F1F0-ATP synthase. Binding of delta subunit to a 22-residue peptide mimicking the N-terminal region of alpha subunit

The stator in F(1)F(0)-ATP synthase resists strain generated by rotor torque. In Escherichia coli the b(2)delta subunit complex comprises the stator, bound to subunit a in F(0) and to alpha(3)beta(3) hexagon of F(1). Proteolysis and cross-linking had suggested that N-terminal residues of alpha subunit are involved in binding delta. Here we demonstrate that a synthetic peptide consisting of the first 22 residues of alpha ("alpha N1-22") binds specifically to isolated wild-type delta subunit with high affinity (K(d) = 130 nm), accounting for a major portion of the binding energy when delta-depleted F(1) and isolated delta bind together (K(d) = 1.4 nm). Stoichiometry of binding of alpha N1-22 to delta at saturation was 1/1, showing that in intact F(1)F(0)-ATP synthase only one of the three alpha subunits is involved in delta binding. When alpha N1-22 was incubated with delta subunits containing mutations in helices 1 or 5 on the F(1)-binding face of delta, peptide binding was impaired as was binding of delta-depleted F(1). Residues alpha 6-18 are predicted to be helical, and a potential helix capping box occurs at residues alpha 3-8. Circular dichroism measurements showed that alpha N1-22 had significant helical content. Hypothetically a helical region of residues alpha N1-22 packs with helices 1 and 5 on the F(1)-binding face of delta, forming the alpha/delta interface.     

Assembly of the stator in Escherichia coli ATP synthase. Complexation of alpha subunit with other F1 subunits is prerequisite for delta subunit binding to the N-terminal region of alpha

Alpha subunit of Escherichia coli ATP synthase was expressed with a C-terminal 6-His tag and purified. Pure alpha was monomeric, was competent in nucleotide binding, and had normal N-terminal sequence. In F1 subunit dissociation/reassociation experiments it supported full reconstitution of ATPase, and reassociated complexes were able to bind to F1-depleted membranes with restoration of ATP-driven proton pumping. Therefore interaction between the stator delta subunit and the N-terminal residue 1-22 region of alpha occurred normally when pure alpha was complexed with other F1 subunits. On the other hand, three different types of experiments showed that no interaction occurred between pure delta and isolated alpha subunit. Unlike in F1, the N-terminal region of isolated alpha was not susceptible to trypsin cleavage. Therefore, during assembly of ATP synthase, complexation of alpha subunit with other F1 subunits is prerequisite for delta subunit binding to the N-terminal region of alpha. We suggest that the N-terminal 1-22 residues of alpha are sequestered in isolated alpha until released by binding of beta to alpha subunit. This prevents 1/1 delta/alpha complexes from forming and provides a satisfactory explanation of the stoichiometry of one delta per three alpha seen in the F1 sector of ATP synthase, assuming that steric hindrance prevents binding of more than one delta to the alpha3/beta3 hexagon. The cytoplasmic fragment of the b subunit (bsol) did not bind to isolated alpha. It might also be that complexation of alpha with beta subunits is prerequisite for direct binding of stator b subunit to the F1-sector.     

Quantitative determination of direct binding of b subunit to F1 in Escherichia coli F1F0-ATP synthase

The stator in F(1)F(0)-ATP synthase resists strain generated by rotor torque. In Escherichia coli, the b(2)delta subunit complex comprises the stator, bound to subunit a in F(0) and to the alpha(3)beta(3) hexagon of F(1). To quantitatively characterize binding of b subunit to the F(1) alpha(3)beta(3) hexagon, we developed fluorimetric assays in which wild-type F(1), or F(1) enzymes containing introduced Trp residues, were titrated with a soluble portion of the b subunit (b(ST34-156)). With five different F(1) enzymes, K(d)(b(ST34-156)) ranged from 91 to 157 nm. Binding was strongly Mg(2+)-dependent; in EDTA buffer, K(d)(b(ST34-156)) was increased to 1.25 microm. The addition of the cytoplasmic portion of the b subunit increases the affinity of binding of delta subunit to delta-depleted F(1). The apparent K(d)(b(ST34-156)) for this effect was increased from 150 nm in Mg(2+) buffer to 1.36 microm in EDTA buffer. This work demonstrates quantitatively how binding of the cytoplasmic portion of the b subunit directly to F(1) contributes to stator resistance and emphasizes the importance of Mg(2+) in stator interactions.     

