The “Second Stalk” of Escherichia coli ATP Synthase: Structure of the Isolated Dimerization Domain † , ‡ (original) (raw)
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Subunit b-Dimer of the Escherichia coli ATP Synthase Can Form Left-Handed Coiled-Coils
Biophysical Journal, 2008
One remaining challenge to our understanding of the ATP synthase concerns the dimeric coiled-coil stator subunit b of bacterial synthases. The subunit b-dimer has been implicated in important protein interactions that appear necessary for energy conservation and that may be instrumental in energy conservation during rotary catalysis by the synthase. Understanding the stator structure and its interactions with the rest of the enzyme is crucial to the understanding of the overall catalytic mechanism. Controversy exists on whether subunit b adopts a classic left-handed or a presumed right-handed dimeric coiled-coil and whether or not staggered pairing between nonhomologous residues in the homodimer is required for intersubunit packing. In this study we generated molecular models of the Escherichia coli subunit b-dimer that were based on the wellestablished heptad-repeat packing exhibited by left-handed, dimeric coiled-coils by employing simulated annealing protocols with structural restraints collected from known structures. In addition, we attempted to create hypothetical right-handed coiledcoil models and left-and right-handed models with staggered packing in the coiled-coil domains. Our analyses suggest that the available structural and biochemical evidence for subunit b can be accommodated by classic left-handed, dimeric coiled-coil quaternary structures.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2009
The structure and functional role of the dimeric external stalk of F o F 1 -ATP synthases have been very actively researched over the last years. To understand the function, detailed knowledge of the structure and protein packing interactions in the dimer is required. In this paper we describe the application of structural prediction and molecular modeling approaches to elucidate the structural packing interaction of the cyanobacterial ATP synthase external stalk. In addition we present biophysical evidence derived from ESR spectroscopy and site directed spin labeling of stalk proteins that supports the proposed structural model. The use of the heterodimeric bb′ dimer from a cyanobacterial ATP synthase (Synechocystis sp. PCC 6803) allowed, by specific introduction of spin labels along each individual subunit, the evaluation of the overall tertiary structure of the subunits by calculating inter-spin distances. At defined positions in both b and b′ subunits, reporter groups were inserted to determine and confirm inter-subunit packing. The experiments showed that an approximately 100 residue long section of the cytoplasmic part of the bb′-dimer exists mostly as an elongated α-helix. The distant C-terminal end of the dimer, which is thought to interact with the δ-subunit, seemed to be disordered in experiments using soluble bb′ proteins. A left-handed coiled coil packing of the dimer suggested from structure prediction studies and shown to be feasible in molecular modeling experiments was used together with the measured inter-spin distances of the inserted reporter groups determined in ESR experiments to support the hypothesis that a significant portion of the bb′ structure exists as a left-handed coiled coil.
Structure of the Cytosolic Part of the Subunit b-Dimer of Escherichia coli F0F1-ATP Synthase
Biophysical Journal, 2008
The structure of the external stalk and its function in the catalytic mechanism of the F 0 F 1 -ATP synthase remains one of the important questions in bioenergetics. The external stalk has been proposed to be either a rigid stator that binds F 1 or an elastic structural element that transmits energy from the small rotational steps of subunits c to the F 1 sector during catalysis. We employed proteomics, sequence-based structure prediction, molecular modeling, and electron spin resonance spectroscopy using site-directed spin labeling to understand the structure and interfacial packing of the Escherichia coli b -subunit homodimer external stalk. Comparisons of bacterial, cyanobacterial, and plant b-subunits demonstrated little sequence similarity. Supersecondary structure predictions, however, show that all compared b-sequences have extensive heptad repeats, suggesting that the proteins all are capable of packing as left-handed coiled-coils. Molecular modeling subsequently indicated that b 2 from the E. coli ATP synthase could pack into stable left-handed coiled-coils. Thirty-eight substitutions to cysteine in soluble b-constructs allowed the introduction of spin labels and the determination of intersubunit distances by ESR. These distances correlated well with molecular modeling results and strongly suggest that the E. coli subunit b-dimer can stably exist as a left-handed coiled-coil.
Journal of Molecular Biology, 2001
Bovine IF 1 is a basic, 84 amino acid residue protein that inhibits the hydrolytic action of the F 1 F 0 ATP synthase in mitochondria under anaerobic conditions. Its oligomerization state is dependent on pH. At a pH value below 6.5 it forms an active dimer. At higher pH values, two dimers associate to form an inactive tetramer. Here, we present the solution structure of a C-terminal fragment of IF 1 (44-84) containing all ®ve of the histidine residues present in the sequence. Most unusually, the molecule forms an anti-parallel coiled-coil in which three of the ®ve histidine residues occupy key positions at the dimer interface.
