Ignacio Arechaga

The rotary mechanism of ATP synthase

Abstract Since the chemiosmotic theory was proposed by Peter Mitchell in the 1960s, a major objective has been to elucidate the mechanism of coupling of the transmembrane proton motive force, created by respiration or photosynthesis, to the synthesis of ATP from ADP and inorganic phosphate. Recently, significant progress has been made towards establishing the complete structure of ATP synthase and revealing its mechanism. The X-ray structure of the F1 catalytic domain has been completed and an electron density map of the F1–c10 subcomplex has provided a glimpse of the motor in the membrane domain. Direct microscopic observation of rotation has been extended to F1-ATPase and F1Fo-ATPase complexes.

Coexistence of multivalent and monovalent TCRs explains high sensitivity and wide range of response

A long-standing paradox in the study of T cell antigen recognition is that of the high specificity–low affinity T cell receptor (TCR)–major histocompatibility complex peptide (MHCp) interaction. The existence of multivalent TCRs could resolve this paradox because they can simultaneously improve the avidity observed for monovalent interactions and allow for cooperative effects. We have studied the stoichiometry of the TCR by Blue Native–polyacrylamide gel electrophoresis and found that the TCR exists as a mixture of monovalent (αβγɛδɛζζ) and multivalent complexes with two or more ligand-binding TCRα/β subunits. The coexistence of monovalent and multivalent complexes was confirmed by electron microscopy after label fracture of intact T cells, thus ruling out any possible artifact caused by detergent solubilization. We found that although only the multivalent complexes become phosphorylated at low antigen doses, both multivalent and monovalent TCRs are phosphorylated at higher doses. Thus, the multivalent TCRs could be responsible for sensing low concentrations of antigen, whereas the monovalent TCRs could be responsible for dose-response effects at high concentrations, conditions in which the multivalent TCRs are saturated. Thus, besides resolving TCR stoichiometry, these data can explain how T cells respond to a wide range of MHCp concentrations while maintaining high sensitivity.

Characterisation of new intracellular membranes in Escherichia coli accompanying large scale over-production of the b subunit of F1Fo ATP synthase

Recombinant membrane proteins in Escherichia coli are either expressed at relatively low level in the cytoplasmic membrane or they accumulate as inclusion bodies. Here, we report that the abundant over‐production of subunit b of E. coli F1Fo ATP synthase in the mutant host strains E. coli C41(DE3) and C43(DE3) is accompanied by the proliferation of intracellular membranes without formation of inclusion bodies. Maximal levels of proliferation of intracellular membranes were observed in C43(DE3) cells over‐producing subunit b. The new proliferated membranes contained all the over‐expressed protein and could be recovered by a single centrifugation step. Recombinant subunit b represented up to 80% of the protein content of the membranes. The lipid:protein ratios and phospholipid compositions of the intracellular membranes differ from those of bacterial cytoplasmic membranes, and they are particularly rich in cardiolipin.

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.

Self‐assembly of ATP synthase subunit c rings

Subunit c of the H+ transporting ATP synthase is an essential part of its membrane domain that participates in transmembrane proton conduction. The annular architecture of the subunit c from different species has been previously reported. However, little is known about the type of interactions that affect the formation of c‐rings in the ATPase complex. Here we report that subunit c over‐expressed in Escherichia coli and purified in non‐ionic detergent solutions self‐assembles into annular structures in the absence of other subunits of the complex. The results suggest that the ability of subunit c to form rings is determined by its primary structure.

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.

The Mitochondrial Uncoupling Protein UCP1: A Gated Pore

The uncoupling protein UCP1 is a member of a superfamily of homologous proteins formed by the mitochondrial metabolite transporters. Although they act in vivo as carriers, under specific experimental conditions some of these transporters have been shown to behave as channels. This dual transport operation suggests that these carriers are likely to be formed by two differentiated functional and structural domains. The kinetic model termed “single binding center gated pore” is well suited to understand the behaviour of these carriers. It proposes that in the protein core there must exist a hydrophilic translocation pore whose access is controlled by gates. It is highly likely that the hydrophilic channel is formed by the transmembrane α‐helices and that loops contribute to the formation of the gates. UCP1 is regulated physiologically by fatty acids and purine nucleotides. Nucleotides maintain the proton conductance inhibited while fatty acids act as cytosolic second messengers of noradrenaline to active UCP1. Based on photoaffinity labeling and mutagenesis data, we propose a structural model for the localization of the binding site. The nucleotide enters through a gate in the cytosolic side and binds deep inside the protein. The three matrix loops contribute to the formation of a hydrophobic binding pocket that would accommodate the purine moiety. Three arginine residues (in helices II, IV, and VI) would interact with the phosphate groups. His214 and Glu190 have been involved in the pH regulation of the nucleotide binding but because they are on the cytosolic side of the protein, we propose that their state of protonation will determine the access of the nucleotide to the binding center.

ATPase Activity and Oligomeric State of TrwK, the VirB4 Homologue of the Plasmid R388 Type IV Secretion System

Type IV secretion systems (T4SS) mediate the transfer of DNA and protein substrates to target cells. TrwK, encoded by the conjugative plasmid R388, is a member of the VirB4 family, comprising the largest and most conserved proteins of T4SS. VirB4 was suggested to be an ATPase involved in energizing pilus assembly and substrate transport. However, conflicting experimental evidence concerning VirB4 ATP hydrolase activity was reported. Here, we demonstrate that TrwK is able to hydrolyze ATP in vitro in the absence of its potential macromolecular substrates and other T4SS components. The kinetic parameters of its ATPase activity have been characterized. The TrwK oligomerization state was investigated by analytical ultracentrifugation and electron microscopy, and its effects on ATPase activity were analyzed. The results suggest that the hexameric form of TrwK is the catalytically active state, much like the structurally related protein TrwB, the conjugative coupling protein.

Over-expression of Escherichia coli F1Fo–ATPase subunit a is inhibited by instability of the uncB gene transcript

Little is known about the stability of transcripts encoding membrane proteins in strong expression systems and its effect on membrane protein over‐production. We have expressed all the genes encoding subunits of the membrane domain Fo of the ATP synthase in a T7 RNA polymerase‐based system. All of them but uncB (subunit a) were expressed separately at very high levels in the bacterial hosts Escherichia coli C41(DE3) and C43(DE3). However, expression of uncB was extremely toxic to the bacteria. Northern blot analysis showed that the level of accumulation of the mRNA from uncB was very low. Deletion of uncB in combination with gene fusion experiments demonstrated that the middle region of the gene, encoding amino acids 92–171, exhibited a dominant toxic phenotype associated with a very poor level of expression. Green fluorescent protein fusions with N‐ and C‐ends of uncB helped to stabilize the mRNA and to obtain high yields of protein.