Structural characterization of the interaction of the delta and alpha subunits of the Escherichia coli F1F0-ATP synthase by NMR spectroscopy

A critical point of interaction between F(1) and F(0) in the bacterial F(1)F(0)-ATP synthase is formed by the alpha and delta subunits. Previous work has shown that the N-terminal domain (residues 3-105) of the delta subunit forms a 6 alpha-helix bundle [Wilkens, S., Dunn, S. D., Chandler, J., Dahlquist, F. W., and Capaldi, R. A. (1997) Nat. Struct. Biol. 4, 198-201] and that the majority of the binding energy between delta and F(1) is provided by the interaction between the N-terminal 22 residues of the alpha- and N-terminal domain of the delta subunit [Weber, J., Muharemagic, A., Wilke-Mounts, S., and Senior, A. E. (2003) J. Biol. Chem. 278, 13623-13626]. We have now analyzed a 1:1 complex of the delta-subunit N-terminal domain and a peptide comprising the N-terminal 22 residues of the alpha subunit by heteronuclear protein NMR spectroscopy. A comparison of the chemical-shift values of delta-subunit residues with and without alpha N-terminal peptide bound indicates that the binding interface on the N-terminal domain of the delta subunit is formed by alpha helices I and V. NOE cross-peak patterns in 2D (12)C/(12)C-filtered NOESY spectra of the (13)C-labeled delta-subunit N-terminal domain in complex with unlabeled peptide verify that residues 8-18 in the alpha-subunit N-terminal peptide are folded as an alpha helix when bound to delta N-terminal domain. On the basis of intermolecular contacts observed in (12)C/(13)C-filtered NOESY experiments, we describe structural details of the interaction of the delta-subunit N-terminal domain with the alpha-subunit N-terminal alpha helix.     

Second stalk of ATP synthase. Cross-linking of gamma subunit in F1 to truncated Fob subunit prevents ATP hydrolysis

ATP synthase consists of two portions, F(1) and F(o), connected by two stalks: a central rotor stalk containing gamma and epsilon subunits and a peripheral, second stalk formed by delta and two copies of F(o)b subunits. The second stalk is expected to keep the stator subunits from spinning along with the rotor. We isolated a TF(1)-b'(2) complex (alpha(3)beta(3)gammadeltaepsilonb'(2)) of a thermophilic Bacillus PS3, in which b' was a truncated cytoplasmic fragment of F(o)b subunit, and introduced a cysteine at its N terminus (bc'). Association of b'(2) or bc'(2) with TF(1) did not have significant effect on ATPase activity. A disulfide bond between the introduced cysteine of bc' and cysteine 109 of gamma subunit was readily formed, and this cross-link caused inactivation of ATPase. This implies that F(o)b subunit bound to stator subunits of F(1) with enough strength to resist rotation of gamma subunit and to prevent catalysis. Contrary to this apparent tight binding, some detergents such as lauryldodecylamine oxide tend to cause release of b'(2) from TF(1).     

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.     

Quantitative determination of binding affinity of delta-subunit in Escherichia coli F1-ATPase: effects of mutation, Mg2+, and pH on Kd

To study the stator function in ATP synthase, a fluorimetric assay has been devised for quantitative determination of binding affinity of delta-subunit to Escherichia coli F(1)-ATPase. The signal used is that of the natural tryptophan at residue delta28, which is enhanced by 50% upon binding of delta-subunit to alpha(3)beta(3)gammaepsilon complex. K(d) for delta binding is 1.4 nm, which is energetically equivalent (50.2 kJ/mol) to that required to resist the rotor strain. Only one site for delta binding was detected. The deltaW28L mutation increased K(d) to 4.6 nm, equivalent to a loss of 2.9 kJ/mol binding energy. While this was insufficient to cause detectable functional impairment, it did facilitate preparation of delta-depleted F(1). The alphaG29D mutation reduced K(d) to 26 nm, equivalent to a loss of 7.2 kJ/mol binding energy. This mutation did cause serious functional impairment, referable to interruption of binding of delta to F(1). Results with the two mutants illuminate how finely balanced is the stator resistance function. delta' fragment, consisting of residues delta1-134, bound with the same K(d) as intact delta, showing that, at least in absence of F(o) subunits, the C-terminal domain of delta contributes zero binding energy. Mg(2+) ions had a strong effect on increasing delta binding affinity, supporting the possibility of bridging metal ion involvement in stator function. High pH environment greatly reduced delta binding affinity, suggesting the involvement of protonatable side-chains in the binding site.     

