Citation for published version (APA): Chaban, Y. (2005). Subunit topology in the V type ATPase and related enzymes. s.n.

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1 University of Groningen Subunit topology in the V type ATPase and related enzymes Chaban, Yuriy IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2005 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Chaban, Y. (2005). Subunit topology in the V type ATPase and related enzymes. s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date:

2 CHAPTER I Introduction An increasing number of studies is dedicated to an intriguing family of enzymes consisting of the A-type and F-type ATP synthases, and V-type ATPases. These enzymes are key players in the energy metabolism of the organisms in all three kingdoms of life. They are universal energy converters and the smallest known molecular engines, which makes them interesting not only for biochemistry and biophysics but also for the fast-developing field of nanotechnology. A fast progress in the topic was recently achieved, leading to understanding of rotational catalysis in the F ATP synthase (Walker, 1998; Boyer, 2000). At the same time, studies on the close relatives of the F ATP synthase, the A-type ATP synthase and V-ATPase have raised new questions concerning mechanisms of energy transduction and regulation in these enzymes. A growing amount of evidence suggests that V-ATPase, in particular, has a number of specific and elegant mechanisms of regulation (Ratajczak, 2000; Murata et al., 2001; Nelson et al., 2002; Inoue et al., 2003; Kane & Smardon, 2003; Kluge et al., 2003; Sun-Wada et al., 2003; Wieczorek et al., 2003). Interest to the V-ATPase is especially high due to the crucial role of this enzyme in important processes like protein processing and degradation, intracellular trafficking, receptor-mediated endocytosis, osmoregulation, ph homeostasis, remnant storage and cell defence (Taiz, 1992; Nishi & Forgac, 2002; Moriyama et al., 2003), maintaining of the blood ph and renal acidification (Gluck et al., 1996; Wagner et al., 2004), bone resorption (Laitala-Leinonen et al., 1999; Li et al., 1999; Toyomura et al., 2003), ph homeostasis and secondary transport processes (Brisseau et al., 1996; Nelson & Harvey, 1999; Wieczorek et al., 2000). Therefore, understanding of the structure, mechanisms of energy conversion and regulation of V-ATPase will promote creating new tools in treatment of a number of severe metabolic disorders. It also can be useful for developing a fineregulated artificial molecular engine for nanotechnology. The A-type ATP synthases are at the moment the least studied group of the family. At the same time, the A-type ATP synthases are considered the most ancient group, being the closest relative of the common ancestor of the V-ATPases and F-type ATP synthases (for review see Müller & Grüber, 2003). Therefore, studies of A-ATPases create a background for understanding of the evolution of these unique energy converters. 10 I-1. Definitions To avoid misunderstanding it is worthwhile to explain our view on the nomenclature and relationships in the family of the A- and F-type ATP synthases, and V-ATPases. Historically these enzymes are also called ATPases, since relatively easy detection of the ATPase activity became a popular tool for their identification. Later it became clear that in many organisms in vivo the main function of the F- and A-ATPase is the synthesis of ATP, despite the fact that at certain conditions these enzymes are reversible. Therefore, in most cases the F- and A-type ATPases should be called ATP synthase. In contrast, the V-ATPases are not capable to any substantial ATP synthesis at physiological conditions and, most probably, evolved as true ATPases. Nevertheless, for convenience, in this manuscript we routinely designate F- and A-type ATP synthases as F- and A- ATPases, respectively.

3 A second remark concerns definitions of the A-, F- and V-ATPase subfamilies themselves. We shall start with the group of A-ATPases, despite it is less explored than its F- and V-type relatives. A-ATPases (A 1 A 0 -ATPases, A-type ATP- or A 1 A 0 -ATP synthases) are membrane-bound protein complexes found in Archaea, which are functioning in µη + or µna + coupled ATP synthesis and/or active proton or Na + pumping at the expense of ATP hydrolysis. The A-ATPase consists of two domains, a membrane-embedded, hydrophobic A 0 - and a hydrophilic A 1 -part, which protrudes into the cell matrix (Coskun et al., 2004a). A- and F-type ATPases (F- or F 1 F 0 -ATPases; F-type ATP- or F 1 F 0 -ATP synthases) have the same functions and similar gross structure, but F-ATPases are found in chloroplasts and mitochondria of the eukaryotic cells and in the majority of Bacteria. There is at least one piece of evidence for the presence of F-ATPases in the Archaea: a sequence encoding an F-ATPase was obtained from the archaeon Methanosarcina barkeri (Sumi et al., 1992, 1997). Unlike A- and F-ATPases, V-ATPases (V-type ATPases; V 1 V 0 -ATPases) are only capable of proton or Na + pumping at the expense of ATP hydrolysis, at least at physiological µη + (Müller & Grüber, 2003). V-ATPases are constitutive enzymes of eukaryotic cells and were found in a number of Eubacteria (Lolkema et al., 2003). Due to the multiple structural and functional similarities it was proposed that A-, V- and F- ATPases share a common ancestor. Especially the A- and V-ATPases have a very similar subunit composition and, probably, similar fine structure. However, the A- and F-ATPases seems to be more related functionally. Nevertheless, there are some significant differences within a group, such as between eukaryotic and bacterial V-ATPases. First, the eukaryotic V-ATPase possesses two additional subunits, C and H (see below), which are thought to play a regulatory role. Second, the relative sequence similarity between eukaryotic and bacterial V-ATPases is lower than between bacterial V-ATPases and A-ATPases (Olendzenski et al., 2000; Lolkema et al., 2003). At last, eukaryotic V-ATPases are capable to reversibly disassembly V 1 and V 0 parts (Wieczorek et al., 1999; Kane, 2000), while this was never yet demonstrated for bacterial V-ATPases. In a very recent review on V-ATPases, it is even proposed to split the group of V-ATPases into two parts: the true V-ATPases, typically found in eukaryotic cells, and the so-called A/V-ATPases, which only include prokaryotic V-ATPases found in the kingdom of Bacteria (Müller & Grüber, 2003). Moreover, a recent report on the 3-D structure of ATP synthase of the bacterium Thermus thermophilus, previously designated as V-ATPase (Yokoyama et al., 2000), refers to this enzyme as an A-ATPase (Bernal & Stock, 2004). At the same time, among V-ATPases only the T. thermophilus enzyme has the ability to synthesize ATP at physiological conditions (Yokoyama et al., 1998) and, to our opinion, might be assigned as a true A-ATPase (Müller et al., 2004). We suggest that for more precise distinction of the A- and V-ATPases additional data on structure, mechanism and genetics of A- and V-ATPases are necessary. Therefore in this work we keep the old nomenclature for the V- ATPases, designating, where necessary, eukaryotic and prokaryotic (bacterial) enzymes. I-2. F-type ATP-synthase F-type ATP synthases (F-ATPases) are the best understood enzymes in the family of A-, F- and V-ATPases. The mechanisms involved in the ATP synthesis/hydrolysis and translocation of the coupling ion as well as F-ATPase structure have been extensively studied*. Data obtained on F- * For recent reviews see: Futai et al., 2000; Richter et al., 2000; Stock et al., 2000; Kaim, 2001; Noji & Yoshida, 2001; Boyer, 2001, 2002; Capaldi & Aggeler, 2002; Senior et al., 2002; Weber & Senior,

