Elena Cabezón

Ectopic |[beta]|-chain of ATP synthase is an apolipoprotein A-I receptor in hepatic HDL endocytosis

The effect of high-density lipoprotein (HDL) in protecting against atherosclerosis is usually attributed to its role in ‘reverse cholesterol transport’. In this process, HDL particles mediate the efflux and the transport of cholesterol from peripheral cells to the liver for further metabolism and bile excretion. Thus, cell-surface receptors for HDL on hepatocytes are chief partners in the regulation of cholesterol homeostasis. A high-affinity HDL receptor for apolipoprotein A-I (apoA-I) was previously identified on the surface of hepatocytes. Here we show that this receptor is identical to the β-chain of ATP synthase, a principal protein complex of the mitochondrial inner membrane. Different experimental approaches confirm this ectopic localization of components of the ATP synthase complex and the presence of ATP hydrolase activity at the hepatocyte cell surface. Receptor stimulation by apoA-I triggers the endocytosis of holo-HDL particles (protein plus lipid) by a mechanism that depends strictly on the generation of ADP. We confirm this effect on endocytosis in perfused rat liver ex vivo by using a specific inhibitor of ATP synthase. Thus, membrane-bound ATP synthase has a previously unsuspected role in modulating the concentrations of extracellular ADP and is regulated by a principal plasma apolipoprotein.

The Bacterial Conjugation Protein Trwb Resembles Ring Helicases and F1-ATPase

The transfer of DNA across membranes and between cells is a central biological process; however, its molecular mechanism remains unknown. In prokaryotes, trans-membrane passage by bacterial conjugation, is the main route for horizontal gene transfer. It is the means for rapid acquisition of new genetic information, including antibiotic resistance by pathogens. Trans-kingdom gene transfer from bacteria to plants or fungi and even bacterial sporulation are special cases of conjugation. An integral membrane DNA-binding protein, called TrwB in the Escherichia coli R388 conjugative system, is essential for the conjugation process. This large multimeric protein is responsible for recruiting the relaxosome DNA–protein complex, and participates in the transfer of a single DNA strand during cell mating. Here we report the three-dimensional structure of a soluble variant of TrwB. The molecule consists of two domains: a nucleotide-binding domain of α/β topology, reminiscent of RecA and DNA ring helicases, and an all-α domain. Six equivalent protein monomers associate to form an almost spherical quaternary structure that is strikingly similar to F1-ATPase. A central channel, 20 Å in width, traverses the hexamer.

GENETIC EVIDENCE OF A COUPLING ROLE FOR THE TRAG PROTEIN FAMILY IN BACTERIAL CONJUGATION

Abstract The ability of conjugative plasmids from six different incompatibility groups to mobilize a set of mobilizable plasmids was examined. The mobilization frequencies of plasmids RSF1010, ColE1, ColE3, and CloDF13 varied over seven orders of magnitude, depending on the helper conjugative plasmid used. Mobilization of CloDF13 was unique in that it did not require TrwB, TraG or TraD (all members of the TraG family) for mobilization by R388, RP4 or F, respectively. CloDF13 itself codes for an essential mobilization protein (MobB) which is also a TraG homolog, only requiring a source of the genes for pilus formation. Besides, CloDF13 was mobilized efficiently by all conjugative plasmids, suggesting that TraG homologs are the primary determinants of the mobilization efficiency of a plasmid, interacting differentialy with the various relaxosomes. Previous results indicated that TraG and TrwB were interchangeable for mobilization of RSF1010 and ColE1 by PILW (the pilus system of IncW plasmids) but TraG could not complement conjugation of trwB mutants, suggesting that additional interactions were taking place between TrwB and oriT(R388) that were not essential for mobilization. To further test this hypothesis, we analyzed the mobilization frequencies of ColE1 and RSF1010 by the P, W, and F pili in the presence of alternative TraG homologs. The results obtained indicated that the frequency of mobilization was determined both by the particular TraG-like protein used and by the pilus system. Thus, TraG-like proteins are not generally interchangeable for mobilization. Therefore we suggest that the factors that determine the frequencies of transfer of different MOB regions are the differential interactions of TrwB with pilus and relaxosome.

