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Conserved arginine residues implicated in ATP hydrolysis, nucleotide-sensing, and inter-subunit interactions in AAA and AAA+ ATPases
Journal of
Structural Biology Journal of Structural Biology 146 (2004) 106–112 www.elsevier.com/locate/yjsbi
Mini Review
Conserved arginine residues implicated in ATP hydrolysis, nucleotide-sensing, and inter-subunit interactions in AAA and AAAþ ATPases Teru Ogura,a,* Sidney W. Whiteheart,b and Anthony J. Wilkinsonc a
Division of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 862-0976, Japan b Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY, USA c Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5YW, UK Received 27 August 2003, and in revised form 6 November 2003
Abstract Arginines are a recurrent feature of the active sites and subunit interfaces of the ATPase domains of AAA and AAAþ proteins. In particular family members these residues occupy two or more, of four key sites in the vicinity of the ATP cofactor, where they transduce the chemical events of ATP binding and hydrolysis into a mechanochemical outcome. Structural and biochemical analyses have led to the proposal of molecular mechanisms in which these conserved arginines play crucial roles. Comparative studies, however, point to functional divergence for each of these conserved arginines. In this review, we will discuss what is known about these critical arginines and what can be concluded about their role in the function of AAA and AAAþ proteins. Ó 2003 Elsevier Inc. All rights reserved. Keywords: AAA family; ATPase; ATP hydrolysis; Inter-subunit interactions
1. Introduction AAAþ family proteins are involved in a variety of cellular activities including membrane fusion, proteolysis, assembly and disassembly of protein complexes, DNA replication, recombination, and transcriptional regulation (Neuwald et al., 1999; Ogura and Wilkinson, 2001). They share similarity both in sequence and 3D structure. The AAA proteins form a subfamily of the AAAþ family, their distinguishing characteristic being a highly conserved motif in the ATPase domain called the second region of homology (SRH) (Lupas and Martin, 2002; Ogura and Wilkinson, 2001). AAAþ proteins usually form hexameric rings of homo- or hetero-oligomers. Crystallographic and electron microscopic studies of representative members of the family have identified common structural features and it is thought that AAAþ proteins may follow a similar molecular
* Corresponding author. Fax: +81-96-373-6582. E-mail address: [email protected] (T. Ogura).
1047-8477/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2003.11.008
mechanism in achieving their functions. The ATPbinding pockets lie at subunit interfaces in the oligomer, suggesting that cooperation between adjacent subunits is integral to their mechanism of action. In this paper, we focus on four conserved arginine residues in AAA and AAAþ ATPase domains, which lie either in the ATP-binding pocket or at an inter-subunit interface. These are referred to here as position 1–4 arginines as indicated in Fig. 1. Arginine has an extended and flexible side chain with a planar and positively charged guanidino group at its extremity. The positive charge is distributed over three side chain nitrogens each of which can form hydrogen bonding/electrostatic interactions with groups of opposite charge/polarity, especially phosphate groups. Its multidentate character enables arginine to mediate long-range interactions in proteins connecting disparate residues/domains. It is therefore an ideal component of molecular motors and phosphorylation-driven signal transduction systems, where domain/subunit dynamics must be coupled to the changing phosphorylation state of adenine nucleotides or amino acid side chains, respectively.
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Fig. 1. Multiple sequence alignment of AAAþ ATPases highlighting conserved arginines. The alignment is according to Lupas et al. (1997), Neuwald et al. (1999), and Karata et al. (2001). Sequences are grouped into subfamilies. Walker A and B motifs and the second region of homology (SRH) are indicated. Conserved arginines are colored blue.
Fig. 2. Arginine nucleotide juxtaposition. The structures of full length p97 (p97-full; 1OZ4; DeLaBarre and Brunger, 2003), the N-D1 domains of p97 (p97-D1; 1E32; Zhang et al., 2000), and HslU (1G41; Sousa et al., 2000) were superposed by least squares minimization procedures applied to the Ca atoms of the P-loop residues. (A) The nucleotide (ADP for p97-full and p97-D1 or ATP for HslU) is displayed together with nearby arginines from the neighboring subunit in the case of Arg359 and Arg362 of p97-full and p97-D1 and Arg325 of HslU, or from the same subunit in the case of Arg393 in HslU. (B) Juxtaposition of the SRH arginines of p97-full and p97-D1 with ATP. The c-phosphate of the nucleotide is colored yellow to indicate that it has been modeled onto the ADP moiety in the crystal structure of p97-full. The panels were produced using the program MOLSCRIPT (Kraulis, 1991).