4 ATPases formed the basis for understanding the structure and mechanism of V- and A-ATPases. Many experiments successfully performed for F-ATPases have been used as templates for studies of the V- and A-ATPase. Therefore here we shall briefly summarize the accumulated knowledge of the structure and principal mechanism of the F-ATPases. ATP is an universal energy currency in all living organisms. It is consumed in the vast number of energy requiring chemical processes in the cell. According to estimation, a human utilizes 40 kg of ATP in normal daily living. Assuming the pool of nucleotides is 100 mmol, each molecule of ADP in the body must be phosphorylated and the product ATP dephosphorylated on average 1000 times per day (Capaldi & Aggeler, 2002). In the same time, the F-ATP synthase enzyme is responsible for the production of nearly 90% of the ATP in the human body! The F-ATP synthase in eukaryotic cells is localized in the inner membrane of mitochondria and in addition in plants in thylakoid membranes of chloroplasts. In bacteria the enzyme resides in the cytoplasmic membrane and, probably, the thylakoid membrane of cyanobacteria. In photosynthetic organisms, it utilizes the proton motive force built up by the photosynthetic light reactions in a process called photophoshorylation. In mitochondria, it uses the proton motive force originating from the four respiratory chain complexes. Hence, this process is called oxidative phosphorylation (Mitchell, 1979; Skulachev, 1989) and ATP synthase is occasionally called Complex V. In some cases, a Na + gradient is utilized (Laubinger & Dimroth, 1989; Kluge & Dimroth, 1993a, b; Reidlinger & Muller, 1994; Neumann et al., 1998). Moreover, the enzyme can work in the reversed direction and as an ATPase it can generate H + or Na + electrochemical gradient at the expense of ATP hydrolysis. 12 I-2.1. Subunit composition and structure of F-ATP synthase The F-ATP synthase complex has eight different subunits in prokaryotes and plant chloroplasts, and subunits in mammals. The molecular weight of the fully assembled complex varies between 550 and 650 kda, depending on species. The subunit nomenclature of the F-ATPase is not consistent, despite the fact that all subunits crucial for the enzyme assembly and function share high levels of homology (Table I-1). The subunits of Escherichia coli F-ATPase migrate on denaturating SDS PAGE in the order α, β, γ, a, δ, b, ε, c and have approximate molecular weights of 55, 50, 31.5, 30, 19, 17, 15, and 8 kda, respectively (Senior, 1988; Ishmukhametov et al., 2005). Early experiments demonstrated that F-ATPase can be easily spited into two parts: F 1 and F 0 (Bragg & Hou, 1972; Futai et al., 1974). The F 1 part contains subunits α, β, γ, ε, and δ with a stoichiometry of α 3 β 3 γδ (Foster & Fillingame, 1979, 1982). The F 0 part consists of subunits a, b and c, with a proposed stoichiometry ab 2 c 10, in case of Escherichia coli (Jiang et al., 2001; Wehrle et al., 2002a also see Table I-1). The release of F 1 is favored by lowering the ionic strength of the medium or by extraction with chloroform-toluene mixtures, which suggests hydrophobic interaction between the F 1 -sector and the F 0 -sector (Amzel & Pedersen, 1983). At the same time binding of subunits b and δ appears to be ionic (Walker et al., 1982a) and depends on divalent cations since addition of EDTA to the medium strongly enhances the release of the F 1 -sector from its membrane-bound form (Amzel & Pedersen, 1983). A number of structural and functional studies on F-ATPase subunits and subcomplexes revealed the character and meaning of interactions in this complicated protein complex. It has been proposed that the F-ATPase acts as a motor, in which a rotary movement of the γ subunit of F 1 is coupled to a similar movement of subunits c of the F 0 part. This movement is coupled to proton or sodium transport across the membrane by F 0 (Cox et al., 1986; Boyer, 1993;

