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Three-dimensional structure of the glycine-betaine transporter BetP by cryo electron crystallography
(2008)
The soil bacterium Corynebacterium glutamicum has five secondary transporters for compatible solutes allowing it to cope with osmotic stress. The most abundant of them, the transporter BetP, performs a high affinity uptake of glycine-betain when encountering hyperosmotic stress. BetP belongs to the betaine/carnitine/choline/transporter (BCCT) family, and is predicted to have twelve transmembrane helices with both termini facing the cytoplasm. The goal of this thesis is to facilitate understanding of BetP function by determining a three dimensional (3D) model of its structure. Two-dimensional (2D) crystallization of wild-type (WT) BetP has been successfully performed by reconstitution into a mixture of E. coli lipids and bovine cardiolipin, which resulted in vesicular crystals diffracting to 7.5 Å resolution (Ziegler, Morbach et al. 2004). Diffraction patterns of these crystals however showed unfocused spots, generally due to high mosaicity. Better results were obtained by using the constitutively active mutant BetPdeltaC45 in which the first 45 amino acids of the positively charged C-terminus were removed. BetPdeltaC45 crystals obtained under the same conditions for BetP WT were concluded to be pseudo crystals, based on the inconsistence of symmetry. These crystals had BetPdeltaC45 molecules randomly up/downwards inserted into membrane crystals, and cannot be used for structure determination, even though they diffracted up to 7 Å. The problem of pseudo crystal formation could be solved by changing the lipids used for 2D crystallization to a native lipid extract from C. glutamicum cells. This change of lipids improved the crystals to well-ordered packing with exclusive p121_b symmetry. To understand the role of lipids in crystal packing and order, lipids were extracted at different stages during crystallization, and identified by using multiple precursor ion scanning mass spectrometry. The results show that phosphatidyl glycerol (PG) 16:0-18:1 is the most dominant lipid species in C. glutamicum membranes, and that BetP has a preference for the fatty acid moieties 16:0-18:1. Crystallization with synthetic PG 16:0-18:1 proved that an excess of this lipid prevents pseudo crystal formation, but these crystals did not reach the quality as previously achieved by using the C. glutamicum lipids. Apart from the effect of lipids in crystallinity, the concentration and type of salts influenced crystal growth and morphology. High salt conditions (>400 mM LiCl or KCl) yielded tubular crystals, whereas low salt conditions (<300 mM LiCl, NaCl or KCl) led to formation of up to 10 µm large sheet-like crystals. The intermediate concentration gave a mixture of sheet-like and tubular crystals. In terms of resolution, sheets diffracted better than tubes. The sheet-like crystals used for 3D map reconstruction were obtained from a dialysis buffer containing 200 mM NaCl combined with using C. glutamicum lipids. Electron microscopic images were taken from frozen-hydrated crystals using a helium-cooled JEOL 300 SFF microscope or a liquid nitrogen-cooled FEI Tecnai G2 microscope at 300 kV, which allowed optimal data collection and minimized radiation damage to the sample. More than 1000 images of tilt angles up to 50° were taken and evaluated using optical diffraction of a laser beam. The best 200 images were processed with the MRC image processing software package, and 79 images from different tilt angles were merged to the final data set used for calculation of a 3D map at a planar resolution of 8 Å. The structure shows BetPdeltaC45 as a trimer with each monomer consisting of 12 transmembrane alpha-helices. Protein termini and loop regions could not be determined due to the limited resolution of the map. Six of the twelve helices line a central cavity forming a potential substrate-binding chamber. Each monomer shows a central cavity in different sizes and shapes. Thus, the constitutively active BetPdeltaC45 thus forms an unusual asymmetric homotrimer. BetP most likely reflects three different conformational states of secondary transporters: the cytoplasmically open (C), the occluded (O), and the periplasmically open (P) states. The C and O states are similar to BetP WT projection structure, while the P state is discrepant and highly flexible due to the shape and size of the central cavity as well as the lowest intensity of the density. The observation of the P state corresponds well to the constitutively active property of BetPdeltaC45. For the high resolution structure of the C and O states are available, this work presents the first structural information of the P state of a secondary transporter.
