A ferredoxin bridge connects the two arms of plant mitochondrial complex I.

Mitochondrial complex I is the main site for electron transfer to the respiratory chain and generates much of the proton gradient across the inner mitochondrial membrane. Complex I is composed of two arms, which form a conserved L-shape. We report the structures of the intact, 47-subunit mitochondrial complex I from Arabidopsis thaliana and the 51-subunit complex I from the green alga Polytomella sp., both at around 2.9 Šresolution. In both complexes, a heterotrimeric γ-carbonic anhydrase domain is attached to the membrane arm on the matrix side. Two states are resolved in A. thaliana complex I, with different angles between the two arms and different conformations of the ND1 (NADH dehydrogenase subunit 1) loop near the quinol binding site. The angle appears to depend on a bridge domain, which links the peripheral arm to the membrane arm and includes an unusual ferredoxin. We propose that the bridge domain participates in regulating the activity of plant complex I.

Membrane perforation by the pore-forming toxin pneumolysin.

Pneumolysin (PLY), a major virulence factor of Streptococcus pneumoniae, perforates cholesterol-rich lipid membranes. PLY protomers oligomerize as rings on the membrane and then undergo a structural transition that triggers the formation of membrane pores. Structures of PLY rings in prepore and pore conformations define the beginning and end of this transition, but the detailed mechanism of pore formation remains unclear. With atomistic and coarse-grained molecular dynamics simulations, we resolve key steps during PLY pore formation. Our simulations confirm critical PLY membrane-binding sites identified previously by mutagenesis. The transmembrane ß-hairpins of the PLY pore conformation are stable only for oligomers, forming a curtain-like membrane-spanning ß-sheet. Its hydrophilic inner face draws water into the protein-lipid interface, forcing lipids to recede. For PLY rings, this zone of lipid clearance expands into a cylindrical membrane pore. The lipid plug caught inside the PLY ring can escape by lipid efflux via the lower leaflet. If this path is too slow or blocked, the pore opens by membrane buckling, driven by the line tension acting on the detached rim of the lipid plug. Interestingly, PLY rings are just wide enough for the plug to buckle spontaneously in mammalian membranes. In a survey of electron cryo-microscopy (cryo-EM) and atomic force microscopy images, we identify key intermediates along both the efflux and buckling pathways to pore formation, as seen in the simulations.

LptC from Anabaena sp. PCC 7120: Expression, purification and crystallization.

Lipopolysaccharides are central elements of the outer leaflet of the outer membrane of Gram-negative bacteria and as such, of cyanobacteria. In the past, the structural analysis of the system in proteobacteria like Escherichia coli has contributed to a deep understanding of the transport of lipopolysaccharides from plasma membrane to the outer membrane. While many components of the transport system are conserved between proteobacteria and cyanobacteria, the periplasmic LptC appears to be distinct. The cyanobacterial proteins are twice as long as the proteobacterial proteins or proteins from firmicutes. This prompted the question whether the structure of the cyanobacterial proteins is comparable the one of the proteobacterial proteins. To address this question, we expressed LptC from Anabaena sp. PCC 7120 in E. coli as truncated protein without the transmembrane segment. We purified the protein utilizing HIS-tag based affinity chromatography and polished the protein after removal of the tag by size exclusion chromatography. The purified recombinant protein was crystallized by the sitting-drop vapor diffusion technique and best crystals, despite being twinned, diffracted to a resolution of 2.6 Å.

Modifying the morphology and properties of aligned CNT foams through secondary CNT growth.

In this work, we report for the first time, growth of secondary carbon nanotubes (CNTs) throughout a three-dimensional assembly of CNTs. The assembly of nanotubes was in the form of aligned CNT/carbon (ACNT/C) foams. These low-density CNT foams were conformally coated with an alumina buffer layer using atomic layer deposition. Chemical vapor deposition was further used to grow new CNTs. The CNT foam's extremely high porosity allowed for growth of secondary CNTs inside the bulk of the foams. Due to the heavy growth of new nanotubes, density of the foams increased more than 2.5 times. Secondary nanotubes had the same graphitic quality as the primary CNTs. Microscopy and chemical analysis revealed that the thickness of the buffer layer affected the diameter, nucleation density as well as growth uniformity across the thickness of the foams. The effects of secondary nanotubes on the compressive mechanical properties of the foams was also investigated.

Alternating-access mechanism in conformationally asymmetric trimers of the betaine transporter BetP.