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Fig. 3. The ATP-binding site and intermolecular interactions involving conserved arginine residues. The figure shows a ribbon tracing of selected secondary structure elements taken from a pair of adjacent molecules from the modeled E. coli FtsH hexamer (1LV7; Krzywda et al., 2002). ATP and its associated magnesium ion, and a catalytic water molecule are drawn in yellow, green, and magenta, respectively. Conserved arginines (P1–P4; positions 1–4) and interacting acidic residues are drawn as sticks and colored blue (with the P3 Arg in cyan) and red, respectively. The Ala362 residue, which corresponds to position 3 arginine (P3 Arg) in AAAþ members (Fig. 1), is mutated to Arg in silico to mimic the position 3 arginineÕs interaction with the c-phosphate group of ATP. Dotted lines represent hydrogen bonds. Some helices and loops surrounding the core b-structure have been omitted for clarity.
Accumulating evidence suggests, not surprisingly, that the four conserved arginines of the AAAþ family are critical for activity. One is conserved in most subfamilies of the AAAþ family (position 1), two are conserved only in the AAA subfamily (positions 2 and 4), and the remaining one (position 3) is conserved in most subfamilies but not in the AAA subfamily (Fig. 1). As we discuss here, emerging data from studies of various family members indicate that, even though these arginines are strongly conserved, there is significant divergence in their roles in, and contributions to, ATP
binding, ATP hydrolysis and the coupling of these steps to the downstream mechanochemical events.
2. Position 1 arginine The arginine at position 1 is highly conserved in most AAAþ proteins with only minor exceptions. In the AAA subfamily, this is one of the highly conserved residues of the SRH (Fig. 1). Although there are two closely spaced arginines (positions 1 and 2) in the SRH, superposition
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of the p97 (AAA) and HslU (AAAþ ) hexamers suggests that the C-terminal arginine in the SRH (Arg362 in p97) corresponds to Arg325 in HslU (Fig. 2A). In the replication factor C (RFC; deltaÕ and gamma in Fig. 1) and minichromosome maintenance (MCM; Mcm3p in Fig. 1) subfamilies, it corresponds to the conserved arginine in the SRC (Ser–Arg–Cys) or SRF (Ser–Arg–Phe) motif. To avoid confusion it should be noted that the SRH in the AAA subfamily does not refer to a Ser–Arg– His sequence, instead it denotes the second region of homology as described above. The SRH spans 15–20 amino acid residues as shown in Fig. 1. In the crystal structures of AAAþ oligomers, the position 1 arginine is located in the ATP-binding pocket in such a way that it forms interactions with the c-phosphate of ATP bound to the adjacent subunit (Fig. 3). Two crystal structures of the p97 hexamer have been solved (DeLaBarre and Brunger, 2003; Zhang et al., 2000). In these structures, the orientations of the two conserved arginines (positions 1 and 2) in the SRH are significantly different (Fig. 2B). This indicates that the side chains of these arginines are highly flexible, and perhaps that their functions are overlapping and interchangeable as discussed later. To simplify the discussion in respect of comparisons with AAAþ proteins, we adopt the structure in which the position 1 arginine is closer to the c-phosphate. The interaction of the position 1 arginine with the c-phosphate of ATP bound to the adjacent subunit is reminiscent of the interactions of arginine ÔfingerÕ residues in GTPase-activating proteins (GAPs) with the c-phosphate of GTP bound to a small G protein partner. The arginine finger in some sense completes a GTPase active site, contributing to catalysis of GTP hydrolysis by forming intermolecular interactions with the phosphate groups in the transition state of this reaction. Thus, it was hypothesized that this arginine may perform a similar function in ATP hydrolysis mediated by AAAþ ATPases. This hypothesis was examined using site-directed mutagenesis for FtsH and NtrC. In both cases the results clearly established that the position 1 arginine is essential for ATP hydrolysis but not ATP binding (Table 1; Karata et al., 1999; Rombel et al., 1999), leading to the proposal of an intersubunit catalysis model for ATP hydrolysis. OÕDonnell and his colleagues explored the mechanism of ATP hydrolysis in the c complex (bacterial RFC homologs) and MCM proteins by site-directed mutagenesis of the position 1 arginines in the c-subunit (Johnson and OÕDonnell, 2003) and Mcm3p (Davey et al., 2003), respectively, and obtained unambiguous evidence to support inter-subunit catalysis of the ATPase reaction (Table 1). In particular, the mutation in Mcm3p abolished ATP hydrolysis by the partner Mcm7p. In HslU, the position 1 arginine may contribute to stabilization of subunit–subunit interactions in addition to ATP hydrolysis (Table 1; Song et al., 2000). Although the con-
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servation of the position 1 arginine was first noted in the AAAþ family (Lupas et al., 1997), it is a defining feature of all ring-forming ATPases with a RecA fold (Lupas and Martin, 2002). An interesting result was obtained following mutation of this arginine in N -ethylmaleimide-sensitive factor (NSF) (Matveeva et al., 2002). The mutant NSF retained ATPase activity and this activity was stimulated by the presence of soluble NSF attachment proteins (SNAP) to the same extent as that of the wild type protein, implying that the mutation of this arginine to an alanine does not interfere with ATP hydrolysis by NSF. Nevertheless, the mutant NSF was inactive in SNAP receptor (SNARE) disassembly. These results indicate that the position 1 arginine of NSF is not essential for ATP hydrolysis but that it is critical for biological activity, suggesting that it senses nucleotides and, perhaps, contributes to the coupling of the free energy of ATP hydrolysis to the conformational changes necessary to achieve biological function (Table 1).