Self-inactivating gammaretroviral vectors for the gene therapy of chronic granulomatous disease
(2008)
Chronic granulomatous disease (CGD) is a rare inherited primary immunodeficiency characterized by defective intracellular oxidative killing of ingested invading microbes by PMN and monocytes. It is caused by mutations in one of the four genes coding for the essential subunits of the NADPH oxidase (gp91phox, p47phox, p67phox and p22phox). Approximately 75% of the CGD cases are due to mutations in the gp91phox gene. If regular care and conventional therapy fail, the recommended therapy is allogeneic bone marrow transplantation (BMT), but only if a matched donor is available. A therapeutic option for patients lacking suitable donors is the genetic modification of autologous hematopoietic stem cells. The gene therapy offers an interesting alternative to BMT since it implies a less invasive treatment and represents a possibly unique curative option for patients with no suitable donor. Gammaretroviral vectors were already used in some gene therapy trials for CGD and other immunodeficiencies showing relevant clinical benefit. However, these trials uncovered an unexpected mutagenic side effect. If the retrovial integration ocurrs near to, or into proto-oncogenes this might lead to clonal dominance or even malignant transformation (Hacein-Bey-Abina et al., 2003a; Ott et al., 2006). Therefore, there was a need to further improve the safety of these vectors and to this end the self-inactivating gammaretroviral vectors were engineered. Non essential sequences for virus infectivity and integration, which might influence the surrounding gene expression, were deleted in these vectors. In the first set of experiments, a series of SIN gamma retroviral vectors was cloned driving the expression of the wild-type gp91phox cDNA under the control of a viral constitutive SFFV promoter. However initial studies with these vectors failed because the titers of the virus produced by transient transfection protocols were extremely low (<5x105 TU/ml). Therefore, a codon optimization of the gp91phox cDNA was considered as an alternative. The codon optimized synthetic gp91phox gene was used to construct a SIN gammaretroviral vector, again under the control of the SFFV promoter (Schambach et al., 2006c). With this vector an increase in titer was observed compared to the native gp91phox sequence, which was due to the improved transcription in 293T transfected cells. The enhancement of the synthetic gp91phox transcription led to a higher internal transcript production and protein expression. An enhanced superoxide production in transduced myelomonocytic X-CGD PLB-985 populations was also detected. All these data indicate that the synthetic gp91phox might represent an excellent alternative to those former constructs expressing the native gp91phox transgene. Since it was postulated that the SFFV promoter could still cause transactivation of neighboring genes due to its strength (Modlich et al., 2006), three different non-viral promoters were tested, one constitutive (the EFs promoter) and two myeloid-specific promoters (the c-fes and MRP8 promoter). The three SIN gammaretroviral vectors were able to generate high titers after transient transfection of 293T packaging cells, to efficiently transduce the X-CGD PLB-985 cell line and to reconstitute the NADPH oxidase activity to a high degree. In mouse transplantation experiments, the EFs promoter showed a high variable transgene expression in the different lineages analyzed, and the c-fes promoter showed also a ubiquitinous expression. In contrast, the MRP8 promoter showed a high myeloid specificity since gp91phox expression in mSca-1+ cells and lymphoid B cells from transplanted mice was extremly low and even absent. However, the lowest levels of transgene expression were observed in the myeloid populations both in bone marrow and peripheral blood with this vector. When the oxidase reconstitution ability of these promoters was tested, the numbers of superoxide producing cells obtained were similar than those observed in the clinical X-CGD trial conducted by the groups of Dr. M. Grez and Prof. R. A. Seger (over 35% in one patient and ~15% in the second), which led to the eradication of therapy refractory infections (Ott et al., 2006). Between the three constructs, the MRP8 promoter was less effective in restoring the NADPH oxidase activity than the EFs and c-fes promoters. The c-fes promoter reached the highest levels of DHR reactive cells in the highest number of mice. Overall, these data showed that between all constructs tested, the c-fes containing construct in combination with the codon optimized gp91phox sequence showed the best performance within the SIN gammaretroviral backbone. It generated the highest titers in combination with a better NADPH oxidase reconstituting ability. One main goal in the development of SIN gammaretroviral vectors is reducing the genotoxic effect due to random vector integration. An improved gene transfer and expression, and a constant performance are also highly desirable. The present study shows that the c-fes SIN vector in combination with the synthetic gp91phox may be considered as an effective gene therapy strategy for the restoration of the NADPH oxidase activity in CGD. It allows the use of a cellular promoter generating adequate physiological levels of the therapeutic protein and reduces the number of vector copies required for a therapeutic effect.
