Gabriele Deckers-Hebestreit

Prolonged Survival and Cytoplasmic pH Homeostasis of Helicobacter pylori at pH 1

ABSTRACT In the presence of urea, Helicobacter pylori survived for at least 3 h at pH 1. Under these conditions, the cells maintained their cytoplasmic pH at 5.8. De novo protein synthesis during acid shock was not essential for survival of H. pylori at pH 1.

Constant c10 ring stoichiometry in the Escherichia coli ATP synthase analyzed by cross-linking.

The subunit c stoichiometry of Escherichia coli ATP synthase was studied by intermolecular cross-linking via oxidation of bi-cysteine-substituted subunit c (cA21C/cM65C). Independent of the carbon source used for growth and independent of the presence of other FoF1 subunits, an equal pattern of cross-link formation stopping at the formation of decamers was obtained.

The ATP synthase of Escherichia coli: structure and function of F0 subunits

In this review we discuss recent work from our laboratory concerning the structure and/or function of the F(0) subunits of the proton-translocating ATP synthase of Escherichia coli. For the topology of subunit a a brief discussion gives (i) a detailed picture of the C-terminal two-thirds of the protein with four transmembrane helices and the C terminus exposed to the cytoplasm and (ii) an evaluation of the controversial results obtained for the localization of the N-terminal region of subunit a including its consequences on the number of transmembrane helices. The structure of membrane-bound subunit b has been determined by circular dichroism spectroscopy to be at least 75% alpha-helical. For this purpose a method was developed, which allows the determination of the structure composition of membrane proteins in proteoliposomes. Subunit b was purified to homogeneity by preparative SDS gel electrophoresis, precipitated with acetone, and redissolved in cholate-containing buffer, thereby retaining its native conformation as shown by functional coreconstitution with an ac subcomplex. Monoclonal antibodies, which have their epitopes located within the hydrophilic loop region of subunit c, and the F(1) part are bound simultaneously to the F(0) complex without an effect on the function of F(0), indicating that not all c subunits are involved in F(1) interaction. Consequences on the coupling mechanism between ATP synthesis/hydrolysis and proton translocation are discussed.

Orientation of subunit c of the ATP synthase of Escherichia coli — a study with peptide-specific antibodies

Antibodies were raised against a peptide of subunit c of the ATP synthase from Escherichia coli obtained by cleavage with cyanogen bromide. This peptide comprises the amino acid residues Gly-18 to Met-57 and contains the highly conserved, hydrophilic stretch of subunit c. Several conformation-specific populations of antibodies recognized this region both in isolated subunit c and in the intact F0 complex. In antibody binding studies with membrane vesicles of different orientations, recognition occurred only after incubation with everted membrane vesicles, independent of the presence or absence of F1, although a higher membrane protein concentration was necessary to observe the same antibody binding in the presence of the F1 part. From these results we conclude that the hydrophilic region of subunit c is exposed to the cytoplasmic side of the membrane.

The Holo-form of the Nucleotide Binding Domain of the KdpFABC Complex from Escherichia coli Reveals a New Binding Mode

P-type ATPases are ubiquitously abundant enzymes involved in active transport of charged residues across biological membranes. The KdpB subunit of the prokaryotic Kdp-ATPase (KdpFABC complex) shares characteristic regions of homology with class II-IV P-type ATPases and has been shown previously to be misgrouped as a class IA P-type ATPase. Here, we present the NMR structure of the AMP-PNP-bound nucleotide binding domain KdpBN of the Escherichia coli Kdp-ATPase at high resolution. The aromatic moiety of the nucleotide is clipped into the binding pocket by Phe377 and Lys395 via a π-π stacking and a cation-π interaction, respectively. Charged residues at the outer rim of the binding pocket (Arg317, Arg382, Asp399, and Glu348) stabilize and direct the triphosphate group via electrostatic attraction and repulsion toward the phosphorylation domain. The nucleotide binding mode was corroborated by the replacement of critical residues. The conservative mutation F377Y produced a high residual nucleotide binding capacity, whereas replacement by alanine resulted in low nucleotide binding capacities and a considerable loss of ATPase activity. Similarly, mutation K395A resulted in loss of ATPase activity and nucleotide binding affinity, even though the protein was properly folded. We present a schematic model of the nucleotide binding mode that allows for both high selectivity and a low nucleotide binding constant, necessary for the fast and effective turnover rate realized in the reaction cycle of the Kdp-ATPase.

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.

Detection and localization of the i protein in Escherichia coli cells using antibodies.

Using antibodies raised against the purifiedi protein, the expression of the chromosomaluncI gene was demonstrated. Thei protein was identified as a component of the cytoplasmic membrane and shown to be present in preparations of F0 or F1F0. The protein is not associated with the F1 moiety.