Aqueous access channels in subunit a of rotary ATP synthase

The role of subunit a in proton translocation by the Escherichia coli F(1)F(o) ATP synthase is poorly understood. In the membrane-bound F(o) sector of the enzyme, H(+) binding and release occurs at Asp(61) in the middle of the second transmembrane helix (TMH) of subunit c. Protons are thought to reach Asp(61) via an aqueous access pathway formed at least in part by one or more of the five TMHs of subunit a. In this report, we have substituted Cys into a 19-residue span of the fourth TMH of subunit a and used chemical modification to obtain information about the aqueous accessibility of residues along this helix. Residues 206, 210, and 214 are N-ethylmaleimide-accessible from the cytoplasmic side of the membrane and may lie on the H(+) transport route. Residues 215 and 218 on TMH4, as well as residue 245 on TMH5, are Ag(+)-accessible but N-ethylmaleimide-inaccessible and may form part of an aqueous pocket extending from Asp(61) of subunit c to the periplasmic surface.     

Coupling factor ATPase from Escherichia coli. An uncA mutant (uncA401) with defective alpha subunit.

Inactive coupling factor ATPase (F1) was prepared from an uncoupled mutant (uncA401) of Escherichia coli. Reconstitution of ATPase activity was observed when alpha subunit from wild-type F1 was added to the dissociated inactive F1 and the mixture was dialyzed against buffer containing ATP and Mg2+. ATPase was also reconstituted when the mixture of alpha subunit (wild type) and crude extract from the mutant was dialyzed against the same buffer. These results indicate that the mutant is defective in alpha subunit, suggesting that the uncA401 locus carries the structural gene for alpha subunit, and that this polypeptide plays an essential role in ATPase activity in F1 molecule.     

Both rotor and stator subunits are necessary for efficient binding of F1 to F0 in functionally assembled Escherichia coli ATP synthase

In F1F0-ATP synthase, the subunit b2delta complex comprises the peripheral stator bound to subunit a in F0 and to the alpha3beta3 hexamer of F1. During catalysis, ATP turnover is coupled via an elastic rotary mechanism to proton translocation. Thus, the stator has to withstand the generated rotor torque, which implies tight interactions of the stator and rotor subunits. To quantitatively characterize the contribution of the F0 subunits to the binding of F1 within the assembled holoenzyme, the isolated subunit b dimer, ab2 subcomplex, and fully assembled F0 complex were specifically labeled with tetramethylrhodamine-5-maleimide at bCys64 and functionally reconstituted into liposomes. Proteoliposomes were then titrated with increasing amounts of Cy5-maleimide-labeled F1 (at gammaCys106 and analyzed by single-molecule fluorescence resonance energy transfer. The data revealed F1 dissociation constants of 2.7 nm for the binding of F0 and 9-10 nm for both the ab2 subcomplex and subunit b dimer. This indicates that both rotor and stator components of F0 contribute to F1 binding affinity in the assembled holoenzyme. The subunit c ring plays a crucial role in the binding of F1 to F0, whereas subunit a does not contribute significantly.     

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.     

F1ATPase of Escherichia coli: a mutation (uncA401) located in the middle of the alpha subunit affects the conformation essential for F1 activity