Betaine and Na(+) symport has been extensively studied in the osmotically regulated transporter BetP from Corynebacterium glutamicum, a member of the betaine/choline/carnitine transporter family, which shares the conserved LeuT-like fold of two inverted structural repeats. BetP adjusts its transport activity by sensing the cytoplasmic K(+) concentration as a measure for hyperosmotic stress via the osmosensing carboxy-terminal domain. BetP needs to be in a trimeric state for communication between individual protomers through several intratrimeric interaction sites. Recently, crystal structures of inward-facing BetP trimers have contributed to our understanding of activity regulation on a molecular level. Here we report new crystal structures, which reveal two conformationally asymmetric BetP trimers, capturing among them three distinct transport states. We observe a total of four new conformations at once: an outward-open apo and an outward-occluded apo state, and two closed transition states--one in complex with betaine and one substrate-free. On the basis of these new structures, we identified local and global conformational changes in BetP that underlie the molecular transport mechanism, which partially resemble structural changes observed in other sodium-coupled LeuT-like fold transporters, but show differences we attribute to the osmolytic nature of betaine, the exclusive substrate specificity and the regulatory properties of BetP.

Electrogenic Cation Binding in the Electroneutral Na+/H+ Antiporter of Pyrococcus abyssi.

Na+/H+ antiporters in the CPA1 branch of the cation proton antiporter family drive the electroneutral exchange of H+ against Na+ ions and ensure pH homeostasis in eukaryotic and prokaryotic organisms. Although their transport cycle is overall electroneutral, specific partial reactions are electrogenic. Here, we present an electrophysiological study of the PaNhaP Na+/H+ antiporter from Pyrococcus abyssi reconstituted into liposomes. Positive transient currents were recorded upon addition of Na+ to PaNhaP proteoliposomes, indicating a reaction where positive charge is rapidly displaced into the proteoliposomes with a rate constant of k >200 s-1 We attribute the recorded currents to an electrogenic reaction that includes Na+ binding and possibly occlusion. Subsequently, positive charge is transported out of the cell associated with H+ binding, so that the overall reaction is electroneutral. We show that the differences in pH profile and Na+ affinity of PaNhaP and the related MjNhaP1 from Methanocaldococcus jannaschii can be attributed to an additional negatively charged glutamate residue in PaNhaP. The results are discussed in the context of the physiological function of PaNhaP and other microbial Na+/H+ exchangers. We propose that both, electroneutral and electrogenic Na+/H+ antiporters, represent a carefully tuned self-regulatory system, which drives the cytoplasmic pH back to neutral after any deviation.

Structural evidence for functional lipid interactions in the betaine transporter BetP.

Bilayer lipids contribute to the stability of membrane transporters and are crucially involved in their proper functioning. However, the molecular knowledge of how surrounding lipids affect membrane transport is surprisingly limited and despite its general importance is rarely considered in the molecular description of a transport mechanism. One reason is that only few atomic resolution structures of channels or transporters reveal a functional interaction with lipids, which are difficult to detect in X-ray structures per se. Overcoming these difficulties, we report here on a new structure of the osmotic stress-regulated betaine transporter BetP in complex with anionic lipids. This lipid-associated BetP structure is important in the molecular understanding of osmoregulation due to the strong dependence of activity regulation in BetP on the presence of negatively charged lipids. We detected eight resolved palmitoyl-oleoyl phosphatidyl glycerol (PG) lipids mimicking parts of the membrane leaflets and interacting with key residues in transport and regulation. The lipid-protein interactions observed here in structural detail in BetP provide molecular insights into the role of lipids in osmoregulated secondary transport.

The thermodynamic signature of ligand binding to histone deacetylase-like amidohydrolases is most sensitive to the flexibility in the L2-loop lining the active site pocket.