5 Dimroth et al, 1999). In our present understanding, a functional division of F-ATPase into rotor and stator components is more relevant than its nominal division on F 1 and F 0 parts (see Table I-1). The rotor and stator are closely interacting during the rotary catalysis of ATP hydrolysis or synthesis (for details see below). The rotor consists of subunits γεс (10-14) and rotates relatively to the stator made of subunits α 3 β 3 δb 2 a (Figure I-1A, p. 23). The rotation of the rotor is driven by the proton- (or Na + -) flow across the channel located in the static F 0 subunit a and leads to the conformational changes in α 3 β 3 catalytic moiety propelled by the γ subunit rotation (F 1 -part) and resulting in ATP synthesis. The rotation of the rotor is reversed when driven by the ATP hydrolysis in α 3 β 3 hexamer, which results in proton- (or Na + -) pumping across the transmembrane channel in subunit a in the direction opposite to that during ATP synthesis (Figure I-1B, p. 23). From this scheme, it is obvious that for functioning of the F-ATP synthase/ ATPase complex a connection between static parts of F 1 and F 0 is necessary. This connection is made of subunits δ and b and gives the stator stability against idle rotation during ATP synthesis/ hydrolysis. Due to its function and location in the complex this connection is often designated as stator- or peripheral stalk, respectively. In contrast, the central subunits γ and ε are designated as the rotor (Figure I-1, p. 23). Table I-1. Subunit composition of F-ATP synthase*. Subcomplex F 1 F 0 Homologue subunits (stoichiometry) Function in Bacteria Chloroplasts Mitochondria catalysis mechanics α (3) α (3) α (3) ATP, ADP and P i binding, β (3) β (3) β (3) ATP synthesis/ hydrolysis δ (1) δ (1) OSCP (1) connects F 1 and F 0 static parts γ (1) γ (1) γ (1) coupling of H + (Na + ) transport and ATP synthesis/ hydrolysis ε (1) ε (1) δ (1) regulation of F 1 -ATPase activity**, rotor stabilization (?) ε (1) rotor stabilization (?) IF 1 inhibits ATPase activity when bound c (10-12) III (14) c (10) H + or Na + binding a (1) IV (1) a (1) H + or Na + channel b (2) I (1), II (1) b (2) connects F 1 and F 0 static parts d (1) F 6 (1) e (1) f (1) not known*** g (1) A6L (1) stator rotor stator * Based on Table 1 from Böttcher et al., 2000, with modifications. ** In mitochondrial F 1 F 0, an intrinsic inhibitor protein controls ATPase activity (van Raaij M. et al., 1996), and δ subunit is blocked from reacting with the α and β subunits by its close interaction with a subunit (unfortunately called ε) (Gibbons et al., 2000), which is not present in bacterial and chloroplast enzymes. *** But see Straffon et al., 1998; Papa et al., 2000; Velours et al., 2000; Osanai et al., 2001; Arselin et al., 2004; Carbajo et al., 2004; Hong & Pedersen, 2004; Rubinstein et al.,

6 14 I-2.2. Structure- and function relationships in F-ATPase I Structure and mechanism of F 1 -ATPase When F-ATP synthase is fully assembled and embedded in the membrane it is capable to utilize the energy stored in the proton-, or in some cases Na + transmembrane electrochemical gradient, and synthesize ATP from ADP and P i (Sone et al., 1977; Schmidt & Gräber, 1985, 1987; Bokranz et al., 1985; Slooten & Vandenbranden, 1989; Dmitriev et al., 1993; Krenn et al., 1993; Fischer et al., 1994; Groth & Walker, 1996; van Walraven et al., 1996; Ishmukhametov et al., 2005). On the other hand, the isolated enzyme in detergent solution is only capable of ATP hydrolysis, because the proton gradient cannot be utilized. A remarkable feature of the ATP hydrolysis by the fully assembled F 1 F 0 -ATPase is its high sensitivity to DCCD (Foster & Fillingame, 1979, Ishmukhametov et al., 2005). A disassembled F 1 -part keeps ATPase activity (F 1 -ATPase), while the membrane embedded F 0 becomes a passive proton or Na + pore (Schneider & Altendorf, 1985; Kluge & Dimroth, 1992; Kaim & Dimroth, 1998b). Experiments on assembly of F-ATPase demonstrated that only F 1 subunits α and β are required for ATP hydrolysis (reviewed in Kagawa & Hamamoto, 1997; Weber & Senior, 1997; Kagawa, 1999). Purified α and β subunits have ATP-binding sites but do not have ATPase activity (Ohta et al., 1980; Issartel & Vignais, 1984; Rao et al., 1988a, b; Mills & Richter, 1991). E. coli needs at least a complex of αβ subunits to get significant activity (al-shawi et al., 1990). Another group demonstrated the catalytic activity of the α 1 β 1 protomer from the thermophilic Bacillus PS3 F 1 -ATPase (TF 1 ) (Ohta et al., 1990). The reconstituted α 3 β 3 complex of TF 1 exhibited a much higher activity, but it was still lower than in the case of the complete TF 1 (Miwa & Yoshida, 1989). The TF 1 α 3 β 3 complex was highly unstable, and easily lost ATPase activity, which could be restored by addition of the γ-subunit (Kagawa et al., 1989). Reconstitution experiments with isolated E. coli F 1 subunits showed that the minimal complex able to catalyze ATP hydrolysis at physiological rate is an α 3 β 3 γ complex (Dunn & Futai, 1980; Futai et al., 1980). This complex resembled F 1 showing inhibition by azide (al-shawi et al., 1990) and the ability to bind and hydrolyze sub-stoichiometric amounts of ATP (Noumi et al., 1988). Kinetic experiments demonstrated that F 1 -ATPase (and ATP synthase) displayed negative cooperativity in substrate binding and positive cooperativity in catalysis (Cross et al., 1982). Incubation of the mitochondrial F 1 with sub-stoichiometric amounts of substrate MgATP leads to very tight binding (dissociation constant of < M) and slow hydrolysis (V max = 10-3 s -1 ) at a catalytic site of the enzyme (Grubmeyer, 1982). At higher substrate concentrations, catalytic turnover with the concurrent release of product ADP from this site is increased by several orders of magnitude. This apparent cooperativity is assumed to result from conformational changes induced by binding of nucleotide at a second catalytic site. Catalysis at the first site has been referred to as uni-site catalysis, whereas catalysis involving more than one site under steady state conditions in the presence of higher substrate concentrations is thought to involve at least two or more catalytic sites and referred to as multi-site catalysis (Richter et al., 2000). A number of experiments suggested an important role of γ subunit in the cooperative catalysis. Truncation (Futai & Kanazawa, 1983) and cross-linking (Kandpal & Boyer, 1987) of γ resulted in loss of ATPase activity. These experiments formed the basis for the hypothesis of the binding change mechanism of catalysis (Boyer, 1993). It was proposed that each catalytic site alternates sequentially between three different states: the tight state (where catalysis occurs), the loose state (where substrates are bound), and the open state (with low affinity for nucleotide) (Figure I-2A). As summarized by Cross, 1992, the structural asymmetry