The structure of bovine F1-ATPase in complex with its regulatory protein IF1

In mitochondria, the hydrolytic activity of ATP synthase is prevented by an inhibitor protein, IF1. The active bovine protein (84 amino acids) is an α-helical dimer with monomers associated via an antiparallel α-helical coiled coil composed of residues 49–81. The N-terminal inhibitory sequences in the active dimer bind to two F1-ATPases in the presence of ATP. In the crystal structure of the F1−IF1 complex at 2.8 Å resolution, residues 1–37 of IF1 bind in the αDP-βDP interface of F1-ATPase, and also contact the central γ subunit. The inhibitor opens the catalytic interface between the αDP and βDP subunits relative to previous structures. The presence of ATP in the catalytic site of the βDP subunit implies that the inhibited state represents a pre-hydrolysis step on the catalytic pathway of the enzyme.

Modulation of the Oligomerization State of the Bovine F1-ATPase Inhibitor Protein, IF1, by pH

Bovine IF1, a basic protein of 84 amino acids, is involved in the regulation of the catalytic activity of the F1 domain of ATP synthase. At pH 6.5, but not at basic pH values, it inhibits the ATP hydrolase activity of the enzyme. The oligomeric state of bovine IF1 has been investigated at various pH values by sedimentation equilibrium analytical ultracentrifugation and by covalent cross-linking. Both techniques confirm that the protein forms a tetramer at pH 8, and below pH 6.5, the protein is predominantly dimeric. By covalent cross-linking, it has been found that at pH 8.0 the fragment of IF1 consisting of residues 44–84 forms a dimer, whereas the fragment from residues 32–84 is tetrameric. Therefore, some or all of the residues between positions 32 and 43 are necessary for tetramer formation and are involved in the pH-sensitive interconversion between dimer and tetramer. One important residue in the interconversion is histidine 49. Mutation of this residue to lysine abolishes the pH-dependent activation-inactivation, and the mutant protein is active and dimeric at all pH values investigated. It is likely from NMR studies that the inhibitor protein dimerizes by forming an antiparallel α-helical coiled-coil over its C-terminal region and that at high pH values, where the protein is tetrameric, the inhibitory regions are masked. The mutation of histidine 49 to lysine is predicted to abolish coiled-coil formation over residues 32–43 preventing interaction between two dimers, forcing the equilibrium toward the dimeric state, thereby freeing the N-terminal inhibitory regions and allowing them to interact with F1.

Characterization of ATP and DNA binding activities of TrwB, the coupling protein essential in plasmid R388 conjugation

TrwB is the conjugative coupling protein of plasmid R388. TrwBΔN70 contains the soluble domain of TrwB. It was constructed by deletion of trwB sequences containing TrwB N-proximal transmembrane segments. Purified TrwBΔN70 protein bound tightly the fluorescent ATP analogue TNP-ATP (K s = 8.7 μm) but did not show measurable ATPase or GTPase activity. A single ATP binding site was found per TrwB monomer. An intact ATP-binding site was essential for R388 conjugation, since a TrwB mutant with a single amino acid alteration in the ATP-binding signature (K136T) was transfer-deficient. TrwBΔN70 also bound DNA nonspecifically. DNA binding enhanced TrwC nic cleavage, providing the first evidence that directly links TrwB with conjugative DNA processing. Since DNA bound by TrwBΔN70 also showed increased negative superhelicity (as shown by increased sensitivity to topoisomerase I), nic cleavage enhancement was assumed to be a consequence of the increased single-stranded nature of DNA aroundnic. The mutant protein TrwB(K136T)ΔN70 was indistinguishable from TrwBΔN70 with respect to the above properties, indicating that TrwB ATP binding activity is not required for them. The reported properties of TrwB suggest potential functions for conjugative coupling proteins, both as triggers of conjugative DNA processing and as motors in the transport process.

The structure of bovine IF1, the regulatory subunit of mitochondrial F-ATPase

In mitochondria, the hydrolytic activity of ATP synthase is regulated by an inhibitor protein, IF1. Its binding to ATP synthase depends on pH, and below neutrality, IF1 is dimeric and forms a stable complex with the enzyme. At higher pH values, IF1 forms tetramers and is inactive. In the 2.2 Å structure of the bovine IF1 described here, the four monomers in the asymmetric unit are arranged as a dimer of dimers. Monomers form dimers via an antiparallel α‐helical coiled coil in the C‐terminal region. Dimers are associated into oligomers and form long fibres in the crystal lattice, via coiled‐coil interactions in the N‐terminal and inhibitory regions (residues 14–47). Therefore, tetramer formation masks the inhibitory region, preventing IF1 binding to ATP synthase.