3. Position 2 arginine In the AAA subfamily, there is a second highly conserved arginine residue within the SRH adjacent to the arginine finger (Fig. 1) as described above. Mutations of this position 2 arginine in FtsH also abolished ATPase activity. A mutation of the position 2 arginine in NSF abolished the SNARE disassembling activity without having any effect on ATP hydrolysis, a phenotype similar to that of the position 1 mutant. Thus, it could function as a second arginine finger as discussed in more detail later. The position 2 arginine is usually separated by two amino acid residues, often Pro–Gly, from the position 1 arginine finger (Fig. 1). In some AAA proteins, such as katanin and Vps4p/ SKD1, the Pro–Gly sequence is absent and the two arginines are adjacent (Fig. 1). Deletion of the Pro–Gly sequence of FtsH did not significantly affect activity (Karata et al., 2001). Modeling studies suggested that deletion of these residues will not significantly alter the conformation of the ATP-binding pocket region or of the two conserved arginines. A temperature-sensitive mutant of Drosophila NSF1 (comtTP7 ), has been found to have a Ser substitution at the Pro residue of the Pro–Gly sequence (Kawasaki et al., 1998), implying that the Pro to Ser substitution may alter the conformation of this region, perhaps affecting the orientation of the position 1 and/or 2 arginines. The position 2 arginine is usually absent in other AAAþ subfamilies (Fig. 1). The functional importance of this arginine in those AAAþ members that do possess it (class 1 members of the Clp/Hsp100 subfamily; Fig. 1) has not been examined.
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Table 1 Functions of the conserved arginine residues in the AAA and AAAþ modules Positions (motifs)
Mutations
Phenotypes
References
Functions
Position 1 (SRH in AAA, SRC/SRF in RFC and MCM)
R315A/L/K of FtsH
Loss of ATP hydrolysis
Karata et al. (1999)
ATP hydrolysis (arginine finger; stabilization of transition state of ATP)
R169A of gamma
Loss of ATP hydrolysis
R542A of Mcm3p
Loss of ATP hydrolysis by Mcm7p Loss of ATP hydrolysis but normal oligomerization Loss of ATP hydrolysis Dominant negative Loss of ATP hydrolysis and hexamerization Loss of SNARE disassembly but not ATP hydrolysis
Johnson and OÕDonnell (2003) Davey et al. (2003)
R294C of NtrC R170A/K of TmRuvB R174H of RuvB R325E of HslU R388A of NSF Position 2 (second Arg in SRH)
Position 3 (sensor 2)
Putnam et al. (2001) Iwasaki et al. (2000) Song et al. (2000) Matveeva et al. (2002)
Loss of ATP hydrolysis
R385A of NSF
Loss of SNARE disassembly Matveeva et al. (2002) but not ATP hydrolysis
R334A/H of DnaA
Loss of ATP hydrolysis, overinitiation Loss of ATP hydrolysis Loss of ATP hydrolysis Loss or decrease of ATP hydrolysis Dominant negative Loss of ATP hydrolysis, ATP-independent nonfunctional multimerization Reduced affinity for ATP and ADP Loss of ATP binding Loss of ATP binding Loss or decrease of ATP binding Inefficient MCM loading
Nishida et al. (2002) and SuÕetsugu et al. (2001) Song et al. (2000) Hansson et al. (2002) Putnam et al. (2001)
ATP hydrolysis or sensing?