7 required to induce different conformational states at each of the nucleotide binding sites must result from an interaction between the α 3 β 3 hexamer and the γ subunit, since chloroplast F 1 lacking the δ and ε subunits retains full catalytic cooperativity (Hu et al., 1993), whereas the α 3 β 3 hexamer does not (Gao et al., 1995). According to the mechanism presented in Duncan et al., 1995 (Figure I-2A) ATP binds to the O (open and empty) site to convert it into a T (tight and ATP-occupied) site. After bond cleavage, the T site is converted into the L (loose and ADP+P i -occupied) site, from which the products can escape to recover the O state. At any one time, the three catalytic sites are in the O, T and L states, respectively. The concerted switching of states in each of the sites results in the hydrolysis (or synthesis) of one ATP molecule, and a rotation of the rotor, composed of subunit γ and associated subunits, with step of 120 (Boyer, 1993; Duncan et al., 1995; Weber & Senior, 2003). In fact, further developments in the field have confirmed this binding change theory (see below). It is now generally accepted that the transition between the different catalytic states of the β-subunits is the result of a physical rotation of the γ subunit relative to the α 3 β 3 sector, although a different point of view on this topic also exists (McCarty et al., 2000). A * B ** Figure I-2. Scheme for Boyer s alternating site hypothesis in ATP synthase. Explanations see in text. A crucial break through in the understanding of the structure and function relationships in F 1 - ATPase came from X-ray crystallography studies. A remarkable study of bovine F 1 -ATPase (Abrahams et al., 1994) revealed the structure of the AMP-PNP inhibited F 1 -ATPase molecule. The F 1 molecule consisted of alternating α and β subunits creating a shaft. An N-terminus of the γ- subunit was partly located inside this shaft, while the C-terminus was protruding about 45 Å out of the α 3 β 3 complex. Further studies demonstrated that the ε subunit binds to the protruding C-terminus of γ subunit and forms a foot connected to ring of c subunits of the F 0 part (Stock et al., 1999). These results created a strong support for the rotational catalysis mechanism. Moreover, analysis of the α 3 β 3 γ (Abrahams et al., 1994) revealed that the complex has a significant structural asymmetry with three different catalytic sites on the interface between α and β subunits. Depending on nucleotide occupancy of the catalytic sites, subunits were designated as β E, α E (non-occupied site, assigned to the open state), β DP, α DP (site occupied by ADP, assigned to the loose state) and β TP, α TP (site occupied by AMP-PNP, assigned to the tight state) in accordance with the hypothesis of Boyer (Abrahams et al., 1994). Nevertheless, successive structural studies of F 1 resulted in a reevaluation of the binding change mechanism of ATPase catalysis (Menz et al., 2001; Ren & Allison, 2000; Boyer, 2001; Capaldi & Aggeler, 2002; Weber & Senior, 2000, 2003). Figure I-2B * From Capaldi & Aggeler, 2002, with modifications. ** From Menz et al., 2001, with modifications. 15

8 shows a scheme based on the bovine crystal structures. Three new intermediate catalytic states (called T*, L and L ) where proposed (Menz et al., 2001; see also Ren & Allison, 2000; Weber & Senior, 2003). ATP binding to the open catalytic site (O) produces a conformational change to a half-closed state (L'), which has not yet been observed crystallographically but is probably similar to the β ADP+Pi conformation. Conformational changes induced by ATP binding result in the committed hydrolysis of ATP at an adjacent catalytic site (T*), and cyclic interconversion of the sites. The T* site becomes a half-open site (L"), the L site becomes a T site, and the L' site becomes an L site (Menz et al., 2001). On the basis of the structural data currently available, it is possible to propose a model for the generation of the γ subunit rotation (Figure I-3, p. 23). The central feature of this model is a high degree of cooperativity between conformational changes occurring simultaneously in two of the three catalytic β subunits, as in the catalytic scheme proposed by Allison (Ren & Allison, 2000). Commencing with the point at which product (ADP+Pi) has just been released from one of the β subunits (Figure I-3A, p. 23), the next step in the catalytic pathway is the binding of substrate (ATP) to the empty β subunit. The positive binding energy of ATP is used to generate a conformational change in this subunit, initially from the open conformation (β E ) to a half-closed conformation similar to β ADP+Pi (Figure I-3B, p. 23). This partial closure of the β E subunit produces small conformational changes in the α E subunit, which are related to the catalytic site of the adjacent β DP subunit. This converts the β DP subunit catalytic site to a state committed to ATP hydrolysis. The energy released by the hydrolysis of ATP on the β DP subunit drives this subunit toward an open conformation, and simultaneously the binding energy of ATP promotes the closure of the β E subunit. The concerted movement of the C-terminal domains of these two β subunits is responsible for the rotation of the γ subunit; the β DP C-terminal domain moves outwards from the axis of the assembly, while that of the β E subunit moves in toward this axis (Figures I-3B and I-3C). Van der Waal's interactions between residues in these C-terminal domains (β385-β395) and the γ subunit (γ12-19, γ25-30, and γ ) are primarily responsible for the movement of the γ subunit. The cooperativity between the catalytic sites arises because γ subunit rotation is dependent upon conformational changes in both β subunits; neither ATP hydrolysis alone nor ATP binding alone is sufficient to generate the rotation (Menz et al., 2001). 16 I F 0 domain In contrast to the detailed structural information available for the F 1 part, little is known about the structural details of F 0. Electron spectroscopic imaging and atomic force microscopy together with recent X-ray crystallography data of the mitochondrial yeast F 1 c subcomplex suggest an overall structure of F 0 consisting of a ring of c subunits which is flanked on one side by the a and the two b subunits, as shown on Figure I-1, p. 23 (Birkenhäger et al., 1995; Singh et al., 1996; Stock et al., 1999; Takeyasu et al., 1996). The a and c subunits are in close contact and protons are thought to be translocated through the interface between them (Jiang & Fillingame, 1998; Vik et al., 2000). I Subunit c Monomeric subunit c is a small hydrophobic membrane protein, which is also called the proteolipid subunit due to its solubility in organic solvent mixtures. The c subunit of different organisms comprises amino acids with molecular masses between 8 and 10 kda. The c

