Structure and regulatory role of the C-terminal winged helix domain of the archaeal minichromosome maintenance complex

The minichromosome maintenance complex (MCM) represents the replicative DNA helicase both in eukaryotes and archaea. Here, we describe the solution structure of the C-terminal domains of the archaeal MCMs of Sulfolobus solfataricus (Sso) and Methanothermobacter thermautotrophicus (Mth). Those domains consist of a structurally conserved truncated winged helix (WH) domain lacking the two typical ‘wings’ of canonical WH domains. A less conserved N-terminal extension links this WH module to the MCM AAA+ domain forming the ATPase center. In the Sso MCM this linker contains a short α-helical element. Using Sso MCM mutants, including chimeric constructs containing Mth C-terminal domain elements, we show that the ATPase and helicase activity of the Sso MCM is significantly modulated by the short α-helical linker element and by N-terminal residues of the first α-helix of the truncated WH module. Finally, based on our structural and functional data, we present a docking-derived model of the Sso MCM, which implies an allosteric control of the ATPase center by the C-terminal domain.     

Amputation of a C-terminal helix of the γ subunit increases ATP-hydrolysis activity of cyanobacterial F1 ATP synthase

F₁ is a soluble part of F_oF₁-ATP synthase and performs a catalytic process of ATP hydrolysis and synthesis. The γ subunit, which is the rotary shaft of F₁ motor, is composed of N-terminal and C-terminal helices domains, and a protruding Rossman-fold domain located between the two major helices parts. The N-terminal and C-terminal helices domains of γ assemble into an antiparallel coiled-coil structure, and are almost embedded into the stator ring composed of α₃β₃ hexamer of the F₁ molecule. Cyanobacterial and chloroplast γ subunits harbor an inserted sequence of 30 or 39 amino acids length within the Rossman-fold domain in comparison with bacterial or mitochondrial γ. To understand the structure–function relationship of the γ subunit, we prepared a mutant F₁-ATP synthase of a thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1, in which the γ subunit is split into N-terminal α-helix along with the inserted sequence and the remaining C-terminal part. The obtained mutant showed higher ATP-hydrolysis activities than those containing the wild-type γ. Contrary to our expectation, the complexes containing the split γ subunits were mostly devoid of the C-terminal helix. We further investigated the effect of post-assembly cleavage of the γ subunit. We demonstrate that insertion of the nick between two helices of the γ subunit imparts resistance to ADP inhibition, and the C-terminal α-helix is dispensable for ATP-hydrolysis activity and plays a crucial role in the assembly of F₁-ATP synthase.     

Sequence Conservation in Plasmodium falciparum α-Helical Coiled Coil Domains Proposed for Vaccine Development

Background

The availability of the P. falciparum genome has led to novel ways to identify potential vaccine candidates. A new approach for antigen discovery based on the bioinformatic selection of heptad repeat motifs corresponding to α-helical coiled coil structures yielded promising results. To elucidate the question about the relationship between the coiled coil motifs and their sequence conservation, we have assessed the extent of polymorphism in putative α-helical coiled coil domains in culture strains, in natural populations and in the single nucleotide polymorphism data available at PlasmoDB.

Methodology/Principal Findings

14 α-helical coiled coil domains were selected based on preclinical experimental evaluation. They were tested by PCR amplification and sequencing of different P. falciparum culture strains and field isolates. We found that only 3 out of 14 α-helical coiled coils showed point mutations and/or length polymorphisms. Based on promising immunological results 5 of these peptides were selected for further analysis. Direct sequencing of field samples from Papua New Guinea and Tanzania showed that 3 out of these 5 peptides were completely conserved. An in silico analysis of polymorphism was performed for all 166 putative α-helical coiled coil domains originally identified in the P. falciparum genome. We found that 82% (137/166) of these peptides were conserved, and for one peptide only the detected SNPs decreased substantially the probability score for α-helical coiled coil formation. More SNPs were found in arrays of almost perfect tandem repeats. In summary, the coiled coil structure prediction was rarely modified by SNPs. The analysis revealed a number of peptides with strictly conserved α-helical coiled coil motifs.

Conclusion/Significance

We conclude that the selection of α-helical coiled coil structural motifs is a valuable approach to identify potential vaccine targets showing a high degree of conservation.     

The regulatory C-terminal domain of subunit ε of F₀F₁ ATP synthase is dispensable for growth and survival of Escherichia coli