Iwasaki et al. (2000) Perez-Martin and de Lorenzo (1996)
ATP hydrolysis, sensing? Inter-subunit interaction?
Loss or decrease of protease activity
Karata et al. (2001)
R221A of RuvB R453H of XylR
R826M of Hsp104 R358C/H of NtrC R453C/H of DmpR R453C/H of PspF R332A/E of Cdc6p R263D/E/K of FtsH
Karata et al. (1999)
ATP hydrolysis, inter-subunit interaction Nucleotide sensing, energy coupling
R312A/L/K of FtsH
R393A of HslU R289K of BchI R217A/K of TmRuvB
Position 4 (downstream of Walker B)
Rombel et al. (1999)
Hattendorf and Lindquist (2002) Rombel et al. (1999) Wikstr€ om et al. (2001) Lew and Gralla (2002)
ATP hydrolysis (second arginine finger) Nucleotide sensing, energy coupling ATP hydrolysis
Nucleotide binding, sensing
Schepers and Diffley (2001) and Takahashi et al. (2002) Structural stability, inter-subunit interaction?
For origins (species) of AAAþ proteins listed in this table, refer to references indicated in the table. Since RuvB proteins from two different species are listed in this table, RuvB from E. coli and Thermotoga maritima are indicated as RuvB and TmRuvB to avoid confusion.
4. Position 3 arginine There is a conserved arginine in the C-terminal a-helical domain of most AAAþ members that is generally not present in the AAA subfamily members (Fig. 1). The arginine at this position is referred to as Ôsensor 2Õ (Guenther et al., 1997) by analogy with an arginine in adenylate kinase which mediates conformational changes upon ATP binding (M€ uller and Schulz, 1992). Many mutants of this arginine have been isolated for AAAþ proteins which function as transcriptional activators such as NtrC, PspF, DmpR, and XylR, which remodel r54 and are thus referred to as r54 activators (Table 1). Zhang et al. (2002) have discussed the function of the sensor 2 arginines in r54 activators. Briefly, it
has been shown that mutations in the sensor 2 arginine result in defective ATP hydrolysis, nucleotide-sensing, and/or inter-subunit interaction. Two points here are worthy of note. First, in most cases, mutations at this position diminish ATP binding (Table 1; Lew and Gralla, 2002; Rombel et al., 1999; Wikstr€ om et al., 2001), implying that this arginine is an important part of the ATP-binding pocket. A similar result has also been obtained with a corresponding mutant of Hsp104, in which the affinity for both ATP and ADP was greatly reduced (Hattendorf and Lindquist, 2002). Second, an R453H mutant of XylR oligomerizes in the absence of ATP, though wild type XylR requires ATP for oligomerization (Perez-Martin and de Lorenzo, 1996). However, this oligomer is unable to activate r54 and the
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mutant can bind ATP but not hydrolyze it (Table 1). Results from mutants of the replication initiator protein, DnaA, are interesting. The ATP-bound form of DnaA is active in the initiation of DNA replication, while the ADP-bound form is not. Sensor 2 arginine mutants of DnaA cause overinitiation of replication due to defects in ATP hydrolysis but not ATP binding (Table 1; Nishida et al., 2002; SuÕetsugu et al., 2001). Taken together, these results suggest that the function of the sensor 2 arginine may be rather divergent and that it may play multiple roles including ATP binding, hydrolysis, and inter-subunit interaction as well as nucleotide sensing. Positions 2 and 3 arginines are mutually exclusive in AAAþ proteins (Fig. 1). This is vividly illustrated in class 1 members of the Clp/Hsp100 subfamily, which contain tandem AAAþ modules; the first D1, has a position 2 arginine in common with the AAA subfamily while the second D2, has a position 3 arginine akin to other AAAþ subfamilies (Lupas et al., 1997; Schirmer et al., 1996; http://finley.med.harvard.edu/ubiquitin/). These data point to an interesting mechanical divergence of the two AAAþ modules in the class 1 members of the Clp/Hsp100 subfamily. The above considerations emphasize that AAAþ ATPases usually have a pair of conserved arginines near the ATP-binding pocket, both of which may interact with the c-phosphate of ATP. In a recently proposed mechanism of action for F1 -ATPase, a typical RecA fold ATPase as well as an AAAþ ATPase (Weber and Senior, 2003), it has been suggested that a pair of conserved arginines, one from the b-subunit and the other from the neighboring a-subunit, are involved in ATP hydrolysis by stabilizing the catalytic transition state cooperatively. Thus, the arginine from the a-subunit functions as an arginine finger. It seems reasonable to assume that other AAAþ ATPases use a similar mechanism of ATP hydrolysis. As discussed above, both of these arginines (positions 1 and 2) in the AAA subfamily are located in the SRH of the neighboring subunit. Therefore, both these arginines may function as arginine fingers and their relative positions may be interchangeable as suggested by the two crystal structures of the p97 hexamer as already discussed. Perhaps, in some AAAþ ATPases only one of these two arginines is required for ATP hydrolysis, allowing the other to ÔdivergeÕ in a mechanistic sense and evolve other contributions to function.