9 subunit has been studied by different approaches including chemical modification with dicyclohexylcarbodiimide (DCCD), mutagenesis experiments, ion specificity studies, and others. It was established that in E. coli enzyme residues Asp61 of subunit c and Arg210 of subunit a (in Propionigenium modestum Glu65 and Arg277, respectively) and polar residues in their neighborhood are directly involved in transport of H + or Na + across the membrane (Kaim & Dimroth, 1998a; Cain, 2000; Vik et al., 2000; Fillingame & Dmitriev, 2002; Fillingame et al, 2002; von Ballmoos et al., 2002; Wehrle et al., 2002b). The structure of E. coli subunit c has been determined by NMR in solution at ph 5.0 and 8.0 (Girvin et al., 1998; Rastogi & Girvin, 1999). Monomeric subunit c of E. coli adopts a hairpin-like fold and packs into two antiparallel α-helices of 38 and 33 residues, respectively, which are connected by a polar loop of six or seven amino acids. The main difference in the two structures is a 140 rotation of the C-terminal helix with respect to the N-terminal helix. Upon modeling these structures into the membrane, the H + binding Asp61 residue was placed into the center of the bilayer (Fillingame et al., 2000). However, since both α- helixes are too long to completely insert into the membrane bilayer, alternative membrane boundaries of the protein are conceivable as well. The suggestion, that the critical Asp61 residue could also be located more closely to the membrane surface where it could have direct access to the coupling protons, was supported by epitope mapping of monoclonal antibodies against subunit c of E. coli, demonstrating that the region between positions 31 and 42 is accessible from the cytoplasm (Birkenhäger et al., 1999). The first structure of the F 0 domain came from an X-ray study of F 1 -ATPase associated with a ring of 10 c subunits from S. cerevisiae (Stock et al., 1999). This F 1 c 10 complex was formed from intact ATP synthase during the crystallization process, when other subunits dissociated. The structure contains a number of important features. First, the 10 c protomers appear to have a secondary structure similar to the c protomer structure determined by NMR. The structure also shows that the C-terminal α helices form an outer ring, with the N-terminal α-helices in a second inner ring. Second, the map shows that the extensive footprint of the central stalk sits asymmetrically on the polar loop regions of six c subunits. This arrangement is consistent with the rotation of the central stalk and the c ring as an ensemble. This is in line with data on covalent crosslinking between the E. coli ε and c subunits, which do not affect the enzyme s activity (Watts & Capaldi, 1997; Hermolin et al., 1999; Schulenberg et al., 1999). Third, 10 c subunits are found in the ring, although the loss of subunit copies during crystallization could not be excluded (Stock et al., 2000). The stoichiometry of c subunits in the ring differs among species. In chloroplasts and the Na + translocating F-ATPase of Iliobacter tartaricus 14 and 11 c subunits are assembled into a cylindrical ring, respectively (Seelert et al., 2000; Vonck et al., 2002). Cross-linking and genetic studies in E. coli have been interpreted as showing the presence of c subunits per F 0 (Jones & Fillingame, 1998; Jones et al., 1998; Dmitriev et al., 1999b; Jiang et al., 2001). There is an indication that the c-ring symmetry may also vary within a single species according to physiological conditions (Schemidt et al., 1998). I Subunit a Subunit a is a very hydrophobic integral membrane protein. Structural information on this subunit is limited. Recent topological models anticipate five or six membrane-spanning helixes (Jäger et al., 1998; Long et al., 1998; Valiyaveetil & Fillingame, 1998; Yamada et al., 1996). All models suggest a similar topological arrangement of the two C-terminal α-helices and from 17

10 mutagenesis studies it was proposed that this domain participates in the translocation of the coupling ions (Lightowlers et al., 1987, 1988; Cain & Simoni 1988, 1989). The helices could be packed to permit a channel to be open to the periplasmic side and closed to the cytoplasmic side. Importantly, only Arg227 is highly conserved among all species investigated and even conservative replacements at this position completely abolish ATP synthesis (Hatch et al., 1995). I Interaction between subunits a and c A conceptual breakthrough came from the demonstration that the c-subunit undergoes a large rotation of its C-terminal-helix upon protonation of casp61 (Rastogi & Girvin, 1999). It was proposed that this helical rotation couples the protonation/deprotonation reaction to angular displacement of subunit c versus a, resulting in net proton-driven c-ring rotation. Structural studies of mutant c-subunit (Fillingame & Dmitriev, 2002), and extensive crosslinking of c with a (Vik & Antonio, 1994; Cain, 2000; Fillingame et al., 2002) allowed future development of this concept, such that swiveling of helices of c and a relative to each other is now considered integral to generation of c-ring rotation (Weber & Senior, 2003). The a subunit in the model of ion translocation by F 0 provides access channels, which are needed to allow the ions to move from the membrane surfaces to the binding site residues of the c-subunits (Figure I-1 A, p. 23). The structures of such pathways, involving primarily residues in subunit a, have been proposed. An important contribution to our present understanding of the structure and function of F 0 has been the work of Dimroth and colleagues on the Na + -translocation enzyme from the bacterium Propionigenium modestum (Dimroth et al., 2000). In a number of mutants of Propionigenium modestum Na + translocation is abolished while Li + or H + translocation is retained. In mutants where subunit a had changes in positions Lys220Arg, Val264Glu, Ile278Asn, Na + inhibits ATP hydrolysis as a result of entrapment of one cation in the sodium proton channel (Kaim & Dimroth, 1998a). This gives strong evidence that the F 0 part acts as a single ion channel. Further studies on Na + linked rotation have emphasized the important role of the electrical potential in generating torque force (Kaim & Dimroth, 1999), revealed details of the mode of interplay between residues aarg210 and casp61 (Kaim et al., 1998; Wehrle et al., 2002b), and delineated specific determinants of ion selectivity for Na + versus H + (Kaim et al., 1997). A B Figure I-4 *. Models of the generation of rotation by movement of ions through the F 0 domain of ATP synthase. Current models of proton translocation through F 0 (Figure I-4) show either a single channel at the a-c subunit interface (reviewed by Kaim, 2001) or two half-channels (Junge et al., 1997; Aksimentiev et al., 2004). In the model proposed by Dimroth and coworkers for the P. modestum * from Stock et al., 2000, with modifications. 18