The C-terminal domain of subunit ε of the bacterial F_oF₁ ATP synthase is reported to be an intrinsic inhibitor of ATP synthesis/hydrolysis activity in vitro, preventing wasteful hydrolysis of ATP under low-energy conditions. Mutants defective in this regulatory domain exhibited no significant difference in growth rate, molar growth yield, membrane potential, or intracellular ATP concentration under a wide range of growth conditions and stressors compared to wild-type cells, suggesting this inhibitory domain is dispensable for growth and survival of Escherichia coli.F_oF₁ ATP synthases are ubiquitous enzymes that synthesize ATP using a transmembrane electrochemical potential of protons or proton motive force (PMF) generated by the respiratory chain across the cytoplasmic membrane of bacteria, the thylakoid membrane of chloroplasts, or the mitochondrial inner membrane (4, 5, 37). The enzyme consists of two parts: membrane-embedded F_o subcomplex (a complex of subunits a, b, and c in bacteria) and hydrophilic F₁ subcomplex (composed of subunits α, β, γ, δ, and ε). The enzyme is also known as a molecular motor, which is composed of the stator subcomplex (α, β, δ, a, and b) and the rotor subcomplex (γ, ε, and c), and its rotation is coupled to ATP synthesis and proton flow across the membrane (20, 31, 52). The reaction of the enzyme is reversible; ATP is hydrolyzed into ADP and inorganic phosphate, the rotor subcomplex rotates in reverse, and protons are extruded to the periplasmic side, resulting in the generation of PMF. Although some bacteria utilize the reverse reaction under particular conditions, the primary function of F_oF₁ ATP synthase is generation of ATP from the PMF. Therefore, the direction of the activity of F_oF₁ ATP synthase is regulated to avoid wasteful ATP hydrolysis.Subunit ε in bacterial F_oF₁ has been known to be an intrinsic inhibitor of F₁ and F_oF₁ complex (18, 21, 23) and is proposed to have a regulatory function (10, 11, 42). Although the inhibitory effects of subunit ε vary among species, in general, ε inhibits ATP hydrolysis activity while repressing ATP synthesis activity to a lesser degree (14, 27). This regulatory function of the ε subunit is mediated almost exclusively by the C-terminal region of ε, which is comprised of two antiparallel α-helices (18, 49, 50). Biochemical and crystallographic studies have revealed that the C-terminal helices can adopt two different conformations (34, 46, 47, 48). In the retracted conformation, the α-helices form a hairpin-like structure and sit on the N-terminal β-sandwich domain of the ε subunit. When the ε subunit exhibits an inhibitory effect, it adopts a more extended conformation in which the C-terminal α-helices extend along the γ subunit, which composes the central stalk. It has also been shown that basic, positively charged residues on the second α-helix of the ε subunit interact with negatively charged residues in the DELSEED segment of subunit β to exert the inhibitory effect (12).Escherichia coli mutants deleted in the entire ε subunit exhibit a reduced growth rate and growth yield, and this effect is proposed to be a result of a deficiency in assembly of the F_o and F₁ complexes (21). The N-terminal β-sandwich domain of the ε subunit is responsible for the assembly of F_o and F₁ and is therefore important for efficient coupling between proton translocation through F_o and ATP synthesis/hydrolysis in F₁ (15, 39). Deletion of the ε subunit leads to dissociation of the F_oF₁ complex and wasteful ATP hydrolysis by free (cytoplasmic) F₁ and dissipation of PMF through free F_o (21, 22, 51).While the importance of the entire ε subunit in the whole-cell physiology of E. coli is fairly well established, the role of the regulatory C-terminal region of ε has received little attention and warrants investigation to determine if the regulatory functions (e.g., inhibition of ATP hydrolysis) observed in vitro are manifested in the physiology of E. coli under various growth conditions. To address this question, we constructed isogenic E. coli mutants that were deleted in the C-terminal region of ε subunit (ε^DC) and used these strains to compare physiological properties of wild-type versus ε^DC cells under a wide range of environmental conditions and stressors.     

Mechanism of Inhibition by C-terminal α-Helices of the ? Subunit of Escherichia coli FoF1-ATP Synthase

The ϵ subunit of bacterial F_oF₁-ATP synthase (F_oF₁), a rotary motor protein, is known to inhibit the ATP hydrolysis reaction of this enzyme. The inhibitory effect is modulated by the conformation of the C-terminal α-helices of ϵ, and the “extended” but not “hairpin-folded” state is responsible for inhibition. Although the inhibition of ATP hydrolysis by the C-terminal domain of ϵ has been extensively studied, the effect on ATP synthesis is not fully understood. In this study, we generated an Escherichia coli F_oF₁ (EF_oF₁) mutant in which the ϵ subunit lacked the C-terminal domain (F_oF₁^ϵΔC), and ATP synthesis driven by acid-base transition (ΔpH) and the K⁺-valinomycin diffusion potential (ΔΨ) was compared in detail with that of the wild-type enzyme (F_oF₁^ϵWT). The turnover numbers (k_cat) of F_oF₁^ϵWT were severalfold lower than those of F_oF₁^ϵΔC. F_oF₁^ϵWT showed higher Michaelis constants (K_m). The dependence of the activities of F_oF₁^ϵWT and F_oF₁^ϵΔC on various combinations of ΔpH and ΔΨ was similar, suggesting that the rate-limiting step in ATP synthesis was unaltered by the C-terminal domain of ϵ. Solubilized F_oF₁^ϵWT also showed lower k_cat and higher K_m values for ATP hydrolysis than the corresponding values of F_oF₁^ϵΔC. These results suggest that the C-terminal domain of the ϵ subunit of EF_oF₁ slows multiple elementary steps in both the ATP synthesis/hydrolysis reactions by restricting the rotation of the γ subunit.     