Towards an integrated model of bacterial conjugation

Bacterial conjugation is one of the main mechanisms for horizontal gene transfer. It constitutes a key element in the dissemination of antibiotic resistance and virulence genes to human pathogenic bacteria. DNA transfer is mediated by a membrane-associated macromolecular machinery called Type IV secretion system (T4SS). T4SSs are involved not only in bacterial conjugation but also in the transport of virulence factors by pathogenic bacteria. Thus, the search for specific inhibitors of different T4SS components opens a novel approach to restrict plasmid dissemination. This review highlights recent biochemical and structural findings that shed new light on the molecular mechanisms of DNA and protein transport by T4SS. Based on these data, a model for pilus biogenesis and substrate transfer in conjugative systems is proposed. This model provides a renewed view of the mechanism that might help to envisage new strategies to curb the threating expansion of antibiotic resistance.

The ATPase activity of the DNA transporter TrwB is modulated by protein TrwA.Implications for a common assembly mechanism of DNA translocating motors

Conjugative systems contain an essential integral membrane protein involved in DNA transport called the Type IV coupling protein (T4CP). The T4CP of conjugative plasmid R388 is TrwB, a DNA-dependent ATPase. Biochemical and structural data suggest that TrwB uses energy released from ATP hydrolysis to pump DNA through its central channel by a mechanism similar to that used by F1-ATPase or ring helicases. For DNA transport, TrwB couples the relaxosome (a DNA-protein complex) to the secretion channel. In this work we show that TrwA, a tetrameric oriT DNA-binding protein and a component of the R388 relaxosome, stimulates TrwBΔN70 ATPase activity, revealing a specific interaction between the two proteins. This interaction occurs via the TrwA C-terminal domain. A 68-kDa complex between TrwBΔN70 and TrwA C-terminal domain was observed by gel filtration chromatography, consistent with a 1:1 stoichiometry. Additionally, electron microscopy revealed the formation of oligomeric TrwB complexes in the presence, but not in the absence, of TrwA protein. TrwBΔN70 ATPase activity in the presence of TrwA was further enhanced by DNA. Interestingly, maximal ATPase rates were achieved with TrwA and different types of dsDNA substrates. This is consistent with a role of TrwA in facilitating the interaction between TrwB and DNA. Our findings provide a new insight into the mechanism by which TrwB recruits the relaxosome for DNA transport. The process resembles the mechanism used by other DNA-dependent molecular motors, such as the RuvA/RuvB system, to be targeted to the DNA followed by hexamer assembly.

The Hexameric Structure of a Conjugative VirB4 Protein ATPase Provides New Insights for a Functional and Phylogenetic Relationship with DNA Translocases

Background: VirB4 ATPases are involved in protein transport in T4SS. Results: The structure of the conjugative VirB4 homologue TrwK has been determined by single-particle electron microscopy. Conclusion: TrwK forms hexamers and binds preferentially G4-quadruplex DNA as the coupling protein TrwB. Significance: The results provide structural and biochemical evidence for a common evolutionary scenario between DNA and protein translocases. VirB4 proteins are ATPases essential for pilus biogenesis and protein transport in type IV secretion systems. These proteins contain a motor domain that shares structural similarities with the motor domains of DNA translocases, such as the VirD4/TrwB conjugative coupling proteins and the chromosome segregation pump FtsK. Here, we report the three-dimensional structure of full-length TrwK, the VirB4 homologue in the conjugative plasmid R388, determined by single-particle electron microscopy. The structure consists of a hexameric double ring with a barrel-shaped structure. The C-terminal half of VirB4 proteins shares a striking structural similarity with the DNA translocase TrwB. Docking the atomic coordinates of the crystal structures of TrwB and FtsK into the EM map revealed a better fit for FtsK. Interestingly, we have found that like TrwB, TrwK is able to bind DNA with a higher affinity for G4 quadruplex structures than for single-stranded DNA. Furthermore, TrwK exerts a dominant negative effect on the ATPase activity of TrwB, which reflects an interaction between the two proteins. Our studies provide new insights into the structure-function relationship and the evolution of these DNA and protein translocases.