Modulation of coupling in the Escherichia coli ATP synthase by ADP and Pi: Role of the ε subunit C-terminal domain.

The ε-subunit of ATP-synthase is an endogenous inhibitor of the hydrolysis activity of the complex and its α-helical C-terminal domain (εCTD) undergoes drastic changes among at least two different conformations. Even though this domain is not essential for ATP synthesis activity, there is evidence for its involvement in the coupling mechanism of the pump. Recently, it was proposed that coupling of the ATP synthase can vary as a function of ADP and Pi concentration. In the present work, we have explored the possible role of the εCTD in this ADP- and Pi-dependent coupling, by examining an εCTD-lacking mutant of Escherichia coli. We show that the loss of Pi-dependent coupling can be observed also in the εCTD-less mutant, but the effects of Pi on both proton pumping and ATP hydrolysis were much weaker in the mutant than in the wild-type. We also show that the εCTD strongly influences the binding of ADP to a very tight binding site (half-maximal effect≈1nM); binding at this site induces higher coupling in EFOF1 and increases responses to Pi. It is proposed that one physiological role of the εCTD is to regulate the kinetics and affinity of ADP/Pi binding, promoting ADP/Pi-dependent coupling.

Mutual information without the influence of phylogeny or entropy dramatically improves residue contact prediction.

MOTIVATION: Compensating alterations during the evolution of protein families give rise to coevolving positions that contain important structural and functional information. However, a high background composed of random noise and phylogenetic components interferes with the identification of coevolving positions. RESULTS: We have developed a rapid, simple and general method based on information theory that accurately estimates the level of background mutual information for each pair of positions in a given protein family. Removal of this background results in a metric, MIp, that correctly identifies substantially more coevolving positions in protein families than any existing method. A significant fraction of these positions coevolve strongly with one or only a few positions. The vast majority of such position pairs are in contact in representative structures. The identification of strongly coevolving position pairs can be used to impose significant structural limitations and should be an important additional constraint for ab initio protein folding. AVAILABILITY: Alignments and program files can be found in the Supplementary Information.

Using information theory to search for co-evolving residues in proteins.

MOTIVATION: Some functionally important protein residues are easily detected since they correspond to conserved columns in a multiple sequence alignment (MSA). However important residues may also mutate, with compensatory mutations occurring elsewhere in the protein, which serve to preserve or restore functionality. It is difficult to distinguish these co-evolving sites from other non-conserved sites. RESULTS: We used Mutual Information (MI) to identify co-evolving positions. Using in silico evolved MSAs, we examined the effects of the number of sequences, the size of amino acid alphabet and the mutation rate on two sources of background MI: finite sample size effects and phylogenetic influence. We then assessed the performance of various normalizations of MI in enhancing detection of co-evolving positions and found that normalization by the pair entropy was optimal. Real protein alignments were analyzed and co-evolving isolated pairs were often found to be in contact with each other. AVAILABILITY: All data and program files can be found at http://www.biochem.uwo.ca/cgi-bin/CDD/index.cgi

Interaction of the N- and C-terminal domains of vinculin. Characterization and mapping studies.

The vinculin head to tail intramolecular self-association controls its binding sites for other components of focal adhesions. To study this interaction, the head and tail domains were expressed, purified, and assayed for various characteristics of complex formation. Analytical centrifugation demonstrated a strong interaction in solution and formation of a complex more asymmetric than either of the individual domains. A survey of binding conditions using a solid-phase binding assay revealed characteristics of both electrostatic and hydrophobic forces involved in the binding. In addition, circular dichroism of the individual domains and the complex demonstrated that conformational changes likely occur in both domains during association. The interaction sites were more closely mapped on the protein sequence by deletion mutagenesis. Amino acids 181-226, a basic region within the acidic head domain, were identified as a binding site for the vinculin tail, and residues 1009-1066 were identified as sufficient for binding the head. Moreover, mutation of an acidic patch in the tail (residues 1013-1015) almost completely eliminated its ability to interact with the head domain further supporting the significance of ionic interactions in the binding. Our data indicate that the interaction between the head and tail domains of vinculin occurs through oppositely charged contact sites and results in conformational changes in both domains.

Specific heterodimer formation by the cytoplasmic domains of the b and b' subunits of cyanobacterial ATP synthase.