Accommodating Discontinuities in Dimeric Left-Handed Coiled Coils in ATP Synthase External Stalks

ATP synthases from coupling membranes are complex rotary motors that convert the energy of proton gradients across coupling membranes into the chemical potential of the β-γ anhydride bond of ATP. Proton movement within the ring of c subunits localized in the F₀-sector drives γ and ɛ rotation within the F₁α₃β₃ catalytic core where substrates are bound and products are released. An external stalk composed of homodimeric subunits b₂ in Escherichia coli or heterodimeric bb′ in photosynthetic synthases connects F₀ subunit a with F₁ subunits δ and most likely α. The external stalk resists rotation, and is of interest both functionally and structurally. Hypotheses that the external stalk contributes to the overall efficiency of the reaction through elastic coupling of rotational substeps, and that stalks form staggered, right-handed coiled coils, are investigated here. We report on different structures that accommodate heptad discontinuities with either local or global underwinding. Analyses of the knob-and-hole packing of the E. coli b₂ and Synechocystis bb′ stalks strongly support the possibility that these proteins can adopt conventional left-handed coiled coils.     

Torque Transmission Mechanism via DELSEED Loop of F1-ATPase

F₁-ATPase (F₁) is an ATP-driven rotary motor in which the three catalytic β subunits in the stator ring sequentially induce the unidirectional rotation of the rotary γ subunit. Many lines of evidence have revealed open-to-closed conformational transitions in the β subunit that swing the C-terminal domain inward. This conformational transition causes a C-terminal protruding loop with conserved sequence DELSEED to push the γ subunit. Previous work, where all residues of DELSEED were substituted with glycine to disrupt the specific interaction with γ and introduce conformational flexibility, showed that F₁ still rotated, but that the torque was halved, indicating a remarkable impact on torque transmission. In this study, we conducted a stall-and-release experiment on F₁ with a glycine-substituted DELSEED loop to investigate the impact of the glycine substitution on torque transmission upon ATP binding and ATP hydrolysis. The mutant F₁ showed a significantly reduced angle-dependent change in ATP affinity, whereas there was no change in the equilibrium for ATP hydrolysis. These findings indicate that the DELSEED loop is predominantly responsible for torque transmission upon ATP binding but not for that upon ATP hydrolysis.     

F1-ATPase of Escherichia coli: THE ?-INHIBITED STATE FORMS AFTER ATP HYDROLYSIS,IS DISTINCT FROM THE ADP-INHIBITED STATE,AND RESPONDS DYNAMICALLY TO CATALYTIC SITE LIGANDS*

F₁-ATPase is the catalytic complex of rotary nanomotor ATP synthases. Bacterial ATP synthases can be autoinhibited by the C-terminal domain of subunit ϵ, which partially inserts into the enzyme''s central rotor cavity to block functional subunit rotation. Using a kinetic, optical assay of F₁·ϵ binding and dissociation, we show that formation of the extended, inhibitory conformation of ϵ (ϵ_X) initiates after ATP hydrolysis at the catalytic dwell step. Prehydrolysis conditions prevent formation of the ϵ_X state, and post-hydrolysis conditions stabilize it. We also show that ϵ inhibition and ADP inhibition are distinct, competing processes that can follow the catalytic dwell. We show that the N-terminal domain of ϵ is responsible for initial binding to F₁ and provides most of the binding energy. Without the C-terminal domain, partial inhibition by the ϵ N-terminal domain is due to enhanced ADP inhibition. The rapid effects of catalytic site ligands on conformational changes of F₁-bound ϵ suggest dynamic conformational and rotational mobility in F₁ that is paused near the catalytic dwell position.     

Conformational Transitions of Subunit ? in ATP Synthase from Thermophilic Bacillus PS3

Subunit ɛ of bacterial and chloroplast F_OF₁-ATP synthase is responsible for inhibition of ATPase activity. In Bacillus PS3 enzyme, subunit ɛ can adopt two conformations. In the “extended”, inhibitory conformation, its two C-terminal α-helices are stretched along subunit γ. In the “contracted”, noninhibitory conformation, these helices form a hairpin. The transition of subunit ɛ from an extended to a contracted state was studied in ATP synthase incorporated in Bacillus PS3 membranes at 59°C. Fluorescence energy resonance transfer between fluorophores introduced in the C-terminus of subunit ɛ and in the N-terminus of subunit γ was used to follow the conformational transition in real time. It was found that ATP induced the conformational transition from the extended to the contracted state (half-maximum transition extent at 140 μM ATP). ADP could neither prevent nor reverse the ATP-induced conformational change, but it did slow it down. Acid residues in the DELSEED region of subunit β were found to stabilize the extended conformation of ɛ. Binding of ATP directly to ɛ was not essential for the ATP-induced conformational change. The ATP concentration necessary for the half-maximal transition (140 μM) suggests that subunit ɛ probably adopts the extended state and strongly inhibits ATP hydrolysis only when the intracellular ATP level drops significantly below the normal value.     

Mutations on the N-Terminal Edge of the DELSEED Loop in either the α or β Subunit of the Mitochondrial F1-ATPase Enhance ATP Hydrolysis in the Absence of the Central γ Rotor

