the clear absence of the N-terminal ‘head’ region, the rest of the protein appears to be correctly folded. Analyses of dissociation constants shows that the ΔN159ChlH mutant retains the ability to bind protoporphyrin and the Gun4 enhancer protein, although the addition of I and D subunits yields an extremely impaired active enzyme complex. Addition of the Gun4 enhancer protein, which stimulates MgCH activity significantly especially at low Mg2+ concentrations, partially reactivates the ΔN159ChlH–I–D mutant enzyme complex, suggesting that the binding site or sites for Gun4 on H do not wholly depend on the N-terminal domain

Enzyme Sequence and Its Relationship to Hyperbaric Stability of Artificial and Natural Fish Lactate Dehydrogenases

The cDNAs of lactate dehydrogenase b (LDH-b) from both deep-sea and shallow living fish species, Corphaenoides armatus and Gadus morhua respectively, have been isolated, sequenced and their encoded products overproduced as recombinant enzymes in E. coli. The proteins were characterised in terms of their kinetic and physical properties and their ability to withstand high pressures. Although the two proteins are very similar in terms of their primary structure, only 21 differences at the amino acid level exist between them, the enzyme from the deep-sea species has a significantly increased tolerance to pressure and a higher thermostability. It was possible to investigate whether the changes in the N-terminal or C-terminal regions played a greater role in barophilic adaptation by the construction of two chimeric enzymes by use of a common restriction site within the cDNAs. One of these hybrids was found to have even greater pressure stability than the recombinant enzyme from the deep-living fish species. It was possible to conclude that the major adaptive changes to pressure tolerance must be located in the N-terminal region of the protein. The types of changes that are found and their spatial location within the protein structure are discussed. An analysis of the kinetic parameters of the enzymes suggests that there is clearly a trade off between Km and kcat values, which likely reflects the necessity of the deep-sea enzyme to operate at low temperatures

Probing the quality control mechanism of theEscherichia colitwin-arginine translocase with folding variants of ade novo-designed heme protein

Protein transport across the cytoplasmic membrane of bacterial cells is mediated by either the general secretion (Sec) system or the twin arginine translocase (Tat). The Tat machinery exports folded and cofactor containing proteins from the cytoplasm to the periplasm by using the transmembrane proton motive force as a source of energy. The Tat apparatus apparently senses the folded state of its protein substrates, a quality control mechanism that prevents premature export of nascent unfolded or misfolded polypeptides, but its mechanistic basis has not yet been determined. Here, we investigated the innate ability of the model Escherichia coli Tat system to recognize and translocate de novo-designed protein substrates with experimentally determined differences in the extent of folding. Water-soluble, four-helix bundle maquette proteins were engineered to bind two, one or no heme b cofactors, resulting in a concomitant reduction in the extent of their folding, assessed with temperature-dependent CD spectroscopy and one-dimensional 1H NMR spectroscopy. Fusion of the archetypal N-terminal Tat signal peptide of the E. coli trimethylamine-N-oxide (TMAO) reductase (TorA) to the N-terminus of the protein maquettes was sufficient for the Tat system to recognize them as substrates. The clear correlation between the level of Tat-dependent export and the degree of heme b-induced folding of the maquette protein suggested that the membrane-bound Tat machinery can sense the extent of folding and conformational flexibility of its substrates. We propose that these artificial proteins are ideal substrates for future investigations of the Tat system’s quality control mechanism

Integration of energy and electron transfer processes in the photosynthetic membrane of Rhodobacter sphaeroides

Photosynthesis converts absorbed solar energy to a protonmotive force, which drives ATP synthesis. The membrane network of chlorophyll–protein complexes responsible for light absorption, photochemistry and quinol (QH2) production has been mapped in the purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides using atomic force microscopy (AFM), but the membrane location of the cytochrome bc1 (cytbc1) complexes that oxidise QH2 to quinone (Q) to generate a protonmotive force is unknown. We labelled cytbc1 complexes with gold nanobeads, each attached by a Histidine10 (His10)-tag to the C-terminus of cytc1. Electron microscopy (EM) of negatively stained chromatophore vesicles showed that the majority of the cytbc1 complexes occur as dimers in the membrane. The cytbc1 complexes appeared to be adjacent to reaction centre light-harvesting 1-PufX (RC–LH1–PufX) complexes, consistent with AFM topographs of a gold-labelled membrane. His-tagged cytbc1 complexes were retrieved from chromatophores partially solubilised by detergent; RC–LH1–PufX complexes tended to co-purify with cytbc1 whereas LH2 complexes became detached, consistent with clusters of cytbc1 complexes close to RC–LH1–PufX arrays, but not with a fixed, stoichiometric cytbc1–RC–LH1–PufX supercomplex. This information was combined with a quantitative mass spectrometry (MS) analysis of the RC, cytbc1, ATP synthase, cytaa3 and cytcbb3 membrane protein complexes, to construct an atomic-level model of a chromatophore vesicle comprising 67 LH2 complexes, 11 LH1–RC–PufX dimers & 2 RC–LH1–PufX monomers, 4 cytbc1 dimers and 2 ATP synthases. Simulation of the interconnected energy, electron and proton transfer processes showed a half-maximal ATP turnover rate for a light intensity equivalent to only 1% of bright sunlight. Thus, the photosystem architecture of the chromatophore is optimised for growth at low light intensities

The authority of next-of-kin in explicit and presumed consent systems for deceased organ donation: an analysis of 54 nations

Background. The degree of involvement by the next-of-kin in deceased organ procurement worldwide is unclear. We investigated the next-of-kin’s authority in the procure-ment process in nations with either explicit or presumed consent. Methods. We collected data from 54 nations, 25 with presumed consent and 29 with explicit consent. We char-acterized the authority of the next-of-kin in the decision to donate deceased organs. Specifically, we examined whether the next-of-kin’s consent to procure organs was always required and whether the next-of-kin were able to veto procurement when the deceased had expressed a wish to donate. Results. The next-of-kin are involved in the organ procure-ment process in most nations regardless of the consent principle and whether the wishes of the deceased to be a donor were expressed or unknown. Nineteen of the 25 nations with presumed consent provide a method for individuals to express a wish to be a donor. However, health professionals in only four of these nations responded that they do not override a deceased’s expressed wish because of a family’s objection. Similarly, health profes-sionals in only four of the 29 nations with explicit consent proceed with a deceased’s pre-existing wish to be a donor and do not require next-of-kin’s consent, but caveats still remain for when this is done. Conclusions. The next-of-kin have a considerable influ-ence on the organ procurement process in both presumed and explicit consent nations

NirJ, a radical SAM family member of the d1 heme biogenesis cluster

AbstractNirJ is involved in the transformation of precorrin-2 into heme d1, although its precise role in the process has not been established. The purified protein was found to contain a 4Fe–4S centre, in line with the prediction that it belongs to the radical SAM class of enzymes. This was further confirmed by binding of S-adenosyl-l-methionine (SAM) to dithionite-reduced NirJ, which resulted in a decrease in the signal intensity and in a shift to higher field of the [4Fe–4S]1+ EPR signal. Significantly, though, this approach also led to the appearance of a small but reproducible organic radical signal that was associated with about 2% of the NirJ molecules and was affected by the incorporation of SAM deuterated at the 5′ adenosyl group