The soluble domains of the b and b' subunits of the ATP synthase of the cyanobacterium Synechocystis PCC 6803 were expressed with His tags attached to their N-termini. Following purification, the polypeptides were characterized by chemical cross-linking, analytical ultracentrifugation, and circular dichroism spectroscopy. Treatment of a mixture of the soluble b and b' domains with a chemical cross-linking agent led to substantial formation of cross-linked dimers, whereas similar treatment of either domain by itself resulted in only trace formation of cross-linked species. The molecular weights of the domains of b and b' in solution at 20 degrees C, measured by sedimentation equilibrium, were 17 800+/-700 and 16 300+/-400, respectively, compared to calculated polypeptide molecular weights of 16 635 and 15 422, whereas a mixture of b and b' gave a molecular weight of 29 800+/-800. The sedimentation coefficient of an equimolar mixture was 1.73+/-0.03. The circular dichroism spectra of the individual polypeptides indicated helical contents in the range of 40-50%; the spectrum of the mixture revealed changes indicative of coiled-coil formation and a helical content of 60%. The results indicate that the cytosolic domains of the b and b' subunits exist individually as monomers but form a highly extended heterodimer when they are mixed together.

A re-examination of the structural and functional consequences of mutation of alanine-128 of the b subunit of Escherichia coli ATP synthase to aspartic acid.

The effects of mutation of residue Ala-128 of the b subunit of Escherichia coli ATP synthase to aspartate on the structure of the subunit and its interaction with the F(1) sector were analyzed. Determination of solution molecular weights by sedimentation equilibrium ultracentrifugation revealed that the A128D mutation had little effect on dimerization in the soluble b construct, b(34-156). However, the mutation caused a structural perturbation detected through both a 12% reduction in the sedimentation coefficient and also a reduced tendency to form intersubunit disulfide bonds between cysteine residues inserted at position 132. Unlike the wild-type sequence, the A128D mutant was unable to interact with F(1)-ATPase. These results indicate that the A128D mutation caused a structural change in the C-terminal region of the protein, preventing the binding to F(1) but having little or no effect on the dimeric nature of b.

The second stalk of Escherichia coli ATP synthase.

Two stalks link the F(1) and F(0) sectors of ATP synthase. The central stalk contains the gamma and epsilon subunits and is thought to function in rotational catalysis as a rotor driving conformational changes in the catalytic alpha(3)beta(3) complex. The two b subunits and the delta subunit associate to form b(2)delta, a second, peripheral stalk extending from the membrane up the side of alpha(3)beta(3) and binding to the N-terminal regions of the alpha subunits, which are approx. 125 A from the membrane. This second stalk is essential for binding F(1) to F(0) and is believed to function as a stator during rotational catalysis. In vitro, b(2)delta is a highly extended complex held together by weak interactions. Recent work has identified the domains of b which are essential for dimerization and for interaction with delta. Disulphide cross-linking studies imply that the second stalk is a permanent structure which remains associated with one alpha subunit or alphabeta pair. However, the weak interactions between the polypeptides in b(2)delta pose a challenge for the proposed stator function.

Site-directed cross-linking of b to the alpha, beta, and a subunits of the Escherichia coli ATP synthase.

The b subunit dimer of the Escherichia coli ATP synthase, along with the delta subunit, is thought to act as a stator to hold the alpha(3)beta(3) hexamer stationary relative to the a subunit as the gammaepsilonc(9-12) complex rotates. Despite their essential nature, the contacts between b and the alpha, beta, and a subunits remain largely undefined. We have introduced cysteine residues individually at various positions within the wild type membrane-bound b subunit, or within b(24-156), a truncated, soluble version consisting only of the hydrophilic C-terminal domain. The introduced cysteine residues were modified with a photoactivatable cross-linking agent, and cross-linking to subunits of the F(1) sector or to complete F(1)F(0) was attempted. Cross-linking in both the full-length and truncated forms of b was obtained at positions 92 (to alpha and beta), and 109 and 110 (to alpha only). Mass spectrometric analysis of peptide fragments derived from the b(24-156)A92C cross-link revealed that cross-linking took place within the region of alpha between Ile-464 and Met-483. This result indicates that the b dimer interacts with the alpha subunit near a non-catalytic alpha/beta interface. A cysteine residue introduced in place of the highly conserved arginine at position 36 of the b subunit could be cross-linked to the a subunit of F(0) in membrane-bound ATP synthase, implying that at least 10 residues of the polar domain of b are adjacent to residues of a. Sites of cross-linking between b(24-156)A92C and beta as well as b(24-156)I109C and alpha are proposed based on the mass spectrometric data, and these sites are discussed in terms of the structure of b and its interactions with the rest of the complex.

