Thomas Vorburger

Arginine-induced conformational change in the c-ring/ a-subunit interface of ATP synthase

The rotational mechanism of ATP synthases requires a unique interface between the stator a subunit and the rotating c‐ring to accommodate stability and smooth rotation simultaneously. The recently published c‐ring crystal structure of the ATP synthase of Ilyobacter tartaricus represents the conformation in the absence of subunit a. However, in order to understand the dynamic structural processes during ion translocation, studies in the presence of subunit a are required. Here, by intersubunit Cys–Cys cross‐linking, the relative topography of the interacting helical faces of subunits a and c from the I. tartaricus ATP synthase has been mapped. According to these data, the essential stator arginine (aR226) is located between the c‐ring binding pocket and the cytoplasm. Furthermore, the spatially vicinal residues cT67C and cG68C in the isolated c‐ring structure yielded largely asymmetric cross‐linking products with aN230C of subunit a, suggesting a small, but significant conformational change of binding‐site residues upon contact with subunit a. The conformational change was dependent on the positive charge of the stator arginine or the aR226H substitution. Energy‐minimization calculations revealed possible modes for the interaction between the stator arginine and the c‐ring. These biochemical results and structural restraints support a model in which the stator arginine operates as a pendulum, moving in and out of the binding pocket as the c‐ring rotates along the interface with subunit a. This mechanism allows efficient interaction between subunit a and the c‐ring and simultaneously allows almost frictionless movement against each other.

Central role of the Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) in sodium bioenergetics of Vibrio cholerae.

Abstract Vibrio cholerae is a Gram-negative bacterium that lives in brackish or sea water environments. Strains of V. cholerae carrying the pathogenicity islands infect the human gut and cause the fatal disease cholera. Vibrio cholerae maintains a Na+ gradient at its cytoplasmic membrane that drives substrate uptake, motility, and efflux of antibiotics. Here, we summarize the major Na+-dependent transport processes and describe the central role of the Na+-translocating NADH:quinone oxidoreductase (Na+-NQR), a primary Na+ pump, in maintaining a Na+-motive force. The Na+-NQR is a membrane protein complex with a mass of about 220 kDa that couples the exergonic oxidation of NADH to the transport of Na+ across the cytoplasmic membrane. We describe the molecular architecture of this respiratory complex and summarize the findings how electron transport might be coupled to Na+-translocation. Moreover, recent advances in the determination of the three-dimensional structure of this complex are reported.

The structure of Na+-translocating of NADH:ubiquinone oxidoreductase of Vibrio cholerae: implications on coupling between electron transfer and Na+ transport

Abstract The Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) of Vibrio cholerae is a respiratory complex that couples the exergonic oxidation of NADH to the transport of Na+ across the cytoplasmic membrane. It is composed of six different subunits, NqrA, NqrB, NqrC, NqrD, NqrE, and NqrF, which harbor FAD, FMN, riboflavin, quinone, and two FeS centers as redox co-factors. We recently determined the X-ray structure of the entire Na+-NQR complex at 3.5-Å resolution and complemented the analysis by high-resolution structures of NqrA, NqrC, and NqrF. The position of flavin and FeS co-factors both at the cytoplasmic and the periplasmic side revealed an electron transfer pathway from cytoplasmic subunit NqrF across the membrane to the periplasmic NqrC, and via NqrB back to the quinone reduction site on cytoplasmic NqrA. A so far unknown Fe site located in the midst of membrane-embedded subunits NqrD and NqrE shuttles the electrons over the membrane. Some distances observed between redox centers appear to be too large for effective electron transfer and require conformational changes that are most likely involved in Na+ transport. Based on the structure, we propose a mechanism where redox induced conformational changes critically couple electron transfer to Na+ translocation from the cytoplasm to the periplasm through a channel in subunit NqrB.

Role of the Na(+)-translocating NADH:quinone oxidoreductase in voltage generation and Na(+) extrusion in Vibrio cholerae.

For Vibrio cholerae, the coordinated import and export of Na(+) is crucial for adaptation to habitats with different osmolarities. We investigated the Na(+)-extruding branch of the sodium cycle in this human pathogen by in vivo (23)Na-NMR spectroscopy. The Na(+) extrusion activity of cells was monitored after adding glucose which stimulated respiration via the Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR). In a V. cholerae deletion mutant devoid of the Na(+)-NQR encoding genes (nqrA-F), rates of respiratory Na(+) extrusion were decreased by a factor of four, but the cytoplasmic Na(+) concentration was essentially unchanged. Furthermore, the mutant was impaired in formation of transmembrane voltage (ΔΨ, inside negative) and did not grow under hypoosmotic conditions at pH8.2 or above. This growth defect could be complemented by transformation with the plasmid encoded nqr operon. In an alkaline environment, Na(+)/H(+) antiporters acidify the cytoplasm at the expense of the transmembrane voltage. It is proposed that, at alkaline pH and limiting Na(+) concentrations, the Na(+)-NQR is crucial for generation of a transmembrane voltage to drive the import of H(+) by electrogenic Na(+)/H(+) antiporters. Our study provides the basis to understand the role of the Na(+)-NQR in pathogenicity of V. cholerae and other pathogens relying on this primary Na(+) pump for respiration.