BACKGROUND: The analysis of the thermodynamic driving forces of ligand-protein binding has been suggested to be a key component for the selection and optimization of active compounds into drug candidates. The binding enthalpy as deduced from isothermal titration calorimetry (ITC) is usually interpreted assuming single-step binding of a ligand to one conformation of the target protein. Although successful in many cases, these assumptions are oversimplified approximations of the reality with flexible proteins and complicated binding mechanism in many if not most cases. The relationship between protein flexibility and thermodynamic signature of ligand binding is largely understudied. METHODS: Directed mutagenesis, X-ray crystallography, enzyme kinetics and ITC methods were combined to dissect the influence of loop flexibility on the thermodynamics and mechanism of ligand binding to histone deacetylase (HDAC)-like amidohydrolases. RESULTS: The general ligand-protein binding mechanism comprises an energetically demanding gate opening step followed by physical binding. Increased flexibility of the L2-loop in HDAC-like amidohydrolases facilitates access of ligands to the binding pocket resulting in predominantly enthalpy-driven complex formation. CONCLUSIONS: The study provides evidence for the great importance of flexibility adjacent to the active site channel for the mechanism and observed thermodynamic driving forces of molecular recognition in HDAC like enzymes. GENERAL SIGNIFICANCE: The flexibility or malleability in regions adjacent to binding pockets should be given more attention when designing better drug candidates. The presented case study also suggests that the observed binding enthalpy of protein-ligand systems should be interpreted with caution, since more complicated binding mechanisms may obscure the significance regarding potential drug likeness.

Self-assembly of the general membrane-remodeling protein PVAP into sevenfold virus-associated pyramids.

Viruses have developed a wide range of strategies to escape from the host cells in which they replicate. For egress some archaeal viruses use a pyramidal structure with sevenfold rotational symmetry. Virus-associated pyramids (VAPs) assemble in the host cell membrane from the virus-encoded protein PVAP and open at the end of the infection cycle. We characterize this unusual supramolecular assembly using a combination of genetic, biochemical, and electron microscopic techniques. By whole-cell electron cryotomography, we monitored morphological changes in virus-infected host cells. Subtomogram averaging reveals the VAP structure. By heterologous expression of PVAP in cells from all three domains of life, we demonstrate that the protein integrates indiscriminately into virtually any biological membrane, where it forms sevenfold pyramids. We identify the protein domains essential for VAP formation in PVAP truncation mutants by their ability to remodel the cell membrane. Self-assembly of PVAP into pyramids requires at least two different, in-plane and out-of-plane, protein interactions. Our findings allow us to propose a model describing how PVAP arranges to form sevenfold pyramids and suggest how this small, robust protein may be used as a general membrane-remodeling system.

Unraveling the Pore-Forming Steps of Pneumolysin from Streptococcus pneumoniae.

Pneumolysin (PLY) is the main virulence factor of Streptococcus pneumoniae that causes pneumonia, meningitis, and invasive pneumococcal infection. PLY is produced as monomers, which bind to cholesterol-containing membranes, where they oligomerize into large pores. To investigate the pore-forming mechanism, we determined the crystal structure of PLY at 2.4 Šand used it to design mutants on the surface of monomers. Electron microscopy of liposomes incubated with PLY mutants revealed that several mutations interfered with ring formation. Mutants that formed incomplete rings or linear arrays had strongly reduced hemolytic activity. By high-resolution time-lapse atomic force microscopy of wild-type PLY, we observed two different ring-shaped complexes. Most of the complexes protruded ∼8 nm above the membrane surface, while a smaller number protruded ∼11 nm or more. The lower complexes were identified as pores or prepores by the presence or absence of a lipid bilayer in their center. The taller complexes were side-by-side assemblies of monomers of soluble PLY that represent an early form of the prepore. Our observations suggest a four-step mechanism of membrane attachment and pore formation by PLY, which is discussed in the context of recent structural models. The functional separation of these steps is necessary for the understanding how cholesterol-dependent cytolysins form pores and lyse cells.

Crystal Structure of a Histone Deacetylase Homologue from Pseudomonas aeruginosa.

Despite the recently growing interest in the acetylation of lysine residues by prokaryotic enzymes, the underlying biological function is still not well understood. Deacetylation is accomplished by proteins that belong to the histone deacetylase (HDAC) superfamily. In this report, we present the first crystal structure of PA3774, a histone deacetylase homologue from the human pathogen Pseudomonas aeruginosa that shares a high degree of homology with class IIb HDACs. We determined the crystal structure of the ligand-free enzyme and protein-ligand complexes with a trifluoromethylketone inhibitor and the reaction product acetate. Moreover, we produced loss of function mutants and determined the structure of the inhibitor-free PA3774H143A mutant, the inhibitor-free PA3774Y313F mutant, and the PA3774Y313F mutant in complex with the highly selective hydroxamate inhibitor PFSAHA. The overall structure reveals that the exceptionally long L1 loop mediates the formation of a tetramer composed of two "head-to-head" dimers. The distinctive dimer interface significantly confines the entrance area of the active site, suggesting a crucial role for substrate recognition and selectivity.