All of the four conserved arginines in AAA and AAAþ modules have crucial functions in these ATPases, as expected from their degree of conservation. Their importance can be rationalized by examination of the recently determined crystal structures of several AAA/ AAAþ ATPases. Site-directed mutagenesis studies of the various AAA/AAAþ ATPases discussed above lead to the somewhat surprising conclusion that the catalytic functions of these conserved arginines may not always be identical. The data discussed suggest that these critical residues play divergent, context-specific roles in the mechanochemical cycle of this family of ATPases. Further structural and biochemical studies are necessary. In particular, high resolution crystal structures of different nucleotide states including complexes with transition state analogs and comparative biochemical analysis are needed to elucidate each arginineÕs function. Therefore, it is wise to be cautious in assigning generic functions based on the homology alone even for residues which are highly conserved.
5. Position 4 arginine
Acknowledgments
In the AAA subfamily but not in other AAAþ subfamilies, there is an additional conserved arginine downstream of the Walker B motif (Fig. 1). Although this arginine is remote from the ATP-binding pocket (Fig. 3), mutation of this residue to alanine in FtsH
We thank one of the reviewers for particularly helpful input. This work was supported in part by grants from the Ministry of Education, Culture, Science, Sports and Technology, Japan to T.O., from the Japan Society for the Promotion of Science to T.O., from the National
abolishes ATPase activity (Table 1; Karata et al., 2001). From the modeled hexamer (Fig. 3), it seems likely that this arginine makes a salt-bridge with a conserved aspartate (Asp307 in FtsH) in the SRH of the neighboring subunit. Mutation of Asp307 to alanine also leads to loss of activity. Thus, we propose that the position 4 arginine of FtsH stabilizes the FtsH hexamer and facilitates efficient ATP hydrolysis. In support of this possibility, two mutations in which charge is retained, R263K and D307E, exhibit residual activity (Karata et al., 1999, 2001). In the crystal structure of the ADPbound p97 hexamer (DeLaBarre and Brunger, 2003), the corresponding Arg and Asp residues are not adjacent and thus the proposed Arg–Asp salt-bridge is not formed. However, cryo-EM studies have revealed dynamic conformational changes of p97 during the cycle of ATP hydrolysis (Beuron et al., 2003; Rouiller et al., 2002). At a certain stage of the cycle, they may form a salt-bridge and this interaction may be important for progression of the cycle. Therefore, although both biochemical and modeling data for FtsH support a hexamer stabilization role for the position 4 arginine, this assertion remains speculative.
6. Conclusions
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Institutes of Health (HL56652) to S.W.W., and from the BBSRC, UK (Grant 87/B13998) to A.J.W.