F1ATPase from the Escherichia coli mutant of H+-ATPase, AN120 (uncA401), has less than 1% of the wild type activity and has been shown to be defective in the alpha subunit by in vitro reconstitution experiments. In the present study, the mutation site was located within a domain of the subunit by recombinant DNA technology. For this, a series of recombinant plasmids carrying various portions of the alpha subunit gene were constructed and used for genetic recombination with AN120. Analysis of the recombinants indicated that the mutation site could be located between amino acid residues 370 and 387. The biochemical properties of the mutant F1 were analyzed further using the fluorescent ATP analog DNS-ATP (2'-(5-dimethylaminonaphthalene-1-sulfonyl)-amino-2'-deoxy ATP). The single turnover process of E. coli F1ATPase proposed by Matsuoka et al. [(1982) J. Biochem. 92, 1383-1398.] was compared in the mutant and wild type F1's. Mutant F1 bound DNS-ATP and hydrolyzed it as efficiently as wild type F1. Results showed that binding of ATP to a low affinity site, possibly in the beta subunit, caused decrease of fluorescence of DNS-ATP in the wild type F1 and that this effect of ATP binding was inhibited by DCCD (dicyclohexyl carbodiimide). However, this effect was not inhibited by DCCD in the mutant F1, suggesting that in the proposed process some step(s) after ATP binding to the low affinity site differed in the mutant and wild F1's. When Pi was added to F1 bound to DNS-ATP or to aurovertin, a fluorescent probe capable of binding to the beta subunit, the opposite changes of fluorescence of these probes in the mutant and wild type F1's were observed, suggesting that the conformational change induced by phosphate binding was altered in the mutant F1. On the basis of the estimated mutation site and the biochemical properties of the mutant F1, the correlation of the domain of this site in the alpha subunit with the function of F1 ATPase is discussed.     

Conformational interactions between alpha and beta subunits in the F1 ATPase of Escherichia coli as shown by chemical modification of uncA401 and uncD412 mutant enzymes

In contrast to wild-type F1 adenosine triphosphatase, the beta subunits of soluble ATPase from Escherichia coli mutant strains AN120 (uncA401) and AN939 (uncD412) were not labeled by the fluorescent thiol-specific reagents 5-iodoacetamidofluorescein, 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid or 4-[N-(iodoacetoxy)ethyl-N-methyl]amino-7-nitrobenzo-2-oxa-1,3-diazole. The mutation in the alpha subunit (uncA401) of F1 ATPase thus influences the accessibility of the single cysteinyl residue in the beta subunit. Following reaction of ATPase with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole or N,N'-dicyclohexylcarbodiimide, the alpha and beta subunits of the uncA401, but not of the uncD412 mutant F1 ATPase were intensely labeled by a fluorescent thiol reagent. The mutation in the beta subunit (uncD412) thus influences the accessibility of the cysteinyl residues in the alpha subunit. In other work [Stan-Lotter, H. and Bragg, P.D. (1986) Arch. Biochem. Biophys. 248] we have shown that 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole and 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid react with a different beta subunit from that labeled by N,N'-dicyclohexylcarbodiimide. This asymmetry with respect to modification by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole and N,N'-dicyclohexylcarbodiimide was seen in both mutant enzymes. In addition, the modification of one beta subunit of the uncA401 F1 ATPase induced the previously unreactive sulfhydryl group of another beta subunit to react with 2-(4'-iodoacetamidoanilino-naphthalene-6-sulfonic acid. These results provide evidence for at least three types of conformational interactions of the major subunits of F1 ATPase: from alpha to beta, from beta to alpha, and from beta to beta. As in wild-type ATPase, labeling of membrane-bound unc mutant ATPase by a fluorescent thiol reagent modified the alpha subunits. This suggests that a conformational change of yet a different type occurs when the enzyme binds to the membrane.     

Orientation of alpha-neurotoxin at the subunit interfaces of the nicotinic acetylcholine receptor

The alpha-neurotoxins are three-fingered peptide toxins that bind selectively at interfaces formed by the alpha subunit and its associating subunit partner, gamma, delta, or epsilon of the nicotinic acetylcholine receptor. Because the alpha-neurotoxin from Naja mossambica mossambica I shows an unusual selectivity for the alpha gamma and alpha delta over the alpha epsilon subunit interface, residue replacement and mutant cycle analysis of paired residues enabled us to identify the determinants in the gamma and delta sequences governing alpha-toxin recognition. To complement this approach, we have similarly analyzed residues on the alpha subunit face of the binding site dictating specificity for alpha-toxin. Analysis of the alpha gamma interface shows unique pairwise interactions between the charged residues on the alpha-toxin and three regions on the alpha subunit located around residue Asp(99), between residues Trp(149) and Val(153), and between residues Trp(187) and Asp(200). Substitutions of cationic residues at positions between Trp(149) and Val(153) markedly reduce the rate of alpha-toxin binding, and these cationic residues appear to be determinants in preventing alpha-toxin binding to alpha 2, alpha 3, and alpha 4 subunit containing receptors. Replacement of selected residues in the alpha-toxin shows that Ser(8) on loop I and Arg(33) and Arg(36) on the face of loop II, in apposition to loop I, are critical to the alpha-toxin for association with the alpha subunit. Pairwise mutant cycle analysis has enabled us to position residues on the concave face of the three alpha-toxin loops with respect to alpha and gamma subunit residues in the alpha-toxin binding site. Binding of NmmI alpha-toxin to the alpha gamma interface appears to have dominant electrostatic interactions not seen at the alpha delta interface.     