Site-directed spin-labeling of the catalytic sites yields insight into structural changes within the F0F1-ATP synthase of Escherichia coli.

Electron spin resonance (ESR) spectroscopy using site-specific cysteine spin-labeling of the catalytic nucleotide binding sites of F(1)-ATPase was employed to investigate conformational changes within the nucleotide binding sites of the enzyme. Mutant Escherichia coli F(1) that had been modified at position beta-Y331C with a spin label showed almost normal catalytic activity and enabled us to study the effects of binding of different nucleotides and of the F(o) subunit b on the conformation of the catalytic binding sites. The ESR spectra of the spin-labeled, nucleotide-depleted F(1) indicate asymmetry within the sites as is expected from the structural models of the enzyme. Nucleotide binding to the enzyme clearly affects the conformation of the sites; the most pronounced feature upon nucleotide binding is the formation of catalytic site(s) in a very open conformation. Using the same beta-331 spin-labeled F(1) and a truncated form of F(o) subunit b, b(24)(-)(156), we found that binding of b(24)(-)(156) to spin-labeled F(1) significantly changes the conformation of the catalytic sites. In this paper we present data that for the first time directly show that a conformational binding change takes place upon binding of nucleotides to the nucleotide binding sites and that also show that binding of b(24)(-)(156) strongly affects the conformation of the catalytic sites, most likely by increasing the population of binding sites that are in the open conformation.

Disulfide linkage of the b and delta subunits does not affect the function of the Escherichia coli ATP synthase.

The ATP synthase of Escherichia coli is believed to act through a rotational mechanism in which the b(2)delta subcomplex holds the alphabeta hexamer stationary relative to the rotating gamma and epsilon subunits. We have engineered a disulfide bond between cysteines introduced at position 158 of the delta subunit and at a position just beyond the normal C-terminus of the b subunit. The formation of this disulfide bond verifies that the C-terminal region of b is proximal to residue 158 of delta. The disulfide bond does not affect the ability of the F(1)F(0) complex to hydrolyze ATP, couple ATP hydrolysis to the establishment of a proton gradient, or maintain a proton gradient generated by the electron transport chain. These results are consistent with a permanent association of b(2) with delta as suggested by the rotational model of enzyme function.

Localization of the delta subunit in the Escherichia coli F(1)F(0)-ATPsynthase by immuno electron microscopy: the delta subunit binds on top of the F(1).

The binding site of the delta subunit in the F(1)F(0)-ATPsynthase from Escherichia coli has been determined by electron microscopy of negatively stained, antibody-decorated enzyme molecules. The images show that the antibody is bound at the very top of the F(1) domain indicating that at least part of delta is bound in the dimple formed by the N termini of the alpha and beta subunits. The data may explain why there is only one binding site for delta on the F(1) despite there being three identical alphabeta pairs. The finding also implies that the b subunits of the F(0) have to extend all the way from the membrane surface to the very top of the F(1) domain to make contact with the delta subunit.

The b subunit of Escherichia coli ATP synthase.

The b subunit of ATP synthase is a major component of the second stalk connecting the F1 and F0 sectors of the enzyme and is essential for normal assembly and function. The 156-residue b subunit of the Escherichia coli ATP synthase has been investigated extensively through mutagenesis, deletion analysis, and biophysical characterization. The two copies of b exist as a highly extended, helical dimer extending from the membrane to near the top of F1, where they interact with the delta subunit. The sequence has been divided into four domains: the N-terminal membrane-spanning domain, the tether domain, the dimerization domain, and the C-terminal delta-binding domain. The dimerization domain, contained within residues 60-122, has many properties of a coiled-coil, while the delta-binding domain is more globular. Sites of crosslinking between b and the a, alpha, beta, and delta subunits of ATP synthase have been identified, and the functional significance of these interactions is under investigation. The b dimer may serve as an elastic element during rotational catalysis in the enzyme, but also directly influences the catalytic sites, suggesting a more active role in coupling.

The dimerization domain of the b subunit of the Escherichia coli F(1)F(0)-ATPase.