F₁-ATPase is a rotary molecular machine with a subunit stoichiometry of α₃β₃γ₁δ₁ε₁. It has a robust ATP-hydrolyzing activity due to effective cooperativity between the three catalytic sites. It is believed that the central γ rotor dictates the sequential conformational changes to the catalytic sites in the α₃β₃ core to achieve cooperativity. However, recent studies of the thermophilic Bacillus PS3 F₁-ATPase have suggested that the α₃β₃ core can intrinsically undergo unidirectional cooperative catalysis (T. Uchihashi et al., Science 333:755-758, 2011). The mechanism of this γ-independent ATP-hydrolyzing mode is unclear. Here, a unique genetic screen allowed us to identify specific mutations in the α and β subunits that stimulate ATP hydrolysis by the mitochondrial F₁-ATPase in the absence of γ. We found that the F446I mutation in the α subunit and G419D mutation in the β subunit suppress cell death by the loss of mitochondrial DNA (ρ^o) in a Kluyveromyces lactis mutant lacking γ. In organello ATPase assays showed that the mutant but not the wild-type γ-less F₁ complexes retained 21.7 to 44.6% of the native F₁-ATPase activity. The γ-less F₁ subcomplex was assembled but was structurally and functionally labile in vitro. Phe446 in the α subunit and Gly419 in the β subunit are located on the N-terminal edge of the DELSEED loops in both subunits. Mutations in these two sites likely enhance the transmission of catalytically required conformational changes to an adjacent α or β subunit, thereby allowing robust ATP hydrolysis and cell survival under ρ^o conditions. This work may help our understanding of the structural elements required for ATP hydrolysis by the α₃β₃ subcomplex.     

The Role of the ��DELSEED-loop of ATP Synthase

ATP synthase uses a unique rotational mechanism to convert chemical energy into mechanical energy and back into chemical energy. The helix-turn-helix motif, termed “DELSEED-loop,” in the C-terminal domain of the β subunit was suggested to be involved in coupling between catalysis and rotation. Here, the role of the DELSEED-loop was investigated by functional analysis of mutants of Bacillus PS3 ATP synthase that had 3–7 amino acids within the loop deleted. All mutants were able to catalyze ATP hydrolysis, some at rates several times higher than the wild-type enzyme. In most cases ATP hydrolysis in membrane vesicles generated a transmembrane proton gradient, indicating that hydrolysis occurred via the normal rotational mechanism. Except for two mutants that showed low activity and low abundance in the membrane preparations, the deletion mutants were able to catalyze ATP synthesis. In general, the mutants seemed less well coupled than the wild-type enzyme, to a varying degree. Arrhenius analysis demonstrated that in the mutants fewer bonds had to be rearranged during the rate-limiting catalytic step; the extent of this effect was dependent on the size of the deletion. The results support the idea of a significant involvement of the DELSEED-loop in mechanochemical coupling in ATP synthase. In addition, for two deletion mutants it was possible to prepare an α₃β₃γ subcomplex and measure nucleotide binding to the catalytic sites. Interestingly, both mutants showed a severely reduced affinity for MgATP at the high affinity site.F₁F₀-ATP synthase catalyzes the final step of oxidative phosphorylation and photophosphorylation, the synthesis of ATP from ADP and inorganic phosphate. F₁F₀-ATP synthase consists of the membrane-embedded F₀ subcomplex, with, in most bacteria, a subunit composition of ab₂c₁₀, and the peripheral F₁ subcomplex, with a subunit composition of α₃β₃γδε. The energy necessary for ATP synthesis is derived from an electrochemical transmembrane proton (or, in some organisms, a sodium ion) gradient. Proton flow down the gradient through F₀ is coupled to ATP synthesis on F₁ by a unique rotary mechanism. The protons flow through (half) channels at the interface of the a and c subunits, which drives rotation of the ring of c subunits. The c₁₀ ring, together with F₁ subunits γ and ε, forms the rotor. Rotation of γ leads to conformational changes in the catalytic nucleotide binding sites on the β subunits, where ADP and P_i are bound. The conformational changes result in the formation and release of ATP. Thus, ATP synthase converts electrochemical energy, the proton gradient, into mechanical energy in the form of subunit rotation and back into chemical energy as ATP. In bacteria, under certain physiological conditions, the process runs in reverse. ATP is hydrolyzed to generate a transmembrane proton gradient, which the bacterium requires for such functions as nutrient import and locomotion (for reviews, see Refs. 1–6).F₁ (or F₁-ATPase) has three catalytic nucleotide binding sites located on the β subunits at the interface to the adjacent α subunit. The catalytic sites have pronounced differences in their nucleotide binding affinity. During rotational catalysis, the sites switch their affinities in a synchronized manner; the position of γ determines which catalytic site is the high affinity site (K_d1 in the nanomolar range), which site is the medium affinity site (K_d2 ≈ 1 μm), and which site is the low affinity site (K_d3 ≈ 30–100 μm; see Refs. 7 and 8). In the original crystal structure of bovine mitochondrial F₁ (9), one of the three catalytic sites, was filled with the ATP analog AMP-PNP,² a second was filled with ADP (plus azide) (see Ref. 10), and the third site was empty. Hence, the β subunits are referred to as β_TP, β_DP, and β_E. The occupied β subunits, β_TP and β_DP, were in a closed conformation, and the empty β_E subunit was in an open conformation. The main difference between these two conformations is found in the C-terminal domain. Here, the “DELSEED-loop,” a helix-turn-helix structure containing the conserved DELSEED motif, is in an “up” position when the catalytic site on the respective β subunit is filled with nucleotide and in a “down” position when the site is empty (Fig. 1A). When all three catalytic sites are occupied by nucleotide, the previously open β_E subunit assumes an intermediate, half-closed (β_HC) conformation. It cannot close completely because of steric clashes with γ (11).Open in a separate windowFIGURE 1.The βDELSEED-loop. A, interaction of the β_TP and β_E subunits with theγ subunit.β subunits are shown in yellow andγ in blue. The DELSEED-loop (shown in orange, with the DELSEED motif itself in green)of β_TP interacts with the C-terminal helixγ and the short helix that runs nearly perpendicular to the rotation axis. The DELSEED-loop of β_E makes contact with the convex portion of γ, formed mainly by the N-terminal helix. A nucleotide molecule (shown in stick representation) occupies the catalytic site of β_TP, and the subunit is in the closed conformation. The catalytic site on β_E is empty, and the subunit is in the open conformation. This figure is based on Protein Data Bank file 1e79 (32). B, deletions in the βDELSEED-loop. The loop was “mutated” in silico to represent the PS3 ATP synthase. The 3–4-residue segments that are removed in the deletion mutants are color-coded as follows: ³⁸⁰LQDI³⁸³, pink; ³⁸⁴IAIL³⁸⁷, green; ³⁸⁸GMDE³⁹¹, yellow; ³⁹²LSD³⁹⁴, cyan; ³⁹⁵EDKL³⁹⁸, orange; ³⁹⁹VVHR⁴⁰², blue. Residues that are the most involved in contacts with γ are labeled. All figures were generated using the program PyMOL (DeLano Scientific, San Carlos, CA).The DELSEED-loop of each of the three β subunits makes contact with the γ subunit. In some cases, these contacts consist of hydrogen bonds or salt bridges between the negatively charged residues of the DELSEED motif and positively charged residues on γ. The interactions of the DELSEED-loop with γ, its movement during catalysis, the conservation of the DELSEED motif (see 12–14). Thus, the finding that an AALSAAA mutant in the α₃β₃γ complex of ATP synthase from the thermophilic Bacillus PS3, where several hydrogen bonds/salt bridges to γ are removed simultaneously, could drive rotation of γ with the same torque as the wild-type enzyme (14) came as a surprise. On the other hand, it seems possible that it is the bulk of the DELSEED-loop, more so than individual interactions, that drives rotation of γ. According to a model favored by several authors (6, 15, 16) (see also Refs. 17–19), binding of ATP (or, more precisely, MgATP) to the low affinity catalytic site on β_E and the subsequent closure of this site, accompanied by its conversion into the high affinity site, are responsible for driving the large (80–90°) rotation substep during ATP hydrolysis, with the DELSEED-loop acting as a “pushrod.” A recent molecular dynamics (20) study supports this model and implicates mainly the region around several hydrophobic residues upstream of the DELSEED motif (specifically βI386 and βL387)³ as being responsible for making contact with γ during the large rotation substep.