Involvement of asparagine 118 in the nucleotide specificity of the catalytic subunit of protein kinase CK2

Protein kinase CK2 is a heteromeric enzyme with catalytic (alpha) and regulatory (beta) subunits which form an alpha2beta2 holoenzyme and utilizes both ATP and GTP as nucleotide substrate. Site-directed mutagenesis of CK2alpha subunit was used to study this capacity to use GTP. Deletion of asparagine 118 (alpha(deltaN118)) or the mutant alphaN118E gives a 5-6-fold increase in apparent Km for GTP with little effect on the affinity for ATP. Mutants alphaN118A and alphaD120N did not alter significantly the Km for either nucleotide. CK2alphaN118 has an apparent Ki for inosine 5' triphosphate 5-fold higher than wild-type and is very heat labile. These studies complement recent crystallographic data indicating a role for CK2alpha asparagine 118 in binding the guanine base.     

High-affinity AKAP7delta-protein kinase A interaction yields novel protein kinase A-anchoring disruptor peptides

PKA (protein kinase A) is tethered to subcellular compartments by direct interaction of its regulatory subunits (RI or RII) with AKAPs (A kinase-anchoring proteins). AKAPs preferentially bind RII subunits via their RII-binding domains. RII-binding domains form structurally conserved amphipathic helices with unrelated sequences. Their binding affinities for RII subunits differ greatly within the AKAP family. Amongst the AKAPs that bind RIIalpha subunits with high affinity is AKAP7delta [AKAP18delta; K(d) (equilibrium dissociation constant) value of 31 nM]. An N-terminally truncated AKAP7delta mutant binds RIIalpha subunits with higher affinity than the full-length protein presumably due to loss of an inhibitory region [Henn, Edemir, Stefan, Wiesner, Lorenz, Theilig, Schmidtt, Vossebein, Tamma, Beyermann et al. (2004) J. Biol. Chem. 279, 26654-26665]. In the present study, we demonstrate that peptides (25 amino acid residues) derived from the RII-binding domain of AKAP7delta bind RIIalpha subunits with higher affinity (K(d)=0.4+/-0.3 nM) than either full-length or N-terminally truncated AKAP7delta, or peptides derived from other RII binding domains. The AKAP7delta-derived peptides and stearate-coupled membrane-permeable mutants effectively disrupt AKAP-RII subunit interactions in vitro and in cell-based assays. Thus they are valuable novel tools for studying anchored PKA signalling. Molecular modelling indicated that the high affinity binding of the amphipathic helix, which forms the RII-binding domain of AKAP7delta, with RII subunits involves both the hydrophobic and the hydrophilic faces of the helix. Alanine scanning (25 amino acid peptides, SPOT technology, combined with RII overlay assays) of the RII binding domain revealed that hydrophobic amino acid residues form the backbone of the interaction and that hydrogen bond- and salt-bridge-forming amino acid residues increase the affinity of the interaction.     

On the stator of rotary ATP synthase: the binding strength of subunit delta to (alpha beta)3 as determined by fluorescence correlation spectroscopy.

ATP synthase is conceived as a rotary enzyme. Proton flow drives the rotor (namely, subunits c12 epsilon gamma) relative to the stator (namely, subunits ab2 delta(alpha beta)3) and extrudes spontaneously formed ATP from three symmetrically arranged binding sites on (alpha beta)3 into the solution. We asked whether the binding of subunit delta to (alpha beta)3 is of sufficient strength to hold against the elastic strain, which is generated during the operation of this enzyme. According to current estimates, the elastically stored energy is about 50 kJ/mol. Subunit delta was specifically labeled without impairing its function. Its association with solubilized (alpha beta)3 gamma in detergent-free buffer was studied by fluorescence correlation spectroscopy (FCS). A very strong tendency of delta to dimerize in detergent-free buffer was apparent (K(d) 相似文献   