Subunit Organization of the Stator Part of the F 0 Complex from Escherichia coli ATP Synthase

Membrane-bound ATP synthases (F1F0) catalyze the synthesis of ATP via a rotary catalyticmechanism utilizing the energy of an electrochemical ion gradient. The transmembrane potentialis supposed to propel rotation of a subunit c ring of F0 together with subunits γ and ∈ of F1,hereby forming the rotor part of the enzyme, whereas the remainder of the F1F0 complexfunctions as a stator for compensation of the torque generated during rotation. This reviewfocuses on our recent work on the stator part of the F0 complex, e.g., subunits a and b. Usingepitope insertion and antibody binding, subunit a was shown to comprise six transmembranehelixes with both the N- and C-terminus oriented toward the cytoplasm. By use of circulardichroism (CD) spectroscopy, the secondary structure of subunit b incorporated intoproteoliposomes was determined to be 80% α-helical together with 14% β turn conformation, providingflexibility to the second stalk. Reconstituted subunit b together with isolated ac subcomplexwas shown to be active in proton translocation and functional F1 binding revealing the nativeconformation of the polypeptide chain. Chemical crosslinking in everted membrane vesiclesled to the formation of subunit b homodimers around residues bQ37 to bL65, whereas bA32Ccould be crosslinked to subunit a, indicating a close proximity of subunits a and b near themembrane. Further evidence for the proposed direct interaction between subunits a and b wasobtained by purification of a stable ab2 subcomplex via affinity chromatography using Histags fused to subunit a or b. This ab2 subcomplex was shown to be active in proton translocationand F1 binding, when coreconstituted with subunit c. Consequences of crosslink formationand subunit interaction within the F1F0 complex are discussed.

The ATP synthase (F1F0) of Streptomyces lividans: sequencing of the atp operon and phylogenetic considerations with subunit beta

The DNA encoding the subunits of the ATP synthase (F1F0) of Streptomyces lividans 66 strain 1326 was identified using oligodeoxyribonucleotide probes derived from the N-terminal sequence of subunit gamma of the F1 complex. The complete nucleotide sequence of the operon was determined. The atp operon contains nine genes, atpIBEFHAGDC, encoding the eight structural components of the ATP synthase complex and the i protein, a polypeptide of unknown function. The gene order found is identical to that in other non-photosynthetic eubacteria. The determination of the N-terminal amino acid (aa) sequences of the F1 subunits alpha, beta, gamma, delta and epsilon allowed us to identify the translational start points and to define the primary structures of the proteins. The aa sequence deduced for subunit delta revealed an N-terminal extension of about 90 aa, which is not present in any delta subunit or OSCP (oligomycin sensitivity-conferral protein) of other species studied so far. The phylogenetic relationship of eu- and archaebacteria was investigated using sequencing data of the highly conserved beta subunit of different ATP synthases including that of S. lividans. The calculations revealed that S. lividans beta does not form a phylogenetic group together with the Gram+ taxa of low G+C contents, but is more closely related to the beta subunit of Rhodobacteria.

Individual interactions of the b subunits within the stator of the Escherichia coli ATP synthase.

Background: The peripheral stator stalk of Escherichia coli ATP synthase contains two b subunits. Results: Using disulfide bond formation, one b subunit was cross-linked to a, α, and δ and the other to β. Conclusion: The b subunits adopt distinct positions within the stator to generate stability. Significance: The different positions imply different roles in counteracting the torque generated by the rotor. FOF1 ATP synthases are rotary nanomotors that couple proton translocation across biological membranes to the synthesis/hydrolysis of ATP. During catalysis, the peripheral stalk, composed of two b subunits and subunit δ in Escherichia coli, counteracts the torque generated by the rotation of the central stalk. Here we characterize individual interactions of the b subunits within the stator by use of monoclonal antibodies and nearest neighbor analyses via intersubunit disulfide bond formation. Antibody binding studies revealed that the C-terminal region of one of the two b subunits is principally involved in the binding of subunit δ, whereas the other one is accessible to antibody binding without impact on the function of FOF1. Individually substituted cysteine pairs suitable for disulfide cross-linking between the b subunits and the other stator subunits (b-α, b-β, b-δ, and b-a) were screened and combined with each other to discriminate between the two b subunits (i.e. bI and bII). The results show the b dimer to be located at a non-catalytic α/β cleft, with bI close to subunit α, whereas bII is proximal to subunit β. Furthermore, bI can be linked to subunit δ as well as to subunit a. Among the subcomplexes formed were a-bI-α, bII-β, α-bI-bII-β, and a-bI-δ. Taken together, the data obtained define the different positions of the two b subunits at a non-catalytic interface and imply that each b subunit has a different role in generating stability within the stator. We suggest that bI is functionally related to the single b subunit present in mitochondrial ATP synthase.