TABLE 1

Conservation of residues in the DELSEED-loop Amino acids found in selected species in the turn region of the DELSEED-loop. Listed are all positions subjected to deletions in the present study. Residue numbers refer to the PS3 enzyme. Consensus annotation: p, polar residue; s, small residue; h, hydrophobic residue; –, negatively charged residue; +, positively charged residue.Open in a separate windowIn the present study, we investigated the function of the DELSEED-loop using an approach less focused on individual residues, by deleting stretches of 3–7 amino acids between positions β380 and β402 of ATP synthase from the thermophilic Bacillus PS3. We analyzed the functional properties of the deletion mutants after expression in Escherichia coli. The mutants showed ATPase activities, which were in some cases surprisingly high, severalfold higher than the activity of the wild-type control. On the other hand, in all cases where ATP synthesis could be measured, the rates where below or equal to those of the wild-type enzyme. In Arrhenius plots, the hydrolysis rates of the mutants were less temperature-dependent than those of wild-type ATP synthase. In those cases where nucleotide binding to the catalytic sites could be tested, the deletion mutants had a much reduced affinity for MgATP at high affinity site 1. The functional role of the DELSEED-loop will be discussed in light of the new information.     

ATP Synthase with Its �� Subunit Reduced to the N-terminal Helix Can Still Catalyze ATP Synthesis