Structural requirements of tropomodulin for tropomyosin binding and actin filament capping

Regulation of actin filament dynamics underlies many cellular functions. Tropomodulin together with tropomyosin can cap the pointed, slowly polymerizing, filament end, inhibiting addition or loss of actin monomers. Tropomodulin has an unstructured N-terminal region that binds tropomyosin and a folded C-terminal domain with six leucine-rich repeats. Of tropomodulin 1's 359 amino acids, an N-terminal fragment (Tmod1(1)(-)(92)) suffices for in vitro function, even though the C-terminal domain can weakly cap filaments independent of tropomyosin. Except for one short alpha-helix with coiled coil propensity (residues 24-35), the Tmod1(1)(-)(92) solution structure shows that the fragment is disordered and highly flexible. On the basis of the solution structure and predicted secondary structure, we have introduced a series of mutations to determine the structural requirements for tropomyosin binding (using native gels and CD) and filament capping (by measuring actin polymerization using pyrene fluorescence). Tmod1(1)(-)(92) fragments with mutations of an interface hydrophobic residue, L27G and L27E, designed to destroy the alpha-helix or coiled coil propensity, lost binding ability to tropomyosin but retained partial capping function in the presence of tropomyosin. Replacement of a flexible region with alpha-helical residues (residues 59-61 mutated to Ala) had no effect on tropomyosin binding but inhibited the capping function. A mutation in a region predicted to be an amphipathic helix (residues 65-75), L71D, destroyed the capping function. The results suggest that molecular flexibility and binding to actin via an amphipathic helix are both required for tropomyosin-dependent capping of the pointed end of the actin filament.     

Binding of Mg2+ to the beta subunit or F1 of H+-ATPase from Escherichia coli

The bindings of Mg2+ to the F1 portion of Escherichia coli H+-ATPase and its isolated alpha and beta subunits were studied with 8-anilinonaphthalene-1-sulfonate (ANS). The fluorescence of ANS increased upon addition of F1 or its alpha subunit or beta subunit, as reported previously (M. Hirano, K. Takeda, H. Kanazawa, and M. Futai (1984) Biochemistry 23, 1652-1656). The fluorescence of ANS bound to F1 or its beta subunit increased significantly with further addition of Mg2+, whereas that of the alpha subunit increased only slightly. Ca2+ and Mn2+ had similar effects on the fluorescence of ANS with F1 and its beta subunit. The Mg2+-induced fluorescence enhancement (delta F) was high at an alkaline pH and was lowered by addition of ethylenediaminetetraacetic acid. Dicyclohexylcarbodiimide and azide had no effect on the delta F. Binding analysis showed that the concentration dependence of Mg2+ on the fluorescence enhancement of the beta subunit is similar to that of F1. These results suggest that both the beta subunit and F1 have binding sites for Mg2+ and that the delta F observed with F1 may be due to the binding of Mg2+ to the beta subunit.     

F1 and F0 connections in the bovine mitochondrial ATP synthase: the role of the of alpha subunit N-terminus, oligomycin-sensitivity conferring protein (OCSP) and subunit d.

We have studied the functional effect of limited proteolysis by trypsin of the constituent subunits in the native and reconstituted F1F0 complex and isolated F1 of the bovine heart mitochondrial ATP synthase (EC 3.6.1.34). Chemical cross-linking of oligomycin-sensitivity conferring protein (OSCP) with other subunits of the ATP synthase and the consequent functional effects were also investigated. The results obtained show that the alpha subunit N-terminus is essential for the correct, functional connection of F1 to F0. The alpha-subunit N-terminus contacts OSCP which, in turn, contacts the F0I-PVP(b) and the F0-d subunits. The N-terminus of subunit alpha, OSCP, a segment of subunit d and the C-terminal and central region of F0I-PVP(b) subunits are peripherally located with respect to subunits gamma and delta which are completely shielded in the F1F0 complex against trypsin digestion. This qualifies the N-terminus of subunit alpha, OSCP, subunit d and F0I-PVP(b) as components of the lateral element of the stalk. These subunits, rather than being confined at one side of the complex which would leave most of the central part of the gamma subunit uncovered, surround the gamma and the delta subunits located in the central stalk.