Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. ...

Case Western Reserve University

To determine whether intravenous contrast improves the ability of radiologists to establish the cause of acute abdominal pain after nondiagnostic or normal unenhanced CT.

Is unenhanced CT sufficient for evaluation of acute abdominal pain?

The genus Lantana has many species complexes, and L. camara is one of the aggressive alien weedy species complexes; species delimitation in these complexes is a nightmare for taxonomists. We examined the diversity in the chemical composition of foliar essential oils among morphotypes of Lantana species complexes inhabiting the same ecological gradient, and its taxonomic and ecological significance. The yields of essential oils varied from 0.1 to 0.79% in foliar hydrodistillates of eleven morphotypes, and a total of 39 chemical constituents were detected by GC/MS. The quantitative and qualitative variability in the composition of essential oils among morphotypes was very high, and hence they represent chemotypes. The diversity observed in the composition of essential oils appears to be of genetic origin and thus of taxonomic value. The formation of distinct clusters and sub-clusters at high distance cluster combine values also substantiates that the patterns of distribution of chemical constituents among morphotypes can be used in delimiting species and infraspecific taxa within the species complexes. The presence of beta-caryophyllene and other such compounds, which are known to prevent herbivory, in morphotypes of Lantana species complexes suggest that these compounds may provide selective advantage to Lantana over native species in the invasion of new and disturbed habitats.

Variability in foliar essential oils among different morphotypes of Lantana species complexes, and its taxonomic and ecological significance.

Vacuolar ATPases use the energy derived from ATP hydrolysis, catalyzed in the A(3)B(3) sector of the V(1) ATPase to pump protons via the membrane-embedded V(O) sector. The energy coupling between the two sectors occurs via the so-called central stalk, to which subunit F does belong. Here we present the first low resolution structure of recombinant subunit F (Vma7p) of a eukaryotic V-ATPase from Saccharomyces cerevisiae, analyzed by small angle X-ray scattering (SAXS). The protein is divided into a 5.5nm long egg-like shaped region, connected via a 1.5nm linker to a hook-like segment at one end. Circular dichroism spectroscopy revealed that subunit F comprises of 43% α-helix, 32% β-sheet and a 25% random coil arrangement. To determine the localization of the N- and C-termini in the protein, the C-terminal truncated form of F, F(1-94) was produced and analyzed by SAXS. Comparison of the F(1-94) shape with the one of subunit F showed the missing hook-like region in F(1-94), supported by the decreased D(max) value of F(1-94) (7.0nm), and indicating that the hook-like region consists of the C-terminal residues. The NMR solution structure of the C-terminal peptide, F(90-116), was solved, displaying an α-helical region between residues 103 and 113. The F(90-116) solution structure fitted well in the hook-like region of subunit F. Finally, the arrangement of subunit F within the V(1) ATPase is discussed.

Solution structure of subunit F (Vma7p) of the eukaryotic V(1)V(O) ATPase from Saccharomyces cerevisiae derived from SAXS and NMR spectroscopy.

Invasion of the erythrocyte by the invasive form of the malaria parasite, the merozoite, is a complex process involving numerous parasite proteins. The reticulocyte-binding protein homologues (RH) family of merozoite proteins has been previously shown to play an important role in the invasion process. Previously, it has been shown that the RH proteins of Plasmodium yoelii, Py235, play a role as an ATP/ADP sensor. Binding of Py235 to the erythrocyte surface is increased in the presence of ATP, while ADP has an inhibitory effect. The sensor domain of Py235 is called NBD94 and the segment that has been shown to covalently bind the adenine nucleotide is made up by the residues (483) FNEIKEKLKHYNFDDFVKEE(502) . Here, we report on the solution nuclear magnetic resonance structure of this peptide (NBD94(483-502) ) showing the presence of an α-helix between amino acid residues 485 and 491. The N- and C-terminal segments of the structure bend at tyrosine 493, a residue important for ATP binding. Importantly, erythrocyte-binding assays demonstrate that NBD94(483-502) can directly interfere with the binding of native Py235 to erythrocytes, suggesting a direct role of this region in erythrocyte binding. The data will provide the foundation for future studies to identify new compounds that directly interfere with the invasion process.

NMR solution structure of NBD94(483-502) of the nucleotide-binding domain of the Plasmodium yoelii reticulocyte-binding protein Py235.