In this study a series of N- and/or C-terminal truncations of the cytoplasmic domain of the b subunit of the Escherichia coli F(1)F(0) ATP synthase were tested for their ability to form dimers using sedimentation equilibrium ultracentrifugation. The deletion of residues between positions 53 and 122 resulted in a strongly decreased tendency to form dimers, whereas all the polypeptides that included that sequence exhibited high levels of dimer formation. b dimers existed in a reversible monomer-dimer equilibrium and when mixed with other b truncations formed heterodimers efficiently, provided both constructs included the 53-122 sequence. Sedimentation velocity and (15)N NMR relaxation measurements indicated that the dimerization region is highly extended in solution, consistent with an elongated second stalk structure. A cysteine introduced at position 105 was found to readily form intersubunit disulfides, whereas other single cysteines at positions 103-110 failed to form disulfides either with the identical mutant or when mixed with the other 103-110 cysteine mutants. These studies establish that the b subunit dimer depends on interactions that occur between residues in the 53-122 sequence and that the two subunits are oriented in a highly specific manner at the dimer interface.

Effect of the epsilon-subunit on nucleotide binding to Escherichia coli F1-ATPase catalytic sites.

The influence of the epsilon-subunit on the nucleotide binding affinities of the three catalytic sites of Escherichia coli F1-ATPase was investigated, using a genetically engineered Trp probe in the adenine-binding subdomain (beta-Trp-331). The interaction between epsilon and F1 was not affected by the mutation. Kd for binding of epsilon to betaY331W mutant F1 was approximately 1 nM, and epsilon inhibited ATPase activity by 90%. The only nucleotide binding affinities that showed significant differences in the epsilon-depleted and epsilon-replete forms of the enzyme were those for MgATP and MgADP at the high-affinity catalytic site 1. Kd1(MgATP) and Kd1(MgADP) were an order of magnitude higher in the absence of epsilon than in its presence. In contrast, the binding affinities for MgATP and MgADP at sites 2 and 3 were similar in the epsilon-depleted and epsilon-replete enzymes, as were the affinities at all three sites for free ATP and ADP. Comparison of MgATP binding and hydrolysis parameters showed that in the presence as well as the absence of epsilon, Km equals Kd3. Thus, in both cases, all three catalytic binding sites have to be occupied to obtain rapid (Vmax) MgATP hydrolysis rates.

The b and delta subunits of the Escherichia coli ATP synthase interact via residues in their C-terminal regions.

An affinity resin for the F1 sector of the Escherichia coli ATP synthase was prepared by coupling the b subunit to a solid support through a unique cysteine residue in the N-terminal leader. b24-156, a form of b lacking the N-terminal transmembrane domain, was able to compete with the affinity resin for binding of F1. Truncated forms of b24-156, in which one or four residues from the C terminus were removed, competed poorly for F1 binding, suggesting that these residues play an important role in b-F1 interactions. Sedimentation velocity analytical ultracentrifugation revealed that removal of these C-terminal residues from b24-156 resulted in a disruption of its association with the purified delta subunit of the enzyme. To determine whether these residues interact directly with delta, cysteine residues were introduced at various C-terminal positions of b and modified with the heterobifunctional cross-linker benzophenone-4-maleimide. Cross-links between b and delta were obtained when the reagent was incorporated at positions 155 and 158 (two residues beyond the normal C terminus) in both the reconstituted b24-156-F1 complex and the membrane-bound F1F0 complex. CNBr digestion followed by peptide sequencing showed the site of cross-linking within the 177-residue delta subunit to be C-terminal to residue 148, possibly at Met-158. These results indicate that the b and delta subunits interact via their C-terminal regions and that this interaction is instrumental in the binding of the F1 sector to the b subunit of F0.

Characterization of a b2delta complex from Escherichia coli ATP synthase.

The delta subunit of Escherichia coli ATP synthase has been expressed and purified, both as the intact polypeptide and as delta', a proteolytic fragment composed of residues 1-134. The solution structure of delta' as a five-helix bundle has been previously reported (Wilkens, S., Dunn, S. D., Chandler, J., Dahlquist, F. W., and Capaldi, R. A. (1997) Nat. Struct. Biol. 4, 198-201). The delta subunit, in conjunction with delta-depleted F1-ATPase, was fully capable of reconstituting energy-dependent fluorescence quenching in membrane vesicles that had been depleted of F1. A complex of delta with the cytoplasmic domain of the b subunit of F0 was demonstrated and characterized by analytical ultracentrifugation using bST34-156, a form of the b domain lacking aromatic residues. Molecular weight determination by sedimentation equilibrium supported a b2delta subunit stoichiometry. The sedimentation coefficient of the complex, 2.1 S, indicated a frictional ratio of approximately 2, suggesting that delta and the b dimer are arranged in an end-to-end rather than side-by-side manner. These results indicate the feasibility of the b2delta complex reaching from the membrane to the membrane-distal portion of the F1 sector, as required if it is to serve as a second stalk.

