Bernd Brutschy

DNA Damage in Oocytes Induces a Switch of the Quality Control Factor TAp63α from Dimer to Tetramer

Summary TAp63α, a homolog of the p53 tumor suppressor, is a quality control factor in the female germline. Remarkably, already undamaged oocytes express high levels of the protein, suggesting that TAp63αs activity is under tight control of an inhibitory mechanism. Biochemical studies have proposed that inhibition requires the C-terminal transactivation inhibitory domain. However, the structural mechanism of TAp63α inhibition remains unknown. Here, we show that TAp63α is kept in an inactive dimeric state. We reveal that relief of inhibition leads to tetramer formation with ∼20-fold higher DNA affinity. In vivo, phosphorylation-triggered tetramerization of TAp63α is not reversible by dephosphorylation. Furthermore, we show that a helix in the oligomerization domain of p63 is crucial for tetramer stabilization and competes with the transactivation domain for the same binding site. Our results demonstrate how TAp63α is inhibited by complex domain-domain interactions that provide the basis for regulating quality control in oocytes.

The Archaeal Lsm Protein Binds to Small RNAs

Proteins of the Lsm family, including eukaryotic Sm proteins and bacterial Hfq, are key players in RNA metabolism. Little is known about the archaeal homologues of these proteins. Therefore, we characterized the Lsm protein from the haloarchaeon Haloferax volcanii using in vitro and in vivo approaches. H. volcanii encodes a single Lsm protein, which belongs to the Lsm1 subfamily. The lsm gene is co-transcribed and overlaps with the gene for the ribosomal protein L37e. Northern blot analysis shows that the lsm gene is differentially transcribed. The Lsm protein forms homoheptameric complexes and has a copy number of 4000 molecules/cell. In vitro analyses using electrophoretic mobility shift assays and ultrasoft mass spectrometry (laser-induced liquid bead ion desorption) showed a complex formation of the recombinant Lsm protein with oligo(U)-RNA, tRNAs, and an small RNA. Co-immunoprecipitation with a FLAG-tagged Lsm protein produced in vivo confirmed that the protein binds to small RNAs. Furthermore, the co-immunoprecipitation revealed several protein interaction partners, suggesting its involvement in different cellular pathways. The deletion of the lsm gene is viable, resulting in a pleiotropic phenotype, indicating that the haloarchaeal Lsm is involved in many cellular processes, which is in congruence with the number of protein interaction partners.

An intermediate step in the evolution of ATPases - a hybrid F0–V0 rotor in a bacterial Na+ F1F0 ATP synthase

The Na+ F1F0 ATP synthase operon of the anaerobic, acetogenic bacterium Acetobacterium woodii is unique because it encodes two types of c subunits, two identical 8 kDa bacterial F0‐like c subunits (c2 and c3), with two transmembrane helices, and a 18 kDa eukaryal V0‐like (c1) c subunit, with four transmembrane helices but only one binding site. To determine whether both types of rotor subunits are present in the same c ring, we have isolated and studied the composition of the c ring. High‐resolution atomic force microscopy of 2D crystals revealed 11 domains, each corresponding to two transmembrane helices. A projection map derived from electron micrographs, calculated to 5 Å resolution, revealed that each c ring contains two concentric, slightly staggered, packed rings, each composed of 11 densities, representing 22 transmembrane helices. The inner and outer diameters of the rings, measured at the density borders, are approximately 17 and 50 Å. Mass determination by laser‐induced liquid beam ion desorption provided evidence that the c rings contain both types of c subunits. The stoichiometry for c2/c3 : c1 was 9 : 1. Furthermore, this stoichiometry was independent of the carbon source of the growth medium. These analyses clearly demonstrate, for the first time, an F0–V0 hybrid motor in an ATP synthase.