Human immunodeficiency viruses (HIV) hide themselves in macrophages at the early stage of infection. Delivering drug in a sustained manner from polymeric nanoparticles in those cells could control the disease effectively. The study was intended to develop poly(d,l-lactic-co-glycolic acid)-based nanoparticles containing didanosine and to observe their uptake by macrophages in vitro. Various physicochemical evaluations related to nanoparticles, such as drug-excipient interaction, surface morphology, particle size, zeta potential, polydispersity index, drug loading, in vitro drug release and nanoparticle-uptake by macrophages in vitro were determined. Homogenising speeds and drug-polymer ratio varied drug loading and polydispersity index of nanoparticles, providing sustained drug release. Dimethyl sulphoxide/polyethylene glycol improved drug loading predominantly. Nanoparticle-uptake by macrophages was concentration dependent. Experimental nanoparticles successfully transported didanosine to macrophages in vitro, suggesting reduction of dose, thus minimising toxicity and side effects. Developed nanoparticle may control HIV infection effectively at an early stage.

Submicron-size biodegradable polymer-based didanosine particles for treating HIV at early stage: an in vitro study.

V-ATPases are very complex multi-subunit enzymes which function as proton-pumping rotary nanomotors. The rotary and coupling subunit F (F(1-94)) was crystallized by the hanging-drop vapour-diffusion method. The native crystals diffracted to a resolution of 2.64 Å and belonged to space group C222(1), with unit-cell parameters a = 47.21, b = 160.26, c = 102.49 Å. The selenomethionyl form of the F(1-94) I69M mutant diffracted to a resolution of 2.3 Å and belonged to space group C222(1), with unit-cell parameters a = 47.22, b = 160.83, c = 102.74 Å. Initial phasing and model building suggested the presence of four molecules in the asymmetric unit.

Crystallization and preliminary X-ray crystallographic analysis of subunit F (F(1-94)), an essential coupling subunit of the eukaryotic V(1)V(O)-ATPase from Saccharomyces cerevisiae.

The subunit ε of bacterial F(1)F(O) ATP synthases plays an important regulatory role in coupling and catalysis via conformational transitions of its C-terminal domain. Here we present the first low-resolution solution structure of ε of Mycobacterium tuberculosis (Mtε) F(1)F(O) ATP synthase and the nuclear magnetic resonance (NMR) structure of its C-terminal segment (Mtε(103-120)). Mtε is significantly shorter (61.6 Å) than forms of the subunit in other bacteria, reflecting a shorter C-terminal sequence, proposed to be important in coupling processes via the catalytic β subunit. The C-terminal segment displays an α-helical structure and a highly positive surface charge due to the presence of arginine residues. Using NMR spectroscopy, fluorescence spectroscopy, and mutagenesis, we demonstrate that the new tuberculosis (TB) drug candidate TMC207, proposed to bind to the proton translocating c-ring, also binds to Mtε. A model for the interaction of TMC207 with both ε and the c-ring is presented, suggesting that TMC207 forms a wedge between the two rotating subunits by interacting with the residues W15 and F50 of ε and the c-ring, respectively. T19 and R37 of ε provide the necessary polar interactions with the drug molecule. This new model of the mechanism of TMC207 provides the basis for the design of new drugs targeting the F(1)F(O) ATP synthase in M. tuberculosis.

Variations of subunit {varepsilon} of the Mycobacterium tuberculosis F1Fo ATP synthase and a novel model for mechanism of action of the tuberculosis drug TMC207.

Subunit F of V-ATPases is proposed to undergo structural alterations during catalysis and reversible dissociation from the V1VO complex. Recently, we determined the low resolution structure of F from Saccharomyces cerevisiae V-ATPase, showing an N-terminal egg shape, connected to a C-terminal hook-like segment via a linker region. To understand the mechanistic role of subunit F of S. cerevisiae V-ATPase, composed of 118 amino acids, the crystal structure of the major part of F, F(1-94), was solved at 2.3 Å resolution. The structural features were confirmed by solution NMR spectroscopy using the entire F subunit. The eukaryotic F subunit consists of the N-terminal F(1-94) domain with four-parallel β-strands, which are intermittently surrounded by four α-helices, and the C terminus, including the α5-helix encompassing residues 103 to 113. Two loops (26)GQITPETQEK(35) and (60)ERDDI(64) are described to be essential in mechanistic processes of the V-ATPase enzyme. The (26)GQITPETQEK(35) loop becomes exposed when fitted into the recently determined EM structure of the yeast V1VO-ATPase. A mechanism is proposed in which the (26)GQITPETQEK(35) loop of subunit F and the flexible C-terminal domain of subunit H move in proximity, leading to an inhibitory effect of ATPase activity in V1. Subunits D and F are demonstrated to interact with subunit d. Together with NMR dynamics, the role of subunit F has been discussed in the light of its interactions in the processes of reversible disassembly and ATP hydrolysis of V-ATPases by transmitting movements of subunit d and H of the VO and V1 sector, respectively.