Poster presentation In pharmaceutical research and drug development, machine learning methods play an important role in virtual screening and ADME/Tox prediction. For the application of such methods, a formal measure of similarity between molecules is essential. Such a measure, in turn, depends on the underlying molecular representation. Input samples have traditionally been modeled as vectors. Consequently, molecules are represented to machine learning algorithms in a vectorized form using molecular descriptors. While this approach is straightforward, it has its shortcomings. Amongst others, the interpretation of the learned model can be difficult, e.g. when using fingerprints or hashing. Structured representations of the input constitute an alternative to vector based representations, a trend in machine learning over the last years. For molecules, there is a rich choice of such representations. Popular examples include the molecular graph, molecular shape and the electrostatic field. We have developed a molecular similarity measure defined directly on the (annotated) molecular graph, a long-standing established topological model for molecules. It is based on the concepts of optimal atom assignments and iterative graph similarity. In the latter, two atoms are considered similar if their neighbors are similar. This recursive definition leads to a non-linear system of equations. We show how to iteratively solve these equations and give bounds on the computational complexity of the procedure. Advantages of our similarity measure include interpretability (atoms of two molecules are assigned to each other, each pair with a score expressing local similarity; this can be visualized to show similar regions of two molecules and the degree of their similarity) and the possibility to introduce knowledge about the target where available. We retrospectively tested our similarity measure using support vector machines for virtual screening on several pharmaceutical and toxicological datasets, with encouraging results. Prospective studies are under way.
A high-precision pressure probe is described which allows non-invasive online-monitoring of the water relations of intact leaves. Real-time recording of the leaf water status occurred by data transfer to an Internet server. The leaf patch clamp pressure probe measures the attenuated pressure, Pp, of a leaf patch in response to a constant clamp pressure, Pclamp. Pp is sensed by a miniaturized silicone pressure sensor integrated into the device. The magnitude of Pp is dictated by the transfer function of the leaf, Tf, which is a function of leaf patch volume and ultimately of cell turgor pressure, Pc, as shown theoretically. The power function Tf=f(Pc) theoretically derived was experimentally confirmed by concomitant Pp and Pc measurements on intact leaflets of the liana Tetrastigma voinierianum under greenhouse conditions. Simultaneous Pp recordings on leaflets up to 10 m height above ground demonstrated that changes in Tf induced by Pc changes due to changes of microclimate and/or of the irrigation regime were sensitively reflected in corresponding changes of Pp. Analysis of the data show that transpirational water loss during the morning hours was associated with a transient rise in turgor pressure gradients within the leaflets. Subsequent recovery of turgescence during the afternoon was much faster than the preceding transpiration-induced water loss if the plants were well irrigated. Our data show the enormous potential of the leaf patch clamp pressure probe for leaf water studies including unravelling of the hydraulic communication between neighbouring leaves and over long distances within tall plants (trees).
The Na+,K+-ATPase was discovered more than 50 years ago, but even today the pumpcycle and its partial reactions are still not completely understood. In this thesis, Voltage Clamp Fluorometry was used to monitor the conformational changes that are associated with several electrogenic partial reactions of the Na+,K+-ATPase. The conformational dynamics of the ion pump were analyzed at different concentrations of internal Na+ or of external K+ and the influences on the conformational equilibrium were determined. To probe the effect of the internal Na+ concentration on the Na+ branch of the ion pump, oocytes were first depleted of internal Na+ and then loaded with Na+ using the epithelial sodium channel which can be blocked by amiloride. The conformational dynamics of the K+ branch were studied using different external K+ concentrations in the presence and in the absence of external Na+ to yield additional information on the apparent affinity of K+. The results of our Voltage Clamp Fluorometry experiments demonstrate that lowering the intracellular concentration of Na+ has a comparable effect on the conformational equilibrium as increasing the amount of K+ in the external solution. Both of these changes shift the equilibrium towards the E1/E1(P) conformation. Furthermore, it can be shown that the ratio between external Na+ and K+ ions is also a determinant for the position of the conformational equilibrium: in the absence of external Na+, the K+ dependent shift of the equilibrium towards E1 was observed at a much lower K+ concentration than in the presence of Na+. In addition, indications were found that both external K+ and internal Na+ bind within an ion well. Finally, the crucial role of negatively charged glutamate residues in the 2nd extracellular loop for the control of ion-access to the binding sites could be verified.
