Cyclophilin anaCyp40 regulates photosystem assembly and phycobilisome association in a cyanobacterium.

Cyclophilins, or immunophilins, are proteins found in many organisms including bacteria, plants and humans. Most of them display peptidyl-prolyl cis-trans isomerase activity, and play roles as chaperones or in signal transduction. Here, we show that cyclophilin anaCyp40 from the cyanobacterium Anabaena sp. PCC 7120 is enzymatically active, and seems to be involved in general stress responses and in assembly of photosynthetic complexes. The protein is associated with the thylakoid membrane and interacts with phycobilisome and photosystem components. Knockdown of anacyp40 leads to growth defects under high-salt and high-light conditions, and reduced energy transfer from phycobilisomes to photosystems. Elucidation of the anaCyp40 crystal structure at 1.2-Å resolution reveals an N-terminal helical domain with similarity to PsbQ components of plant photosystem II, and a C-terminal cyclophilin domain with a substrate-binding site. The anaCyp40 structure is distinct from that of other multi-domain cyclophilins (such as Arabidopsis thaliana Cyp38), and presents features that are absent in single-domain cyclophilins.

Crystal structures of phosphatidyl serine synthase PSS reveal the catalytic mechanism of CDP-DAG alcohol O-phosphatidyl transferases.

Phospholipids are the major components of the membrane in all type of cells and organelles. They also are critical for cell metabolism, signal transduction, the immune system and other critical cell functions. The biosynthesis of phospholipids is a complex multi-step process with high-energy intermediates. Several enzymes in different metabolic pathways are involved in the initial phospholipid synthesis and its subsequent conversion. While the "Kennedy pathway" is the main pathway in mammalian cells, in bacteria and lower eukaryotes the precursor CDP-DAG is used in the de novo pathway by CDP-DAG alcohol O-phosphatidyl transferases to synthetize the basic lipids. Here we present the high-resolution structures of phosphatidyl serine synthase from Methanocaldococcus jannaschii crystallized in four different states. Detailed structural and functional analysis of the different structures allowed us to identify the substrate binding site and show how CDP-DAG, serine and two essential metal ions are bound and oriented relative to each other. In close proximity to the substrate binding site, two anions were identified that appear to be highly important for the reaction. The structural findings were confirmed by functional activity assays and suggest a model for the catalytic mechanism of CDP-DAG alcohol O-phosphatidyl transferases, which synthetize the phospholipids essential for the cells.

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.

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.

Ion Binding and Selectivity of the Na+/H+ Antiporter MjNhaP1 from Experiment and Simulation.

Cells employ membrane-embedded antiporter proteins to control their pH, salt concentration, and volume. The large family of cation/proton antiporters is dominated by Na+/H+ antiporters that exchange sodium ions against protons, but homologous K+/H+ exchangers have recently been characterized. We show experimentally that the electroneutral antiporter NhaP1 of Methanocaldococcus jannaschii (MjNhaP1) is highly selective for Na+ ions. We then characterize the ion selectivity in both the inward-open and outward-open states of MjNhaP1 using classical molecular dynamics simulations, free energy calculations, and hybrid quantum/classical (QM/MM) simulations. We show that MjNhaP1 is highly selective for binding of Na+ over K+ in the inward-open state, yet it is only weakly selective in the outward-open state. These findings are consistent with the function of MjNhaP1 as a sodium-driven deacidifier of the cytosol that maintains a high cytosolic K+ concentration in environments of high salinity. By combining experiment and computation, we gain mechanistic insight into the Na+/H+ transport mechanism and help elucidate the molecular basis for ion selectivity in cation/proton exchangers.

Structural properties of an engineered outer membrane protein G mutant, OmpG-16SL, investigated with infrared spectroscopy.

The structural and functional differences between wild type (WT) outer membrane protein G and its two mutants are investigated with Fourier transform infrared spectroscopy. Both mutants have a long extension to the primary sequence to increase the number of ß-strands from 14 (wild type) to 16 in an attempt to enlarge the pore diameter. The comparison among proteins is made in terms of pH-dependent conformational changes and thermal stability. Results show that all proteins respond to pH change but at different degrees. At acidic environment, all proteins contain the same number of residues participated in ß-sheet structure. However, at neutral pH, the mutants have less ordered structure compared to WT porin. Thermal stability tests show that mutants differ significantly from WT porin at neutral pH. Although the transition temperature is directly proportional with the amount of ß-sheet content, the changes in the pre-transition phase that pave the way to structural breakdown are shown to involve interactions among charged residues by two-dimensional correlation spectroscopy analysis. Results of the analysis show that side chain interactions play an active role under increasing temperature. Both mutants have more unordered secondary structure but they respond to pH change in tertiary structure level. Findings of this study provided deeper insight on the active players in structural stability of the WT porin.Communicated by Ramaswamy H. Sarma [Formula: see text].

Rotary substates of mitochondrial ATP synthase reveal the basis of flexible F1-Fo coupling.

F1Fo-adenosine triphosphate (ATP) synthases make the energy of the proton-motive force available for energy-consuming processes in the cell. We determined the single-particle cryo-electron microscopy structure of active dimeric ATP synthase from mitochondria of Polytomella sp. at a resolution of 2.7 to 2.8 angstroms. Separation of 13 well-defined rotary substates by three-dimensional classification provides a detailed picture of the molecular motions that accompany c-ring rotation and result in ATP synthesis. Crucially, the F1 head rotates along with the central stalk and c-ring rotor for the first ~30° of each 120° primary rotary step to facilitate flexible coupling of the stoichiometrically mismatched F1 and Fo subcomplexes. Flexibility is mediated primarily by the interdomain hinge of the conserved OSCP subunit. A conserved metal ion in the proton access channel may synchronize c-ring protonation with rotation.