Crystal and NMR structures give insights into the role and dynamics of subunit F of the eukaryotic V-ATPase from Saccharomyces cerevisiae.

Eukaryotic V1VO-ATPases hydrolyze ATP in the V1 domain coupled to ion pumping in VO. A unique mode of regulation of V-ATPases is the reversible disassembly of V1 and VO, which reduces ATPase activity and causes silencing of ion conduction. The subunits D and F are proposed to be key in these enzymatic processes. Here, we describe the structures of two conformations of the subunit DF assembly of Saccharomyces cerevisiae (ScDF) V-ATPase at 3.1 Å resolution. Subunit D (ScD) consists of a long pair of α-helices connected by a short helix ((79)IGYQVQE(85)) as well as a β-hairpin region, which is flanked by two flexible loops. The long pair of helices is composed of the N-terminal α-helix and the C-terminal helix, showing structural alterations in the two ScDF structures. The entire subunit F (ScF) consists of an N-terminal domain of four β-strands (β1-β4) connected by four α-helices (α1-α4). α1 and β2 are connected via the loop (26)GQITPETQEK(35), which is unique in eukaryotic V-ATPases. Adjacent to the N-terminal domain is a flexible loop, followed by a C-terminal α-helix (α5). A perpendicular and extended conformation of helix α5 was observed in the two crystal structures and in solution x-ray scattering experiments, respectively. Fitted into the nucleotide-bound A3B3 structure of the related A-ATP synthase from Enterococcus hirae, the arrangements of the ScDF molecules reflect their central function in ATPase-coupled ion conduction. Furthermore, the flexibility of the terminal helices of both subunits as well as the loop (26)GQITPETQEK(35) provides information about the regulatory step of reversible V1VO disassembly.

Crystal structure of subunits D and F in complex gives insight into energy transmission of the eukaryotic V-ATPase from Saccharomyces cerevisiae.

In bacteria, an ensemble of alkyl hydroperoxide reductase subunits C (AhpC) and F (AhpF) is responsible for scavenging H2O2. AhpC donates electrons for the reduction of H2O2, which are provided after NADH oxidation by AhpF. The latter contains an N-terminal domain (NTD), catalyzing the electron transfer from NADH via a FAD of the C-terminal domain (CTD) into AhpC. The NADH-bound Escherichia coli AhpF structure revealed that NADH binding brings the substrate to the re-face of the FAD, making the Cys-Cys center of the CTD accessible to the NTD disulfide center for electron transfer (Kamariah et al. (2015) Biochim Biophys Acta 1847, 1139-1152). So far insight into the epitope and mechanism of AhpF and AhpC interaction as well as the electron transfer from the NTD to AhpC have been lacking. Here using NMR spectroscopy, we glean insight into the interaction of the NTD of AhpF with AhpC from E. coli. A coordinated disappearance of EcAhpF NTD peaks was observed in the presence of full length EcAhpC, indicating a long-lived AhpC-AhpF complex. C-terminal truncated EcAhpC resulted in a more dynamic interaction, revealing specific residue chemical shift perturbation and hence the binding epitope of the complex. Combined with docking studies, we have suggested that the C terminus of AhpC binds to the backside groove of the NTD. In addition, AhpC-AhpF formation is abolished under reducing conditions. We propose for the first time a binding mechanism in which the C terminus of AhpC wraps around the NTD, slowing the dissociation rate for an efficient electron transfer process, and a release mechanism mediated by the conformational change of the C terminus of AhpC upon reduction.

Sandip Basak

Department of Physiology and Biophysics

Case Western Reserve University

Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

Sandip Basak

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Physiology and Biophysics

Case Western Reserve University

Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

Department of Physiology and Biophysics