ATP synthase uses a unique rotary mechanism to couple ATP synthesis and hydrolysis to transmembrane proton translocation. As part of the synthesis mechanism, the torque of the rotor has to be converted into conformational rearrangements of the catalytic binding sites on the stator to allow synthesis and release of ATP. The γ subunit of the rotor, which plays a central role in the energy conversion, consists of two long helices inside the central cavity of the stator cylinder plus a globular portion outside the cylinder. Here, we show that the N-terminal helix alone is able to fulfill the function of full-length γ in ATP synthesis as long as it connects to the rest of the rotor. This connection can occur via the ϵ subunit. No direct contact between γ and the c ring seems to be required. In addition, the results indicate that the ϵ subunit of the rotor exists in two different conformations during ATP synthesis and ATP hydrolysis.F₁F_o-ATP synthase is responsible for the bulk of ATP synthesis from ADP and P_i in most organisms. F₁F_o-ATP synthase consists of the membrane-embedded F_o subcomplex with, in most bacteria, a subunit composition of ab₂c_n (with n = 10–15) and the peripheral F₁ subcomplex, with a subunit composition of α₃β₃γδϵ. The energy necessary for ATP synthesis is derived from an electrochemical transmembrane proton (or, in some organisms, sodium ion) gradient. Proton flow, down the gradient, through F_o is coupled to ATP synthesis on F₁ by a unique rotary mechanism. The protons flow through channels at the interface of a and c subunits, which drives rotation of the ring of c subunits. The c_n ring, together with F₁ subunits γ and ϵ, forms the rotor. Rotation of γ leads to conformational changes in the catalytic nucleotide-binding sites on the β subunits, where ADP and P_i are bound. The conformational changes result in formation and release of ATP. Thus, ATP synthase converts electrochemical energy, the proton gradient, into mechanical energy in the form of subunit rotation and back into chemical energy as ATP. In bacteria, under certain physiological conditions, the process can run in reverse. ATP is hydrolyzed to generate a transmembrane proton gradient that the bacterium requires for such functions as nutrient import and locomotion (for reviews, see Refs. 1–6).F₁ (or “F₁-ATPase”) has three catalytic nucleotide-binding sites, located on the β subunits, at the interface with the adjacent α subunit. The catalytic sites have pronounced differences in their nucleotide-binding affinity. During rotational catalysis, the sites switch their affinities in a synchronized manner; the position of γ determines which catalytic site is the high affinity site (K_d1 in the nanomolar range), which site is the medium affinity site (K_d2 ≈ 1 μm), and which site is the low affinity site (K_d3 ≈ 30–100 μm; see Refs. 7, 8). Only the high affinity site is catalytically active (9). In the original crystal structure of bovine mitochondrial F₁ (10), one of the three catalytic sites was filled with the ATP analog AMPPNP,³ a second one with ADP (plus azide; see Ref. 11), and the third site was empty. Hence, the β subunits are referred to as β_TP, β_DP, and β_E, respectively. The high affinity site is located on the β_TP subunit (12).The coupling process between ATP synthesis or hydrolysis on β and rotation of γ is not yet fully understood on a residue level. A number of point mutations at the interfaces between α or β and γ and between γ, ϵ, and c have been described that result in varying degrees of uncoupling (13–17). Some mutations at these interfaces were found that abolish ATP synthesis or hydrolysis activity or both (18–20). Considering the pronounced effect of these point mutations, some of which were even conservative substitutions, it came as a surprise when it was recently shown that an “axle-less” γ, consisting just of the globular portion, with the portions of the N- and C-terminal helices that reach into the α₃β₃ cylinder removed, displayed ATP-driven rotation in the correct direction (21).Some reports have implicated the ϵ subunit (corresponding to the δ subunit in the mitochondrial enzyme) as being involved in coupling (15, 22–25). It was shown that ϵ exists in different conformations that vary in the folding and positioning of the C-terminal domain. The x-ray structure of the mitochondrial enzyme (26) shows the two helices of the C-terminal domain folded back on each other like a hairpin and positioned close to the interface between γ and the c ring (“down” conformation). In the crystal structure of a γϵ complex from Escherichia coli the hairpin is unfolded (27); when integrated into F₁ or F₁F_o, the C terminus would reach “up,” coming close to the DELSEED-loop of the α and/or β subunits. While in this up conformation the angle between both helices of the C-terminal domain of ϵ is ∼90°, it has been postulated that this domain might also exist in a fully extended up conformation, stretching close to the N terminus of γ, with helical regions replacing the turn between the two helices (28). Fixing ϵ in either up conformation by cross-linking to γ has been shown to impair ATP hydrolysis but not synthesis. Freezing ϵ in the down position inhibited neither reaction (29, 30).Here, we report a finding that is arguably just as surprising as the rotation of an axle-less γ. In ATP synthase from the thermophilic bacterium Bacillus PS3, enzymes with γ subunit constructs where the globular domain and the C-terminal helix were eliminated, consisting of just the N-terminal 35 or 42 residues, TF₁F_o(γQ36stop)⁴ and TF₁F_o(γP43stop), were able to catalyze significant rates of ATP synthesis. According to the crystal structure (26), the shorter of the two γ constructs should not make any contact either with c or with ϵ in the down conformation. Thus, the fact that ATP synthesis was observed suggests that ϵ in an up conformation forms a complex with the truncated γ, which is able to catalyze ATP synthesis. Indeed, when the γQ36stop truncation was combined with an ϵ truncation where the C-terminal domain was removed, ATP synthesis was abolished. The functions of the γ and ϵ subunits will be discussed in light of the new findings.     

Central Region of Talin Has a Unique Fold That Binds Vinculin and Actin

Talin is an adaptor protein that couples integrins to F-actin. Structural studies show that the N-terminal talin head contains an atypical FERM domain, whereas the N- and C-terminal parts of the talin rod include a series of α-helical bundles. However, determining the structure of the central part of the rod has proved problematic. Residues 1359–1659 are homologous to the MESDc1 gene product, and we therefore expressed this region of talin in Escherichia coli. The crystal structure shows a unique fold comprised of a 5- and 4-helix bundle. The 5-helix bundle is composed of nonsequential helices due to insertion of the 4-helix bundle into the loop at the C terminus of helix α3. The linker connecting the bundles forms a two-stranded anti-parallel β-sheet likely limiting the relative movement of the two bundles. Because the 5-helix bundle contains the N and C termini of this module, we propose that it is linked by short loops to adjacent bundles, whereas the 4-helix bundle protrudes from the rod. This suggests the 4-helix bundle has a unique role, and its pI (7.8) is higher than other rod domains. Both helical bundles contain vinculin-binding sites but that in the isolated 5-helix bundle is cryptic, whereas that in the isolated 4-helix bundle is constitutively active. In contrast, both bundles are required for actin binding. Finally, we show that the MESDc1 protein, which is predicted to have a similar fold, is a novel actin-binding protein.     