11 enzyme, the carboxyl side chains of the essential residue Glu65 ** in the subunit c ring are negatively charged when they enter the interface between the c ring and subunit a (Figure I-4A). The positive charge of Arg227 *** in subunit a attracts the negative charge of the essential carboxylate in subunit c and also prevents ion leakage. Once this carboxylate has been neutralized by a Na + ion from the periplasm, it will move by thermal vibrations, bringing the next negatively charged carboxylate into the channel. Electrostatic forces strongly bias the rotation, making it effectively unidirectional (Dimroth et al., 1999). In the two-half-channel model (Figure I-4B), each c subunit is protonated at Asp61 through one half-channel when this monomer comes into contact with the a subunit. The protonated c subunit then rotates almost 360 before the proton is released through the second halfchannel when it comes into contact with the a subunit again (Junge et al., 1997). A more detailed explanation of mechanism of ion pumping and coupling obviously requires additional structural information on F 0, as well as knowledge of the number of c subunits in the ring (Elston et al., 1998). I Subunit b Biophysical studies of the b subunit indicated a largely α-helical protein in an extended conformation (Cain, 2000; Dunn et al., 2000; Vogel, 2000; Revington et al., 2002). The b subunits of E. coli form a b 2 homodimer (Howitt et al., 1996; McLachlin & Dunn, 1997), which stretches from the periplasmic side of the membrane to near the top of F 1 where it makes contact with the δ subunit. The elongated C-terminus of the subunit b dimer is essential for the connection of F 0 with F 1 via subunit δ (Steffens et al., 1987; Takeyama et al., 1988; Wilkens et al., 2000). Additional interactions were detected between subunit b and at least one αβ pair (McLachlin et al., 2000). The combined protein-protein interactions between b 2 and the F 1 subunits were found to provide sufficient binding energy, preventing rotation of the α 3 β 3 hexamer due to the rotation of γεc 10 rotor (Cherepanov et al., 1999; McLachlin & Dunn, 2000; Weber et al., 2003). Dunn and coworkers have defined that two distinct functional domains proximal to the carboxyl terminus are necessary for formation of the b 2 dimer and interactions with F 1 (Revington et al., 1999). Recently, the structure of the dimerization domain was reported (Del Rizzo et al., 2002). The tether region roughly corresponds to the peripheral stalk visibly linking F 1 and F 0 in electron micrographs (Wilkens, 2000; Wilkens et al., 2000; Böttcher et al., 1998, Mellwig & Böttcher, 2003; Rubinstein et al., 2003). Relatively large insertions and deletions in the tether region allow retention of F 1 F 0 ATP synthase activity. Therefore a highly flexible structure and inter-subunit interactions in the tether domain was suggested (Sorgen et al., 1998; Sorgen et al., 1999; Motz et al., 2004). Several chemical crosslinks have been demonstrated between a cytoplasmic loop of the a subunit and the b subunit (Long et al., 1998; McLachlin et al., 2000, see also Figure I-1B, p. 23). Mutations affecting Arg36 yielded an uncoupled phenotype suggesting an altered proton channel (Caviston et al., 1998). Dmitriev et al., 1999a, determined the structure of a polypeptide modeling the b subunit membrane domain in an organic solvent by nuclear magnetic resonance spectroscopy. The polypeptide formed an α-helix with a 20 bend resulting from Pro27 and Pro28 located near the cytoplasmic surface of the membrane. A series of cysteine substitutions within the membrane domain suggested that disulfide bridges could be most efficiently formed within the b 2 dimer at positions proximal to the periplasmic side of the membrane. This led to a structural model in which the extreme amino-terminal ends of ** Glu65 is P. modestum analogue of Asp61 in E.coli. *** Arg277 is P. modestum analogue of Arg210 in E.coli 19

12 the b subunits participated in direct interactions, but the two subunits moved apart from one another as the proteins crossed the membrane (Dmitriev et al. 1999a). Recently it has been demonstrated that multiple substitutions on the periplasmic side result in defects in the assembly of the enzyme complex. The mutants had insufficient oxidative phosphorylation to support growth, and biochemical studies showed little F 1 F 0 ATPase and no detectable ATP driven proton-pumping activity. At the same time, single amino acid substitutions had minimal reductions in F 1 F 0 ATP synthase function. This might suggest that the membrane domain of the b subunit has several sites of interaction contributing to the assembly of F 0, and that these interactions are strongest on the periplasmic side of the bilayer (Hardy et al., 2003). 20 I Conformational changes during rotational catalysis The rotation of the F 1 -ATPase central stalk subunits driven by ATP hydrolysis has been observed directly with light microscopy. In these experiments, the F 1 -ATPase was immobilized and a long fluorescent actin filament was attached to the γ subunit. An ATP dependent rotation of the actin filament was detected. (Noji et al., 1997, 1999; Kato-Yamada et al., 1998; Omote et al., 1999; Hisabori et al., 1999). The main characteristics of this rotation are that it is highly efficient in energy usage, that it proceeds in 120 steps (Yasuda et al., 1998) and that the rotation is counterclockwise as viewed from the tip of the central stalk protrusion (Figure I-5). This direction is consisted with a scheme for rotation of the γ subunit deduced from X-ray structures of the F 1 domain (Figure I-3, p. 23). MgATP binding was shown to initiate each step and pausing molecules were concluded to be awaiting a productive collision with substrate. Replacement of actin filaments by 40-nm gold beads with less drag allowed a major advance in time resolution (Yasuda et al., 2001). Now it was observed that upon MgATP binding there occurred a 90 rotation substeps of γ in 0.25 ms, followed by a stationary interval of around 2 ms, followed by a terminating 30º substep (also 0.25 ms duration). Attempts have also been made to observe the rotation in F 1 F 0 -ATPase preparations by attaching actin filaments to the c ring on the surface distal from F 1 (Sambongi et al., 1999; Panke et al., 2000; Figure I-5C). Although technical objections have been put forward concerning these experiments (Tsunoda et al., 2000), they can be reasonably interpreted as showing that the F 1 -c ring rotates as an ensemble in response to ATP hydrolysis in F 1. However, because the detergents used to isolate the complex destabilize interactions of the c ring with the a subunit, these experiments should not be taken as definitive proof of the rotation of the F 1 -c ring in an intact F 1 F 0 complex that is capable of ATP synthesizing, as well as ATP hydrolyzing (Stock et al., 2000). Most recently, fluorescence resonance energy transfer was used to follow MgATP-driven rotation of γ in liposomereconstituted F 1 F 0. It confirmed the rotor stepping (Börsch et al., 2002). The obvious next technical challenge is a demonstration of proton gradient-driven rotation, and the nature of its substeps (Weber & Senior, 2003). Another intriguing question is how rotation of the c-ring is connected to the ATP synthesis/hydrolysis. The three catalytic sites of F 1 can be connected to the F 0 c ring with symmetry mismatch, since values of 10, 14, and 11 c-subunits per ring have been obtained experimentally (Stock et al., 1999; Seelert et al., 2000; Vonck et al., 2002). Lack of three-fold symmetry between c- rings and catalytic sites has been discussed as an advantage for a rotational machine, which helps to prevent sinking into energy minima (Stock et al., 1999) or assists in elastic power transmission between F 0 and F 1 (Junge et al., 2001). The idea of elastic power transmission was proposed as an explanation for the high efficiency of energy transmission during rotation (Cherepanov et al., 1999).