Studying the stoichiometries of membrane proteins by mass spectrometry: microbial rhodopsins and a potassium ion channel

In the present work we demonstrate the advantages of LILBID mass spectrometry in the mass analysis of membrane proteins with emphasis on ion-pumps and channels. Due to their hydrophobic nature, membrane proteins have to be solubilized by detergents. However, these molecules tend to complicate the analysis by mass spectrometry. In LILBID, detergent molecules are readily tolerated which allows for the study of solution phase quaternary structures of membrane proteins. This is shown for the proton-pump bacteriorhodospin and the potassium channel KcsA where in both cases the stoichiometries found by LILBID reflect the known structures from 2D or 3D crystals. With proteorhodopsin we demonstrate a preliminary detergent screening showing different structures in different detergents and the implications for the functionality of this protein. We show that Triton-X 100 prevents the formation of the pentamer of proteorhodopsin. Furthermore, the quaternary structures of proteorhodopsin cloned without the signal peptide and of the cation channel channelrhodopsin-2 were studied. The intrinsic properties of channelrhodopsin-2 allow for mass spectrometric analysis in very high salt concentrations up to 100 mM of NaCl. In summary we demonstrate that LILBID is an alternative mass spectrometric method for the analysis of membrane proteins from solution phase.

Asymmetric ATP Hydrolysis Cycle of the Heterodimeric Multidrug ABC Transport Complex TmrAB from Thermus thermophilus

ATP-binding cassette (ABC) systems translocate a wide range of solutes across cellular membranes. The thermophilic Gram-negative eubacterium Thermus thermophilus, a model organism for structural genomics and systems biology, discloses ∼46 ABC proteins, which are largely uncharacterized. Here, we functionally analyzed the first two and only ABC half-transporters of the hyperthermophilic bacterium, TmrA and TmrB. The ABC system mediates uptake of the drug Hoechst 33342 in inside-out oriented vesicles that is inhibited by verapamil. TmrA and TmrB form a stable heterodimeric complex hydrolyzing ATP with a Km of 0.9 mm and kcat of 9 s−1 at 68 °C. Two nucleotides can be trapped in the heterodimeric ABC complex either by vanadate or by mutation inhibiting ATP hydrolysis. Nucleotide trapping requires permissive temperatures, at which a conformational ATP switch is possible. We further demonstrate that the canonic glutamate 523 of TmrA is essential for rapid conversion of the ATP/ATP-bound complex into its ADP/ATP state, whereas the corresponding aspartate in TmrB (Asp-500) has only a regulatory role. Notably, exchange of this single noncanonic residue into a catalytic glutamate cannot rescue the function of the E523Q/D500E complex, implicating a built-in asymmetry of the complex. However, slow ATP hydrolysis in the newly generated canonic site (D500E) strictly depends on the formation of a posthydrolysis state in the consensus site, indicating an allosteric coupling of both active sites.

Structural study on the architecture of the bacterial ATP synthase Fo motor

We purified the Fo complex from the Ilyobacter tartaricus Na+-translocating F1Fo-ATP synthase and performed a biochemical and structural study. Laser-induced liquid bead ion desorption MS analysis demonstrates that all three subunits of the isolated Fo complex were present and in native stoichiometry (ab2c11). Cryoelectron microscopy of 2D crystals yielded a projection map at a resolution of 7.0 Å showing electron densities from the c11 rotor ring and up to seven adjacent helices. A bundle of four helices belongs to the stator a-subunit and is in contact with c11. A fifth helix adjacent to the four-helix bundle interacts very closely with a c-subunit helix, which slightly shifts its position toward the ring center. Atomic force microscopy confirms the presence of the Fo stator, and a height profile reveals that it protrudes less from the membrane than c11. The data limit the dimensions of the subunit a/c-ring interface: Three helices from the stator region are in contact with three c11 helices. The location and distances of the stator helices impose spatial restrictions on the bacterial Fo complex.