Structural analysis of smooth muscle tropomyosin α and β isoforms

A large number of tropomyosin (Tm) isoforms function as gatekeepers of the actin filament, controlling the spatiotemporal access of actin-binding proteins to specialized actin networks. Residues ∼40–80 vary significantly among Tm isoforms, but the impact of sequence variation on Tm structure and interactions with actin is poorly understood, because structural studies have focused on skeletal muscle Tmα. We describe structures of N-terminal fragments of smooth muscle Tmα and Tmβ (sm-Tmα and sm-Tmβ). The 2.0-Å structure of sm-Tmα81 (81-aa) resembles that of skeletal Tmα, displaying a similar super-helical twist matching the contours of the actin filament. The 1.8-Å structure of sm-Tmα98 (98-aa) unexpectedly reveals an antiparallel coiled coil, with the two chains staggered by only 4 amino acids and displaying hydrophobic core interactions similar to those of the parallel dimer. In contrast, the 2.5-Å structure of sm-Tmβ98, containing Gly-Ala-Ser at the N terminus to mimic acetylation, reveals a parallel coiled coil. None of the structures contains coiled-coil stabilizing elements, favoring the formation of head-to-tail overlap complexes in four of five crystallographically independent parallel dimers. These complexes show similarly arranged 4-helix bundles stabilized by hydrophobic interactions, but the extent of the overlap varies between sm-Tmβ98 and sm-Tmα81 from 2 to 3 helical turns. The formation of overlap complexes thus appears to be an intrinsic property of the Tm coiled coil, with the specific nature of hydrophobic contacts determining the extent of the overlap. Overall, the results suggest that sequence variation among Tm isoforms has a limited effect on actin binding but could determine its gatekeeper function.     

Pivotal Role of Extended Linker 2 in the Activation of Gα by G Protein-coupled Receptor

G protein-coupled receptors (GPCRs) relay extracellular signals mainly to heterotrimeric G-proteins (Gαβγ) and they are the most successful drug targets. The mechanisms of G-protein activation by GPCRs are not well understood. Previous studies have revealed a signal relay route from a GPCR via the C-terminal α5-helix of Gα to the guanine nucleotide-binding pocket. Recent structural and biophysical studies uncover a role for the opening or rotating of the α-helical domain of Gα during the activation of Gα by a GPCR. Here we show that β-adrenergic receptors activate eight Gα_s mutant proteins (from a screen of 66 Gα_s mutants) that are unable to bind Gβγ subunits in cells. Five of these eight mutants are in the αF/Linker 2/β2 hinge region (extended Linker 2) that connects the Ras-like GTPase domain and the α-helical domain of Gα_s. This extended Linker 2 is the target site of a natural product inhibitor of G_q. Our data show that the extended Linker 2 is critical for Gα activation by GPCRs. We propose that a GPCR via its intracellular loop 2 directly interacts with the β₂/β₃ loop of Gα to communicate to Linker 2, resulting in the opening and closing of the α-helical domain and the release of GDP during G-protein activation.     

Crystal structure of N-glycosylated human glypican-1 core protein: structure of two loops evolutionarily conserved in vertebrate glypican-1

Glypicans are a family of cell-surface proteoglycans that regulate Wnt, hedgehog, bone morphogenetic protein, and fibroblast growth factor signaling. Loss-of-function mutations in glypican core proteins and in glycosaminoglycan-synthesizing enzymes have revealed that glypican core proteins and their glycosaminoglycan chains are important in shaping animal development. Glypican core proteins consist of a stable α-helical domain containing 14 conserved Cys residues followed by a glycosaminoglycan attachment domain that becomes exclusively substituted with heparan sulfate (HS) and presumably adopts a random coil conformation. Removal of the α-helical domain results in almost exclusive addition of the glycosaminoglycan chondroitin sulfate, suggesting that factors in the α-helical domain promote assembly of HS. Glypican-1 is involved in brain development and is one of six members of the vertebrate family of glypicans. We expressed and crystallized N-glycosylated human glypican-1 lacking HS and N-glycosylated glypican-1 lacking the HS attachment domain. The crystal structure of glypican-1 was solved using crystals of selenomethionine-labeled glypican-1 core protein lacking the HS domain. No additional electron density was observed for crystals of glypican-1 containing the HS attachment domain, and CD spectra of the two protein species were highly similar. The crystal structure of N-glycosylated human glypican-1 core protein at 2.5 Å, the first crystal structure of a vertebrate glypican, reveals the complete disulfide bond arrangement of the conserved Cys residues, and it also extends the structural knowledge of glypicans for one α-helix and two long loops. Importantly, the loops are evolutionarily conserved in vertebrate glypican-1, and one of them is involved in glycosaminoglycan class determination.     