13 In general terms, the proposal is that energy can be stored during the rotational step by protein conformational torsion. Subsequently, the stored energy is gradually released and translated into ligand binding affinity changes. Flexibility of the b-subunit (Cain, 2000), unwinding of γ, and conformational transitions within β (Oster & Wang, 2000) could provide a physical basis for elastic energy storage. Unwinding of the γ coiled-coil region was predicted to occur during the rotation step (Ma et al., 2002; Cui et al., 2004). Hausrath et al. (2001) observed unwinding of the coiled-coil region of γ, when comparing crystals of E. coli F 1 to the bovine enzyme. It was concluded that unwinding of γ might well represent a physiological state related to elastic energy transmission (Capaldi & Aggeler, 2002). A displacement of ε in E. coli F 1 was also detected (Hausrath et al., 2001), with the structure of ε conforming to that in isolated E. coli γε complex (Rodgers & Wilce, 2000). A relatively large-scale motion in the C-terminal region of ε is predicted from comparison of various X-ray structures, which is consistent with crosslinking and immunological studies (Tsunoda et al., 2001a; Johnson & McCarty, 2002). On this basis the ε-subunit has been proposed to regulate ATP synthesis versus hydrolysis by acting as a ratchet to control the directionality of rotation (Tsunoda et al, 2001b; Capaldi & Aggeler, 2002). Figure I-5 *. Models of experiments on direct observations of rotation of F-ATPase. Explanations see it text. I-2.3. Concluding remarks for the Section I-2. To summarize, the available information on F-ATPase/ATP synthase characterizes this enzyme as a fairly complicated but highly efficient mechanochemical engine. In the case of ATP synthesis, rotation of the c-ring in the hydrophobic part of enzyme is triggered by the transmembrane electrochemical potential of H + or Na +, leading to the rotation of the tightly bound hydrophilic γε part. This triggers conformational changes in the inner part of the α 3 β 3 hexamer, where three alternating nucleotide binding sites are located, allowing ATP synthesis. In the case of ATP hydrolysis the direction of the γε rotation reverses, which allows proton (or Na + ) pumping across the membrane. The stability of the complex is provided by the stator, consisting of subunits δ and b, which span the length of F 1 F 0 from the top of the α 3 β 3 hexamer to the opposite side of the membrane. Extensive studies of the F-ATP synthase have led to understanding of the structural and functional integrity of the F 1 F 0 complex. The data on F-type ATP synthase complex arrangement and subunit interactions became a working model for understanding of the structure and mechanism of the genetically and functionally related A-type ATP synthases and V-ATPases, as will be explained in the next section. * From Stock et al.,

14 22 I-3. V-ATPase Vacuolar-type ATPases (V-ATPases) are membrane-bound proteins functioning in active ion pumping at the expense of ATP hydrolysis. They are present in the membranes of all eukaryotic cells and plasma membrane of some bacteria (Stevens & Forgac, 1997; Nelson & Harvey, 1999; Lolkema et al., 2003). V-ATPases first were described during studies on the energization of catecholamine uptake into chromaffin granules (Kirshner, 1962, Njus & Radda, 1978). It was demonstrated that an ATPase generates electrochemical gradient for their membranes by an ATPdependent proton uptake. Subsequently, the proton gradient drives the accumulation of catecholamines (Kanner & Schuldiner, 1987). Subsequently, it was found that a similar proton pump operates in the vacuoles of fungi and plants (Bowman et al., 1982; Kakinuma et al., 1981; Sze, 1985). A number of biochemical experiments identified the enzyme as the V-ATPase (Cidon & Nelson, 1983, Forgac et al., 1983, Stone et al., 1983). Isolation of the yeast V-ATPase (Uchida et al., 1985) enabled detailed molecular biology studies of its structure and properties (Nelson & Nelson, 1990). Subsequent cloning of genes encoding V-ATPase subunits provided evidence that the F- and V-ATPases are closely related and have evolved from a common ancestor (Nelson & Harvey, 1999). I-3.1. Structure of V-ATPase The structure of the V-ATPase has been intensively studied during the past few years. It has been shown that the overall structure is similar to that of the F-ATPases (Boekema et al., 1999; Wilkens et al., 1999; Domgall et al., 2002). V-ATPase consists of an extramembranous catalytic domain, the headpiece V 1, which is linked by means of a stalk region to a membrane-bound iontranslocating domain V 0 (Figure I-6, p. 36). The structure of the stalk region of the V-ATPase is more complex if compared to the F-ATPase: in V-ATPase it might consist of two or up to four stalks (Boekema et al., 1999; Domgall et al., 2002; Harrison et al., 2003; Wilkens et al., 2004). The nomenclature of the V-ATPase subunits is summarized in Table I-2. The V 1 domain of the V- ATPase contains eight subunits (A-H) in the case of the eukaryotic enzyme and seven subunits (A- G) in the prokaryotic one (Lolkema et al., 2003, Arata et al., 2002a). The stoichiometry of subunits was determined by quantitative amino acid analysis as A 3 B 3 C 1 D 1 E 1 F x G y H z (Arai et al., 1988). Biophysical, biochemical and structural evidence suggests that subunits H and F are present in single copies, while subunit G can form a dimer (Wilkens et al., 2004; Xu et al., 1999; Armbrüster et al., 2003). The ability to dissociate the V 1 subunits from the membrane using chaotropic agents in the absence of detergents identified these subunits as peripheral (Arai et al., 1989; Adachi et al., 1990). The V 1 domain is responsible for ATP hydrolysis. The catalytic nucleotide-binding sites are located on the A subunit, which is analogous to the β subunit of the F-ATPase. An additional nucleotide-binding site has been found in the B subunit, which is an analogue of the α subunit of the F-ATPase (Puopolo et al., 1992a). The stoichiometry and architecture of the A and B subunits in the V-ATPase is also identical to those of β and α subunits of F-ATPase (Figure I-6A, B, p. 36). They form a trimer consisting of pairs of A and B subunits. The precise function and location of other subunits of the V 1 -ATPase remains matter of debate. The V 0 domain is responsible for proton translocation across the membrane (Zhang et al., 1994). The eukaryotic V 0 consists of subunits a, c and d, corresponding to subunits I, K and C, respectively, in prokaryotic V-ATPase (Enterococcus hirae nomenclature). Subunit c (K) is represented in multiple copies; in eukaryotes one or two of copies are replaced by the isoforms c and c (Powell et