Dimerization interactions of the b subunit of the Escherichia coli F1F0-ATPase.

Site-directed mutagenesis and N-terminal truncations were used to examine dimerization interactions in the b subunit of Escherichia coli F1F0-ATPase. Individual cysteine residues were incorporated into bsyn, a soluble form of the protein lacking the membrane-spanning N-terminal domain, in two main areas: the heptad repeat region and the hydrophobic region which begins at residue Val-124. The tendencies of these cysteine residues to form disulfide bonds with the corresponding cysteine in the bsyn dimer were tested using disulfide exchange by glutathione and air oxidation catalyzed by Cu2+. Within the heptad repeat region, only cysteines at residues 59 and 60, which occupy the b and c positions of the heptad repeat, showed significant tendencies to form disulfides, a result inconsistent with a coiled-coil model for bsyn. Mixed disulfide formation most readily occurred with the S60C + L65C and A61C + L65C pairs. Cysteines at positions 124, 128, 132, and 139 showed strong tendencies to form disulfides with their mates in the dimer, suggesting a parallel alpha-helical interaction between the subunits in this region. Deletion of residues N-terminal to either Glu-34 or Asp-53 had no apparent effect on dimerization as determined by sedimentation equilibrium, while deletion of all residues N-terminal to Lys-67 produced a monomeric form. These results imply that residues 53-66 but not 24-52 are essential for bsyn dimerization. Taken together the results are consistent with a model in which the two b subunits interact in more than one region, including a parallel alignment of helices containing residues 124-139.

Epsilon-binding regions of the gamma subunit of Escherichia coli ATP synthase.

The interaction between the gamma and epsilon subunits of the F1-ATPase sector of Escherichia coli ATP synthase has been investigated using monoclonal antibodies directed against the gamma subunit and ligand blotting using 125I-epsilon. Monoclonal antibody (MAb) gamma-1 was able to bind to epsilon-depleted F1-ATPase but not to epsilon-replete F1, implying that epsilon blocked access to the epitope. A ligand blot assay for the binding of 125I-epsilon to gamma was developed. Both MAb gamma-1 and a second antibody, MAb gamma II, inhibited binding of 125I-epsilon to gamma in this assay while two other anti-gamma monoclonal antibodies did not. The epitope recognized by MAb gamma-1 was mapped between residues R49 and R70, quite distant in sequence from that of MAb gamma II, which is located C-terminal to residue K199 of the 286-residue polypeptide. The competition of these antibodies with epsilon for binding to gamma implies that their epitopes, quite separate in sequence, are both located in parts of the subunit involved in binding epsilon.

Solution structure of the N-terminal domain of the delta subunit of the E. coli ATPsynthase.

NMR studies of the delta subunit of the Escherichia coli F1F0-ATPsynthase reveal that it consists of an N-terminal six alpha-helix bundle and a less well ordered C terminus. Both domains are part of one of two separate connections between F1 and F0.

Structural influence of cation binding to recombinant human brain S100b: evidence for calcium-induced exposure of a hydrophobic surface.

The dimeric calcium-binding protein S100b is proposed to undergo a calcium-induced structural change allowing it to interact, via a hydrophobic surface, with other proteins. Previously it has been suggested that calcium binding to S100b leads to the exposure of at least one phenylalanine residue (Mani et al., 1982, 1983). This effect appears to be "reversed" at higher ionic strength, leading to a possible reburying of phenylalanine residues (Mani et al., 1982, 1983). To study these effects, we monitored calcium binding to recombinant human S100b by NMR spectroscopy under different salt (KCI) conditions. 15N-Labeled glycine residues in S100b showed calcium-induced chemical shift changes similar to those reported for the related monomeric protein calbindin D9k, suggesting similar conformational changes are occurring in the calcium-binding loops of these two proteins. Calcium binding to S100b also resulted in a shifting and broadening of several 1H resonances from the Ca-S100b form only including those from the side chains of residues F14, F70, and F73 but not those of residue Y17. This broadening was enhanced with increased ionic strength (KCI). However, small additions ( < 15% v/v) of the hydrophobic solvent trifluoroethanol relieved this phenomenon, leading to narrower line widths. These observations are consistent with the calcium-induced exposure of at least one of these hydrophobic residues, resulting in self-association of the S100b dimer. Trifluoroethanol serves to dissociate these complexes back to the dimeric calcium species. We propose that this cluster of hydrophobic residues which include F14, F73, and F88 may be important for interactions with a target protein.