The novel NhaE-type Na+/H+ antiporter of the pathogenic bacterium Neisseria meningitidis

Neisseria meningitidis is a pathogenic bacterium responsible for meningitis. The mechanisms underlying the control of Na+ transmembrane movement, presumably important to pathogenicity, have been barely addressed. To elucidate the function of the components of the Na+ transport system in N. meningitidis, an open reading frame from the genome of this bacterium displaying similarity with the NhaE type of Na+/H+ antiporters was expressed in Escherichia coli and characterized for sodium transport ability. The N. meningitidis antiporter (NmNhaE) was able to complement an E. coli strain devoid of Na+/H+ antiporters (KNabc) respecting the ability to grow in the presence of NaCl and LiCl. Ion transport assays in everted vesicles prepared from KNabc expressing NmNhaE from a plasmid confirmed its ability to translocate Na+ and Li+. Here is presented the characterization of the first NhaE from a pathogen, an important contribution to the comprehension of sodium ion metabolism in this kind of microorganisms.

Functional role of a conserved aspartic acid residue in the motor of the Na+-driven flagellum from Vibrio cholerae

The flagellar motor consists of a rotor and a stator and couples the flux of cations (H(+) or Na(+)) to the generation of the torque necessary to drive flagellum rotation. The inner membrane proteins PomA and PomB are stator components of the Na(+)-driven flagellar motor from Vibrio cholerae. Affinity-tagged variants of PomA and PomB were co-expressed in trans in the non-motile V. cholerae pomAB deletion strain to study the role of the conserved D23 in the transmembrane helix of PomB. At pH 9, the D23E variant restored motility to 100% of that observed with wild type PomB, whereas the D23N variant resulted in a non-motile phenotype, indicating that a carboxylic group at position 23 in PomB is important for flagellum rotation. Motility tests at decreasing pH revealed a pronounced decline of flagellar function with a motor complex containing the PomB-D23E variant. It is suggested that the protonation state of the glutamate residue at position 23 determines the performance of the flagellar motor by altering the affinity of Na(+) to PomB. The conserved aspartate residue in the transmembrane helix of PomB and its H(+)-dependent homologs might act as a ligand for the coupling cation in the flagellar motor.

Crystallization and preliminary analysis of the NqrA and NqrC subunits of the Na + -translocating NADH:ubiquinone oxidoreductase from Vibrio cholerae

The Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) from Vibrio cholerae is a membrane protein complex consisting of six different subunits NqrA-NqrF. The major domains of the NqrA and NqrC subunits were heterologously expressed in Escherichia coli and crystallized. The structure of NqrA1-377 was solved in space groups C222₁ and P2₁ by SAD phasing and molecular replacement at 1.9 and 2.1 Å resolution, respectively. NqrC devoid of the transmembrane helix was co-expressed with ApbE to insert the flavin mononucleotide group covalently attached to Thr225. The structure was determined by molecular replacement using apo-NqrC of Parabacteroides distasonis as search model at 1.8 Å resolution.

Serine 26 in the PomB subunit of the flagellar motor is essential for hypermotility of Vibrio cholerae.

Vibrio cholerae is motile by means of its single polar flagellum which is driven by the sodium-motive force. In the motor driving rotation of the flagellar filament, a stator complex consisting of subunits PomA and PomB converts the electrochemical sodium ion gradient into torque. Charged or polar residues within the membrane part of PomB could act as ligands for Na+, or stabilize a hydrogen bond network by interacting with water within the putative channel between PomA and PomB. By analyzing a large data set of individual tracks of swimming cells, we show that S26 located within the transmembrane helix of PomB is required to promote very fast swimming of V. cholerae. Loss of hypermotility was observed with the S26T variant of PomB at pH 7.0, but fast swimming was restored by decreasing the H+ concentration of the external medium. Our study identifies S26 as a second important residue besides D23 in the PomB channel. It is proposed that S26, together with D23 located in close proximity, is important to perturb the hydration shell of Na+ before its passage through a constriction within the stator channel.