Structure of p22 headful packaging nuclease

Packaging of viral genomes into preformed procapsids requires the controlled and synchronized activity of an ATPase and a genome-processing nuclease, both located in the large terminase (L-terminase) subunit. In this paper, we have characterized the structure and regulation of bacteriophage P22 L-terminase (gp2). Limited proteolysis reveals a bipartite organization consisting of an N-terminal ATPase core flexibly connected to a C-terminal nuclease domain. The 2.02 Å crystal structure of P22 headful nuclease obtained by in-drop proteolysis of full-length L-terminase (FL-L-terminase) reveals a central seven-stranded β-sheet core that harbors two magnesium ions. Modeling studies with DNA suggest that the two ions are poised for two-metal ion-dependent catalysis, but the nuclease DNA binding surface is sterically hindered by a loop-helix (L₁-α₂) motif, which is incompatible with catalysis. Accordingly, the isolated nuclease is completely inactive in vitro, whereas it exhibits endonucleolytic activity in the context of FL-L-terminase. Deleting the autoinhibitory L₁-α₂ motif (or just the loop L₁) restores nuclease activity to a level comparable with FL-L-terminase. Together, these results suggest that the activity of P22 headful nuclease is regulated by intramolecular cross-talk with the N-terminal ATPase domain. This cross-talk allows for precise and controlled cleavage of DNA that is essential for genome packaging.     

The N-terminal coiled coil of the Rhodococcus erythropolis ARC AAA ATPase is neither necessary for oligomerization nor nucleotide hydrolysis

Deletion mutants of the Rhodococcus erythropolis ARC AAA ATPase were generated and characterized by biochemical analysis and electron microscopy. Based on sequence comparisons the ARC protein was divided into three consecutive regions, the N-terminal coiled coil, the central ARC-specific inter domain and the C-terminal AAA domain. When the ARC AAA domain was expressed separately it formed aggregates of undefined structure. However, when the AAA domain was expressed in conjunction with the preceeding inter domain, but without the N-terminal coiled coil, high-molecular weight-complexes were formed (ARC-DeltaCC) which showed an [Formula: see text] -ethylmaleimide-sensitive ATPase activity. In 2D crystallization experiments the ARC-DeltaCC particles yielded crystals nearly identical to those formed by the wild-type ARC complexes. Thus, the N-terminal coiled coil, which was proposed to have a role in the assembly of and/or interaction between the eukaryotic AAA ATPases in the 26S proteasome, is neither essential for assembly nor for ATP hydrolysis of the ARC ATPase. The N-terminal domain of related AAA ATPases mediates the interaction with substrates or co-factors, suggesting a regulatory function for the N-terminal coiled coil of the ARC ATPase. Surprisingly, the mutant ARC protein ARC-DeltaAAA consisting of the N-terminal coiled coil and the central inter domain, but deleted for the C-terminal AAA domain, was shown to form a dodecameric complex with sixfold symmetry. This suggests an important role of the inter domain for the ordered assembly of the ARC ATPase.     

Characterization of desnutrin functional domains: critical residues for triacylglycerol hydrolysis in cultured cells

Murine desnutrin/human ATGL is a triacylglycerol (TAG) hydrolase with a predicted catalytic dyad within an α-β hydrolase fold in the N-terminal region. In humans, mutations resulting in C-terminal truncation cause neutral lipid storage disease with myopathy. To identify critical functional domains, we measured TAG breakdown in cultured cells by mutated or truncated desnutrin. In vitro, C-terminally truncated desnutrin displayed an even higher apparent V_max than the full-length form without changes in K_m, which may be explained by our finding of an interaction between the C- and N-terminal domains. In live cells, however, C-terminally truncated adenoviral desnutrin had lower TAG hydrolase activity. We investigated a role for the phosphorylation of C-terminal S406 and S430 residues but found that these were not necessary for TAG breakdown or lipid droplet localization in cells. The predicted N-terminal active sites, S47 and D166, were both critical for TAG hydrolysis in live cells and in vitro. We also identified two overlapping N-terminal motifs that predict lipid substrate binding domains, a glycine-rich motif (underlined) and an amphipathic α-helix (bold) within amino acid residues 10–24 (ISFAGCGFLGVYHIG). G14, F17, L18, and V20, but not G16 and G19, were important for TAG hydrolysis, suggesting a potential role for the amphipathic α-helix in TAG binding. This study identifies for the first time critical sites in the N-terminal region of desnutrin and reveals the requirement of the C-terminal region for TAG hydrolysis in cultured cells.     

Disruption of the C-terminal helix by single amino acid deletion is directly responsible for impaired cholesterol efflux ability of apolipoprotein A-I Nichinan

Apolipoprotein A-I (apoA-I) Nichinan, a naturally occurring variant with ΔE235 in the C terminus, is associated with low plasma HDL levels. Here, we investigated the tertiary structure, lipid-binding properties, and ability to induce cellular cholesterol efflux of apoA-I Nichinan and its C-terminal peptide. Thermal and chemical denaturation experiments demonstrated that the ΔE235 mutation decreased the protein stability compared with wild type (WT). ApoA-I Nichinan exhibited capabilities to bind to or solubilize lipid vesicles that are intermediate to that of WT and a L230P/L233P/Y236P variant in which the C-terminal α-helix folding is completely disrupted and forms relatively larger and unstable discoidal complexes, indicating that perturbation of the C-terminal α-helical structure by the ΔE235 mutation leads to reduced lipid binding. Supporting this, apoA-I 209-241/ΔE235 peptide showed significantly decreased ability to form α-helix both in the lipid-free and lipid-bound states, and reduced efficiency to solubilize vesicles. In addition, both apoA-I Nichinan and its C-terminal peptide exhibited reduced activity in ABCA1-mediated cellular cholesterol efflux. Thus, the disruption of the ability of the C-terminal region to form α-helix caused by the E235 deletion appears to be the important determinant of impaired lipid binding and cholesterol efflux ability and, consequently, the low plasma HDL levels of apoA-I Nichinan probands.