Conformational changes in the Escherichia coli ATP synthase b-dimer upon binding to F1-ATPase
Journal of Bioenergetics and Biomembranes, 2008
Conformational changes within the subunit bdimer of the E. coli ATP synthase occur upon binding to the F 1 sector. ESR spectra of spin-labeled b at room temperature indicated a pivotal point in the b-structure at residue 62. Spectra of frozen b ± F 1 and calculated interspin distances suggested that where contact between b 2 and F 1 occurs (above about residue 80), the structure of the dimer changes minimally. Between b-residues 33 and 64 inter-subunit distances in the F 1 -bound b-dimer were found to be too large to accommodate tightly coiled coil packing and therefore suggest a dissociation and disengagement of the dimer upon F 1 -binding. Mechanistic implications of this "bubble" formation in the tether domain of ATP synthase b 2 are discussed.
Crystal structure of the ϵ subunit of the proton-translocating ATP synthase from Escherichia coli
Structure, 1997
Background: Proton-translocating ATP synthases convert the energy generated from photosynthesis or respiration into ATP. These enzymes, termed F 0 F 1-ATPases, are structurally highly conserved. In Escherichia coli, F 0 F 1-ATPase consists of a membrane portion, F 0 , made up of three different polypeptides (a, b and c) and an F 1 portion comprising five different polypeptides in the stoichiometry α 3 β 3 γδε. The minor subunits γ, δ and ε are required for the coupling of proton translocation with ATP synthesis; the ε subunit is in close contact with the α, β, γ and c subunits. The structure of the ε subunit provides clues to its essential role in this complex enzyme. Results: The structure of the E. coli F 0 F 1-ATPase ε subunit has been solved at 2.3 Å resolution by multiple isomorphous replacement. The structure, comprising residues 2-136 of the polypeptide chain and 14 water molecules, refined to an R value of 0.214 (R free = 0.288). The molecule has a novel fold with two domains. The N-terminal domain is a β sandwich with two fivestranded sheets. The C-terminal domain is formed from two α helices arranged in an antiparallel coiled-coil. A series of alanine residues from each helix form the central contacting residues in the helical domain and can be described as an 'alanine zipper'. There is an extensive hydrophobic contact region between the two domains providing a stable interface. The individual domains of the crystal structure closely resemble the structures determined in solution by NMR spectroscopy. Conclusions: Sequence alignments of a number of ε subunits from diverse sources suggest that the C-terminal domain, which is absent in some species, is not essential for function. In the crystal the N-terminal domains of two ε subunits make a close hydrophobic interaction across a crystallographic twofold axis. This region has previously been proposed as the contact surface between the ε and γ subunits in the complete F 1-ATPase complex. In the crystal structure, we observe what is apparently a stable interface between the two domains of the ε subunit, consistent with the fact that the crystal and solution structures are quite similar despite close crystal packing. This suggests that a gross conformational change in the ε subunit, to transmit the effect of proton translocation to the catalytic domain, is unlikely, but cannot be ruled out.
Journal of Structural Biology, 2002
A comprehensive analysis of the sequences of all types of intermediate filament chains has been undertaken with a particular emphasis on those of segment 1A and linker L1. This has been done to assess whether structural characteristics can be recognized in the sequences that would be consistent with the role of each region in the recently proposed "swinging head" hypothesis. The analyses show that linker L1 is the most flexible rod domain region, that it is the most elongated structure (on a per residue basis), and that it is the most variable region as regards sequence and length. Segment 1A has one of the two most highly conserved regions of sequence in the rod domain (the other being at the end of segment 2B), with seven particular residues conserved across all chain types. It also contains one of the very few potential interchain ionic interactions that could be conserved across all chain types. However, the aggregation of chains in segment 1A is specified less precisely overall by interchain ionic interactions than are the other coiled-coil segments. The apolar residue contents in positions a and d of the heptad substructure are the highest of any coiled-coil segment in the intermediate filament family. Segment 1A also displays an amino acid composition atypical of not only coiled-coil segments 1B and 2B, but indeed of two-stranded coiled coils in general. Nonetheless, molecular modeling based on the crystal structure of the monomeric 1A fragment from human vimentin shows that coiled-coil formation is plausible. The most extensive regions of apolar/aromatic residues lie at the C-terminal end of segment 2B in the helix termination motif and in segment 1A in and close to the helix initiation motif. The predicted stability of the individual ␣-helices in segment 1A is greater than in those comprising segments 1B and 2B, though potential intrachain ionic interactions are either lacking or are minimal in number.
Journal of Molecular Biology, 2001
Bovine IF1 is a basic, 84 amino acid residue protein that inhibits the hydrolytic action of the F1F0 ATP synthase in mitochondria under anaerobic conditions. Its oligomerization state is dependent on pH. At a pH value below 6.5 it forms an active dimer. At higher pH values, two dimers associate to form an inactive tetramer. Here, we present the solution structure of a C-terminal fragment of IF1 (44–84) containing all five of the histidine residues present in the sequence. Most unusually, the molecule forms an anti-parallel coiled-coil in which three of the five histidine residues occupy key positions at the dimer interface.