Fold and Function of the InlB B-repeat

Host cell invasion by the facultative intracellular pathogen Listeria monocytogenes requires the invasion protein InlB in many cell types. InlB consists of an N-terminal internalin domain that binds the host cell receptor tyrosine kinase Met and C-terminal GW domains that bind to glycosaminoglycans (GAGs). Met binding and activation is required for host cell invasion, while the interaction between GW domains and GAGs enhances this effect. Soluble InlB elicits the same cellular phenotypes as the natural Met ligand hepatocyte growth factor/scatter factor (HGF/SF), e.g. cell scatter. So far, little is known about the central part of InlB, the B-repeat. Here we present a structural and functional characterization of the InlB B-repeat. The crystal structure reveals a variation of the β-grasp fold that is most similar to small ubiquitin-like modifiers (SUMOs). However, structural similarity also suggests a potential evolutionary relation to bacterial mucin-binding proteins. The B-repeat defines the prototype structure of a hitherto uncharacterized domain present in over a thousand bacterial proteins. Generally, this domain probably acts as a spacer or a receptor-binding domain in extracellular multi-domain proteins. In cellular assays the B-repeat acts synergistically with the internalin domain conferring to it the ability to stimulate cell motility. Thus, the B-repeat probably binds a further host cell receptor and thereby enhances signaling downstream of Met.

Functional production of the Na+ F1FO ATP synthase from Acetobacterium woodii in Escherichia coli requires the native AtpI

The Na+ F1FO ATP synthase of the anaerobic, acetogenic bacterium Acetobacterium woodii has a unique FOVO hybrid rotor that contains nine copies of a FO-like c subunit and one copy of a VO-like c1 subunit with one ion binding site in four transmembrane helices whose cellular function is obscure. Since a genetic system to address the role of different c subunits is not available for this bacterium, we aimed at a heterologous expression system. Therefore, we cloned and expressed its Na+ F1FO ATP synthase operon in Escherichia coli. A Δatp mutant of E. coli produced a functional, membrane-bound Na+ F1FO ATP synthase that was purified in a single step after inserting a His6-tag to its β subunit. The purified enzyme was competent in Na+ transport and contained the FOVO hybrid rotor in the same stoichiometry as in A. woodii. Deletion of the atpI gene from the A. woodii operon resulted in a loss of the c ring and a mis-assembled Na+ F1FO ATP synthase. AtpI from E. coli could not substitute AtpI from A. woodii. These data demonstrate for the first time a functional production of a FOVO hybrid rotor in E. coli and revealed that the native AtpI is required for assembly of the hybrid rotor.

ATP synthases: cellular nanomotors characterized by LILBID mass spectrometry.

Mass spectrometry of membrane protein complexes is still a methodological challenge due to hydrophobic and hydrophilic parts of the species and the fact that all subunits are bound non-covalently together. The present study with the novel laser induced liquid bead ion desorption mass spectrometry (LILBID-MS) reports on the determination of the subunit composition of the F(1)F(o)-ATP synthase from Bacillus pseudofirmus OF4, that of both bovine heart and, for the first time, of human heart mitochondrial F(1)F(o)-ATP synthases. Under selected buffer conditions the mass of the intact F(1)F(o)-ATP synthase of B. pseudofirmus OF4 could be measured, allowing the analysis of complex subunit stoichiometry. The agreement with theoretical masses derived from sequence databases is very good. A comparison of the ATP synthase subunit composition of 5 different ATPases reveals differences in the complexity of eukaryotic and bacterial ATP synthases. However, whereas the overall construction of eukaryotic enzymes is more complex than the bacterial ones, functionally important subunits are conserved among all ATPases.

Recognition of the let-7g miRNA precursor by human Lin28B

Mammalian homologs of lin28: Lin28 and Lin28B block the post‐transcriptional processing of the let‐7 family of miRNAs. We report that in vitro the terminal stem‐loop region of the let‐7g miRNA precursor (pre‐let‐7g) required to bind Lin28B is restricted to 24 nucleotides (nt) including the 3′ GGAG motif. Additionally, full length Lin28B is required for efficient binding to pre‐let‐7g and the stoichiometry of the complex is 1:1. Molecular dynamics (MD) simulations reveal the interactions of the pre‐let‐7g stem‐loop and the GGAG motif in the stem region to the cold shock domain (CSD) and to the zinc knuckle domain (ZKD) of Lin28B, respectively.