Cytochrome c oxidase (CcO), also called Complex IV of the aerobic respiratory chain, is located in the plasma membrane of prokaryotes and in the inner mitochondrial membrane of eukaryotes. The redox energy of dioxygen reduction is used to translocate protons across the membrane resulting in an electrochemical proton gradient. The generated proton gradient is exploited by the adenosine-5’-triphosphate synthase. In this work, bacterial four-subunit aa3-Type CcO from Paracoccus denitrificans (ATCC 13543, 4 SU-wt ATCC CcO) was used for analyses. 1) The recombinant homologously produced 4 SU-wt CcO (4 SU-wt rec CcO) was functionally compared with the native 4 SU-wt ATCC CcO. The 4 SU-wt rec CcO showed functional deficiencies as determined by UV-vis spectroscopy and electron paramagnetic resonance (EPR) studies. Total X-ray Reflection Fluorescence measurements show in both wild type CcOs the same ratio of the redoxactive Fe and Cu (2 Fe : 3 Cu) indicating full complement of the functional metals. If CcO contains only subunit I and II, it loses its functional integrity during continuous turnover activity. The importance of subunit III for integrity of CcO was demonstrated using 2 SU-wt rec CcO. Crystallisation trials of suicide inactivated 2 SU-wt rec CcOs have been ineffective using standard crystallisation conditions. Crystals of active 2 SU-wt rec CcO (positive control) have been obtained under these conditions and this result indicates possible structural changes in suicide inactivated 2 SU-wt rec CcO. The structure of active 2 SU-wt rec CcO was determined to 2.25 Å resolution. 2) Terminal oxidases require four electrons for the cleavage of the dioxygen bond (O=O). In general, the catalytic cycle of CcO is described by the electron input and thus by the different redox states of the metal centres: the O, E, R, P and F state. The two-electron reduced R intermediate is able to donate four electrons for dioxygen reduction forming the P state. The P intermediate is an oxoferryl state implying the lack of an electron for the R -> P transition, because the metal centres can only provide three electrons (Fe+II forms Fe+IV and Cu+II forms Cu+I). The P state, where the dioxygen bond is already broken, shows an oxoferryl state (FeIV=O2-) and a nearby tyrosine is proposed to form a tyrosyl radical representing the donor of the missing electron. H2O2-induced artificial intermediates provide the opportunity to investigated different catalytic intermediates in detail. Mixing equimolar amounts of H2O2 to CcO in the O state induces the "two-electron" reduced PH state at high pH and the electronically equal "two-electron" reduced F• H state at low pH. The addition of an excess amount of H2O2 leads to the three-electron reduced FH state. Functional studies using the 4 SU-wt ATCC CcO have demonstrated a bound peroxide (O- - O-) intermediate during the catalytic cycle. Using EPR it was previously shown that Y167 hosts a radical species in PH/F• H state which suggests that Y167 could provide this "missing electron". While X-ray structural models of CcO and Fourier-transformed infrared (FTIR) measurements of oxygenated ("pulsed") 4 SU-wt ATCC CcO suggest a bound peroxide in the O state, UV-vis and EPR spectroscopic studies indicate that other intermediates may also contain such peroxide species. Equimolar and excess amounts of H2O2 induce the PH/F• H and FH states, respectively and catalase treatment of the FH state leads, contrary to the natural direction of the catalytic cycle, to the apparent transition of the FH -> PH/F• H states, which is accompanied by reappearance of an EPR signal from the Y167• radical. The novel PFH/F• FH states are presented here and we postulate that the FH state hosts a superoxide (or peroxide) adduct at CuB in the binuclear site. In addition, the novel P10 state is also introduced having a maximum at lambda = 612 nm in the difference absorption spectrum (minus the O state). The P10 state is induced by mixing CcO in the O state with a pH 10 buffer. This pH 10 induced state resembles standard P states such as PCO, PH and PR. However, the P10 state evolves out of the O state without addition of reduction equivalents. Using EPR spectroscopy it was shown that Y167 hosts a radical species in the P10 state such as in the PH state. In summary, all functional data presented here provide evidence for a peroxide bound during the O state. Finally, a new model for the natural catalytic cycle is proposed. If the O state contains a peroxide, it is also likely that the E and R state contain this species. Even the oxoferryl intermediates P and F states may complex a peroxide at CuB in the binuclear site. 3) The amino acid residue Y167, which hosts the radical in the PH/F•H states, is not directly part of the binuclear site of CcO. For identification of the primary electron donor, two tryptophan variants of CcO, W272F and W164F, which are located nearby the binuclear site, were produced. Evidence is provided that W272 is a kinetically fast electron donor for the O2 molecule. The electron is replenished by Y167, or probably by Y280 in the natural cycle. The Y167 radical is detectable by EPR spectroscopy after treatment with equimolar amounts of H2O2 in the active variant W164F, but is absent in the inactive variant W272F. 4) CcO contains two proton conducting pathways, the D- and the K-pathway. Proteoliposomes of the variants H28A and D30N, mutations located at the entrance of the D-pathway, both show the identical proton pumping activity as the 4 SU-wt rec CcO (pumped H+/e- = 1). The variant N113D shows abolished proton pumping (pumped H+/e- = 0), but a relative high cytochrome c oxidation activity (63 %). G196D displays no cytochrome c oxidation and proton pumping activity. Overall, the addition or removal of a negative charge within the D-pathway such as in D124N, N131D, N113D and G196D leads to a decoupled phenotype indicating the high degree of electrostatic coupling in CcO.