Ultralight Interconnected Metal Oxide Nanotube Networks.

Record-breaking ultralow density aluminum oxide structures are prepared using a novel templating technique. The alumina structures are unique in that they are comprised by highly aligned and interconnected nanotubes yielding anisotropic behavior. Large-scale network structures with complex form-factors can easily be made using this technique. The application of the low density networks as humidity sensing materials as well as thermal insulation is demonstrated.

A new type of Na(+)-driven ATP synthase membrane rotor with a two-carboxylate ion-coupling motif.

The anaerobic bacterium Fusobacterium nucleatum uses glutamate decarboxylation to generate a transmembrane gradient of Na⁺. Here, we demonstrate that this ion-motive force is directly coupled to ATP synthesis, via an F1F0-ATP synthase with a novel Na⁺ recognition motif, shared by other human pathogens. Molecular modeling and free-energy simulations of the rotary element of the enzyme, the c-ring, indicate Na⁺ specificity in physiological settings. Consistently, activity measurements showed Na⁺ stimulation of the enzyme, either membrane-embedded or isolated, and ATP synthesis was sensitive to the Na⁺ ionophore monensin. Furthermore, Na⁺ has a protective effect against inhibitors targeting the ion-binding sites, both in the complete ATP synthase and the isolated c-ring. Definitive evidence of Na⁺ coupling is provided by two identical crystal structures of the c11 ring, solved by X-ray crystallography at 2.2 and 2.6 Šresolution, at pH 5.3 and 8.7, respectively. Na⁺ ions occupy all binding sites, each coordinated by four amino acids and a water molecule. Intriguingly, two carboxylates instead of one mediate ion binding. Simulations and experiments demonstrate that this motif implies that a proton is concurrently bound to all sites, although Na⁺ alone drives the rotary mechanism. The structure thus reveals a new mode of ion coupling in ATP synthases and provides a basis for drug-design efforts against this opportunistic pathogen.

The c-ring stoichiometry of ATP synthase is adapted to cell physiological requirements of alkaliphilic Bacillus pseudofirmus OF4.

The c-rings of ATP synthases consist of individual c-subunits, all of which harbor a conserved motif of repetitive glycine residues (GxGxGxG) important for tight transmembrane α-helix packing. The c-ring stoichiometry determines the number of ions transferred during enzyme operation and has a direct impact on the ion-to-ATP ratio, a cornerstone parameter of cell bioenergetics. In the extreme alkaliphile Bacillus pseudofirmus OF4, the glycine motif is replaced by AxAxAxA. We performed a structural study on two mutants with alanine-to-glycine changes using atomic force microscopy and X-ray crystallography, and found that mutants form smaller c12 rings compared with the WT c13. The molar growth yields of B. pseudofirmus OF4 cells on malate further revealed that the c12 mutants have a considerably reduced capacity to grow on limiting malate at high pH. Our results demonstrate that the mutant ATP synthases with either c12 or c13 can support ATP synthesis, and also underscore the critical importance of an alanine motif with c13 ring stoichiometry for optimal growth at pH >10. The data indicate a direct connection between the precisely adapted ATP synthase c-ring stoichiometry and its ion-to-ATP ratio on cell physiology, and also demonstrate the bioenergetic challenges and evolutionary adaptation strategies of extremophiles.

Directly Observing the Lipid-Dependent Self-Assembly and Pore-Forming Mechanism of the Cytolytic Toxin Listeriolysin O.

Listeriolysin O (LLO) is the major virulence factor of Listeria monocytogenes and a member of the cholesterol-dependent cytolysin (CDC) family. Gram-positive pathogenic bacteria produce water-soluble CDC monomers that bind cholesterol-dependent to the lipid membrane of the attacked cell or of the phagosome, oligomerize into prepores, and insert into the membrane to form transmembrane pores. However, the mechanisms guiding LLO toward pore formation are poorly understood. Using electron microscopy and time-lapse atomic force microscopy, we show that wild-type LLO binds to membranes, depending on the presence of cholesterol and other lipids. LLO oligomerizes into arc- or slit-shaped assemblies, which merge into complete rings. All three oligomeric assemblies can form transmembrane pores, and their efficiency to form pores depends on the cholesterol and the phospholipid composition of the membrane. Furthermore, the dynamic fusion of arcs, slits, and rings into larger rings and their formation of transmembrane pores does not involve a height difference between prepore and pore. Our results reveal new insights into the pore-forming mechanism and introduce a dynamic model of pore formation by LLO and other CDC pore-forming toxins.