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.

Mechanism of the electroneutral sodium/proton antiporter PaNhaP from transition-path shooting.

Na+/H+ antiporters exchange sodium ions and protons on opposite sides of lipid membranes. The electroneutral Na+/H+ antiporter NhaP from archaea Pyrococcus abyssi (PaNhaP) is a functional homolog of the human Na+/H+ exchanger NHE1, which is an important drug target. Here we resolve the Na+ and H+ transport cycle of PaNhaP by transition-path sampling. The resulting molecular dynamics trajectories of repeated ion transport events proceed without bias force, and overcome the enormous time-scale gap between seconds-scale ion exchange and microseconds simulations. The simulations reveal a hydrophobic gate to the extracellular side that opens and closes in response to the transporter domain motion. Weakening the gate by mutagenesis makes the transporter faster, suggesting that the gate balances competing demands of fidelity and efficiency. Transition-path sampling and a committor-based reaction coordinate optimization identify the essential motions and interactions that realize conformational alternation between the two access states in transporter function.

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.

Structural basis of proton translocation and force generation in mitochondrial ATP synthase.

ATP synthases produce ATP by rotary catalysis, powered by the electrochemical proton gradient across the membrane. Understanding this fundamental process requires an atomic model of the proton pathway. We determined the structure of an intact mitochondrial ATP synthase dimer by electron cryo-microscopy at near-atomic resolution. Charged and polar residues of the a-subunit stator define two aqueous channels, each spanning one half of the membrane. Passing through a conserved membrane-intrinsic helix hairpin, the lumenal channel protonates an acidic glutamate in the c-ring rotor. Upon ring rotation, the protonated glutamate encounters the matrix channel and deprotonates. An arginine between the two channels prevents proton leakage. The steep potential gradient over the sub-nm inter-channel distance exerts a force on the deprotonated glutamate, resulting in net directional rotation.

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.

CryoEM structures of membrane pore and prepore complex reveal cytolytic mechanism of Pneumolysin.

Many pathogenic bacteria produce pore-forming toxins to attack and kill human cells. We have determined the 4.5 Å structure of the ~2.2 MDa pore complex of pneumolysin, the main virulence factor of Streptococcus pneumoniae, by cryoEM. The pneumolysin pore is a 400 Å ring of 42 membrane-inserted monomers. Domain 3 of the soluble toxin refolds into two ~85 Å ß-hairpins that traverse the lipid bilayer and assemble into a 168-strand ß-barrel. The pore complex is stabilized by salt bridges between ß-hairpins of adjacent subunits and an internal α-barrel. The apolar outer barrel surface with large sidechains is immersed in the lipid bilayer, while the inner barrel surface is highly charged. Comparison of the cryoEM pore complex to the prepore structure obtained by electron cryo-tomography and the x-ray structure of the soluble form reveals the detailed mechanisms by which the toxin monomers insert into the lipid bilayer to perforate the target membrane.

Toward Photopharmacological Antimicrobial Chemotherapy Using Photoswitchable Amidohydrolase Inhibitors.

Photopharmacological agents exhibit light-dependent biological activity and may have potential in the development of new antimicrobial agents/modalities. Amidohydrolase enzymes homologous to the well-known human histone deacetylases (HDACs) are present in bacteria, including resistant organisms responsible for a significant number of hospital-acquired infections and deaths. We report photopharmacological inhibitors of these enzymes, using two classes of photoswitches embedded in the inhibitor pharmacophore: azobenzenes and arylazopyrazoles. Although both classes of inhibitor show excellent inhibitory activity (nM IC50 values) of the target enzymes and promising differential activity of the switchable E- and Z-isomeric forms, the arylazopyrazoles exhibit better intrinsic photoswitch performance (more complete switching, longer thermal lifetime of the Z-isomer). We also report protein-ligand crystal structures of the E-isomers of both an azobenzene and an arylazopyrazole inhibitor, bound to bacterial histone deacetylase-like amidohydrolases (HDAHs). These structures not only uncover interactions important for inhibitor binding but also reveal conformational differences between the two photoswitch inhibitor classes. As such, our data may pave the way for the design of improved photopharmacological agents targeting the HDAC superfamily.

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.

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.

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.

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.

Mechanism of Na(+)-dependent citrate transport from the structure of an asymmetrical CitS dimer.

The common human pathogen Salmonella enterica takes up citrate as a nutrient via the sodium symporter SeCitS. Uniquely, our 2.5 Å x-ray structure of the SeCitS dimer shows three different conformations of the active protomer. One protomer is in the outside-facing state. Two are in different inside-facing states. All three states resolve the substrates in their respective binding environments. Together with comprehensive functional studies on reconstituted proteoliposomes, the structures explain the transport mechanism in detail. Our results indicate a six-step process, with a rigid-body 31° rotation of a helix bundle that translocates the bound substrates by 16 Å across the membrane. Similar transport mechanisms may apply to a wide variety of related and unrelated secondary transporters, including important drug targets.