In a combined NMR/MD study, the temperature-dependent changes in the conformation of two members of the RNA YNMG-tetraloop motif (cUUCGg and uCACGg) have been investigated at temperatures of 298, 317 and 325 K. The two members have considerable different thermal stability and biological functions. In order to address these differences, the combined NMR/MD study was performed. The large temperature range represents a challenge for both, NMR relaxation analysis (consistent choice of effective bond length and CSA parameter) and all-atom MD simulation with explicit solvent (necessity to rescale the temperature). A convincing agreement of experiment and theory is found. Employing a principle component analysis of the MD trajectories, the conformational distribution of both hairpins at various temperatures is investigated. The ground state conformation and dynamics of the two tetraloops are indeed found to be very similar. Furthermore, both systems are initially destabilized by a loss of the stacking interactions between the first and the third nucleobase in the loop region. While the global fold is still preserved, this initiation of unfolding is already observed at 317 K for the uCACGg hairpin but at a significantly higher temperature for the cUUCGg hairpin.
Life-threatening fungal infections are becoming increasingly common for immunocompromised patients such as those with AIDS, or those undergoing organ transplantation or chemotheraphy, as well as for other health-vulnerable patients. Excellent targets for antifungal drugs are chitin synthases, which are essential for survival of the fungus and lacking in humans. To design new antifungal drugs, knowledge of the three-dimensional structure and mechanism of action of chitin synthases are crucial. Chitin synthases are members of an important family of enzymes that synthesize structural polysaccharides, such as cellulose, β(1,3)-glucan, β(1,4)-mannan and hyaluronan. Therefore, chitin synthases could be used as a model system to understand these more complex enzymes, which are also of major medical and commercial importance. Chitin synthase 2 from Saccharomyces cerevisiae (ScChS2), the protein under study, is an integral membrane protein that synthesizes the primary septum between mother and daughter cells in budding yeast. It is essential for proper cell separation and expected to be highly regulated. An important aspect is that ScChS2 shows 55% sequence identity and is functionally analogous to chitin synthase 1 from the human opportunistic pathogen Candida albicans, this enzyme is also essential for cell survival (Munro, Winter et al. 2001). ...
Chlamydia are obligate intracellular bacteria that cause variety of human diseases. Host cells infected with Chlamydia are protected against many different apoptotic stimuli. The induction of apoptosis resistance is thought to be an important immune escape mechanism allowing Chlamydia to replicate inside the host cell. Infection with C. trachomatis activates the Raf/MEK/ERK pathway and the PI3K/AKT pathway. Here we show that inhibition of these two pathways by chemical inhibitors sensitized C. trachomatis infected cells to granzyme B-mediated cell death. Infection leads to the Raf/MEK/ERK-mediated up-regulation and PI3K-dependent stabilization of the anti-apoptotic Bcl-2 family member Mcl-1. Consistently, interfering with Mcl-1 up-regulation sensitized infected cells for apoptosis induced via the TNF receptor, DNA damage, granzyme B and stress. Our data suggest that Mcl-1 up-regulation is primarily required to maintain apoptosis resistance in C. trachomatis-infected cells.
The degradation of the poly(A) tail is crucial for posttranscriptional gene regulation and for quality control of mRNA. Poly(A)-specific ribonuclease (PARN) is one of the major mammalian 3’ specific exo-ribonucleases involved in the degradation of the mRNA poly(A) tail, and it is also involved in the regulation of translation in early embryonic development. The interaction between PARN and the m7GpppG cap of mRNA plays a key role in stimulating the rate of deadenylation. Here we report the solution structures of the cap-binding domain of mouse PARN with and without the m7GpppG cap analog. The structure of the cap-binding domain adopts the RNA recognition motif (RRM) with a characteristic a-helical extension at its C-terminus, which covers the b-sheet surface (hereafter referred to as PARN RRM). In the complex structure of PARN RRM with the cap analog, the base of the N7-methyl guanosine (m7G) of the cap analog stacks with the solvent-exposed aromatic side chain of the distinctive tryptophan residue 468, located at the C-terminal end of the second b-strand. These unique structural features in PARN RRM reveal a novel cap-binding mode, which is distinct from the nucleotide recognition mode of the canonical RRM domains.