15 Figure I-1*. Model of E. coli F-ATP synthase/atpase based on X-ray crystallography, NMR data and cross-linking experiments. The enzyme consists of eight subunits α 3 β 3 γδεab 2 c n. F 1 corresponds to α 3 β 3 γδε and F 0 to ab 2 c n. The rotor consists of γε c n and the stator consists of α 3 β 3 δb 2 a. (A) models enzyme working as F-ATP synthase; (B) - as F-ATPase. White rectangles show stator interactions necessary to counteract rotor torque. For more details see text. A B C Figure I-3**. A model for the generation of rotation of the γ subunit during ATP hydrolysis in F 1 - ATPase based on X-ray crystallography data. The structures of the C-terminal domains of the α and β subunits are shown in red and yellow, respectively. The coiled-coil region of the γ subunit in violet. The nucleotide binding sites are shown in blue. For details see text. * From Weber & Senior, 2003, with modifications. ** From Menz et al.,

16 A B V 1 central stalk peripheral stalks Top view V 0 ion channel Side view Bottom view Figure I-6. Structure of V-ATPase based on electron microscopy data. (A) Side- and (B) top views of the V-ATPase from thermophilic bacterium Caloramator fervidus*. (C) Simulation of the bottom view based on 3-D maps of V 0 from bovine clathrin coated vesicles** and the V-ATPase proteolipid ring from the bacterium Enterococcus hirae ***. C * Ubbink-Kok et al., ** Wilkens & Forgac, *** Murata et al.,

17 al., 2000 ; Gibson et al., 2002). The stoichiometry of eukaryotic V 0 domain is a 1 d 1 c 1 (c,c ) 6 (Arai et al., 1988), while in the prokaryotic enzyme subunit K was found in 6-7 copies (Lolkema et al., 2003, Murata et al., 2003). Subunit d of the eukaryotic V 0 is tightly bound to the V 0 domain (Zhang et al., 1994), while the homologous subunit C of the prokaryotic enzyme might be associated with the V 1 domain (Ubbink-Kok et al., 2000). In plants, one other hydrophobic subunit, tentatively named VHA-e, is considered to be part of the V-ATPase (Sze et al., 2002) and in some mammalian tissues the enzyme contains an additional subunit termed Ac45 (Supek et al., 1994). Table I-2. Subunit composition of V-ATPases from different species in comparison to F-ATP synthase * Prokaryota V-ATPase Eukaryota E. hirae C. fervidus S. cerevisiae N. crassa Subunit function F-ATPase counterpart E. coli gene name mass** name mass gene name mass gene name mass name mass ntpa A 66 A 66 vma1 A 69 vma1 A 67 ATP hydrolysis β 55 ntpb B 51 B 51 vma2 B 60 vma2 B 57 interacts with A during catalysis ntpc C 38 C 37 vma6 d 36 vma6 d 41 bridges V 0 and V 1 rotor parts ntpd D 27 D 25.5 vma8 D 32 vma8 D 28 ntpe E 23 E 26 vma4 E 27 vma4 E 26 α not known, could be a coupling subunit γ(?) 31.5 ntpf F 14 F 12 vma10 G 13 vma10 G 13 not known b(?) 19 ntpg G 11 G 10 vma7 F 14 vma7 F 13 not known ntph H Na + /K + -antiport(?) ntpi I 76 I 61 vph1/vtv1 a 95 vph1 a? 98 H + or Na + channel a 30 ntpj J Na + /K + -antiport(?) ntpk K 16 K 17 vma3 c 17 vma3 c vma11 c 17 vma11 c vma16 c 23? c? H + or Na + binding c vma5 C 42? C 47 not known vma13 H 54 vma13 H 50 not known I-3.2. Comparison of F- and V-ATPases Biochemical and structural studies of the V-ATPase show its relation to the F-ATPase (for review see Nelson & Harvey, 1999). Some of the subunits of V-ATPases, namely A and B subunits of V 1, and c, c and c of V 0, are homologous to β, α and c subunits of the F-ATPase, respectively. On the basis of the sequence similarity and overall architecture, it was suggested that the catalytic mechanism of the two enzymes is also the same (Fillingame, 1996; Nishi & Forgac, 2002). ATP * For references see Forgac, 2000; Murata et al., 2001; Lolkema et al., ** Mass of subunits is given in kda. 25