The c-ring ion binding site of the ATP synthase from Bacillus pseudofirmus†OF4 is adapted to alkaliphilic lifestyle.

In the c-ring rotor of ATP synthases ions are shuttled across the membrane during ATP synthesis by a unique rotary mechanism. We investigated characteristics of the c-ring from the alkaliphile Bacillus pseudofirmus OF4 with respect to evolutionary adaptations to operate with protons at high environmental pH. The X-ray structures of the wild-type c13 ring at pH 9.0 and a 'neutralophile-like' mutant (P51A) at pH 4.4, at 2.4 and 2.8 Šresolution, respectively, reveal a dependency of the conformation and protonation state of the proton-binding glutamate (E(54) ) on environmental hydrophobicity. Faster labelling kinetics with the inhibitor dicyclohexylcarbodiimide (DCCD) demonstrate a greater flexibility of E(54) in the mutant due to reduced water occupancy within the H(+) binding site. A second 'neutralophile-like' mutant (V21N) shows reduced growth at high pH, which is explained by restricted conformational freedom of the mutant's E(54) carboxylate. The study directly connects subtle structural adaptations of the c-ring ion binding site to in vivo effects of alkaliphile cell physiology.

Structure of the archaeal Na+/H+ antiporter NhaP1 and functional role of transmembrane helix 1.

We have determined the structure of the archaeal sodium/proton antiporter NhaP1 at 7 Å resolution by electron crystallography of 2D crystals. NhaP1 is a dimer in the membrane, with 13 membrane-spanning α-helices per protomer, whereas the distantly related bacterial NhaA has 12. Dimer contacts in the two antiporters are very different, but the structure of a six-helix bundle at the tip of the protomer is conserved. The six-helix bundle of NhaA contains two partially unwound α-helices thought to harbour the ion-translocation site, which is thus similar in NhaP1. A model of NhaP1 based on detailed sequence comparison and the NhaA structure was fitted to the 7 Å map. The additional N-terminal helix 1 of NhaP1, which appears to be an uncleaved signal sequence, is located near the dimer interface. Similar sequences are present in many eukaryotic homologues of NhaP1, including NHE1. Although fully folded and able to dimerize, NhaP1 constructs without helix 1 are inactive. Possible reasons are investigated and discussed.

IR-spectroscopic characterization of an elongated OmpG mutant.

OmpG is a nonselective, pH dependent outer membrane protein from Escherichia coli. It consists of 281 residues, forming a 14-stranded ß-sheet structure. In this study, OmpG is extended by 38 amino acids to produce a 16-stranded ß-barrel (OmpG-16S). The resulting protein is investigated by IR-spectroscopy. The secondary structure, pH-dependent opening/closing mechanism, buffer accessibility and thermal stability of OmpG-16S are compared to OmpG-WT. The results show that OmpG-16S is responsive to pH change as indicated by the Amide I band shift upon a switch from acidic to neutral pH. This spectral shift is consistent with that observed in OmpG-WT, which confirms the existence of structural differences consistent with the presence of the open or closed state. Secondary structure analysis after curve-fitting of Amide I band revealed that the additional residues do not fold into ß-sheet; rather they are in the form of turns and unordered structure. In thermal stability experiments, OmpG-16S is found to be as stable as OmpG-WT. Additionally, H/D exchange experiments showed no difference in the exchange rate of OmpG-16S between the acidic and alkaline pH, suggesting that the loop L6 is no longer sufficient to block the pore entrance at acidic pH.

Healthcare quality indicators--a systematic review.

PURPOSE: The purpose of this paper is to review the literature on quality model development, validation and limitations. DESIGN/METHODOLOGY/APPROACH: The systematic literature review used online journal indexes between January 1995 and April 2010. International studies focusing on multiple functional domains and those in which development methods were selected. Two reviewers assessed all studies and 18 were shortlisted. FINDINGS: Literature reviews, peer reviews, questionnaires and expert panels are the most frequently used model development methods. Expert judges were widely used to validate the models. The most important limitation was that key indicators were missing. ORIGINALITY/VALUE: Existing healthcare quality models are not comprehensive and there is no consensus on targets, clinical areas or diseases.