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Structural Biology Journal of Structural Biology 146 (2004) 106–112 www.elsevier.com/locate/yjsbi
Mini Review
Conserved arginine residues implicated in ATP hydrolysis, nucleotide-sensing, and inter-subunit interactions in AAA and AAAþ ATPases Teru Ogura,a,* Sidney W. Whiteheart,b and Anthony J. Wilkinsonc a
Division of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 862-0976, Japan b Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY, USA c Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5YW, UK Received 27 August 2003, and in revised form 6 November 2003
Abstract Arginines are a recurrent feature of the active sites and subunit interfaces of the ATPase domains of AAA and AAAþ proteins. In particular family members these residues occupy two or more, of four key sites in the vicinity of the ATP cofactor, where they transduce the chemical events of ATP binding and hydrolysis into a mechanochemical outcome. Structural and biochemical analyses have led to the proposal of molecular mechanisms in which these conserved arginines play crucial roles. Comparative studies, however, point to functional divergence for each of these conserved arginines. In this review, we will discuss what is known about these critical arginines and what can be concluded about their role in the function of AAA and AAAþ proteins. Ó 2003 Elsevier Inc. All rights reserved. Keywords: AAA family; ATPase; ATP hydrolysis; Inter-subunit interactions
1. Introduction AAAþ family proteins are involved in a variety of cellular activities including membrane fusion, proteolysis, assembly and disassembly of protein complexes, DNA replication, recombination, and transcriptional regulation (Neuwald et al., 1999; Ogura and Wilkinson, 2001). They share similarity both in sequence and 3D structure. The AAA proteins form a subfamily of the AAAþ family, their distinguishing characteristic being a highly conserved motif in the ATPase domain called the second region of homology (SRH) (Lupas and Martin, 2002; Ogura and Wilkinson, 2001). AAAþ proteins usually form hexameric rings of homo- or hetero-oligomers. Crystallographic and electron microscopic studies of representative members of the family have identified common structural features and it is thought that AAAþ proteins may follow a similar molecular
* Corresponding author. Fax: +81-96-373-6582. E-mail address: [email protected] (T. Ogura).
1047-8477/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2003.11.008
mechanism in achieving their functions. The ATPbinding pockets lie at subunit interfaces in the oligomer, suggesting that cooperation between adjacent subunits is integral to their mechanism of action. In this paper, we focus on four conserved arginine residues in AAA and AAAþ ATPase domains, which lie either in the ATP-binding pocket or at an inter-subunit interface. These are referred to here as position 1–4 arginines as indicated in Fig. 1. Arginine has an extended and flexible side chain with a planar and positively charged guanidino group at its extremity. The positive charge is distributed over three side chain nitrogens each of which can form hydrogen bonding/electrostatic interactions with groups of opposite charge/polarity, especially phosphate groups. Its multidentate character enables arginine to mediate long-range interactions in proteins connecting disparate residues/domains. It is therefore an ideal component of molecular motors and phosphorylation-driven signal transduction systems, where domain/subunit dynamics must be coupled to the changing phosphorylation state of adenine nucleotides or amino acid side chains, respectively.
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Fig. 1. Multiple sequence alignment of AAAþ ATPases highlighting conserved arginines. The alignment is according to Lupas et al. (1997), Neuwald et al. (1999), and Karata et al. (2001). Sequences are grouped into subfamilies. Walker A and B motifs and the second region of homology (SRH) are indicated. Conserved arginines are colored blue.
Fig. 2. Arginine nucleotide juxtaposition. The structures of full length p97 (p97-full; 1OZ4; DeLaBarre and Brunger, 2003), the N-D1 domains of p97 (p97-D1; 1E32; Zhang et al., 2000), and HslU (1G41; Sousa et al., 2000) were superposed by least squares minimization procedures applied to the Ca atoms of the P-loop residues. (A) The nucleotide (ADP for p97-full and p97-D1 or ATP for HslU) is displayed together with nearby arginines from the neighboring subunit in the case of Arg359 and Arg362 of p97-full and p97-D1 and Arg325 of HslU, or from the same subunit in the case of Arg393 in HslU. (B) Juxtaposition of the SRH arginines of p97-full and p97-D1 with ATP. The c-phosphate of the nucleotide is colored yellow to indicate that it has been modeled onto the ADP moiety in the crystal structure of p97-full. The panels were produced using the program MOLSCRIPT (Kraulis, 1991).
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Fig. 3. The ATP-binding site and intermolecular interactions involving conserved arginine residues. The figure shows a ribbon tracing of selected secondary structure elements taken from a pair of adjacent molecules from the modeled E. coli FtsH hexamer (1LV7; Krzywda et al., 2002). ATP and its associated magnesium ion, and a catalytic water molecule are drawn in yellow, green, and magenta, respectively. Conserved arginines (P1–P4; positions 1–4) and interacting acidic residues are drawn as sticks and colored blue (with the P3 Arg in cyan) and red, respectively. The Ala362 residue, which corresponds to position 3 arginine (P3 Arg) in AAAþ members (Fig. 1), is mutated to Arg in silico to mimic the position 3 arginineÕs interaction with the c-phosphate group of ATP. Dotted lines represent hydrogen bonds. Some helices and loops surrounding the core b-structure have been omitted for clarity.
Accumulating evidence suggests, not surprisingly, that the four conserved arginines of the AAAþ family are critical for activity. One is conserved in most subfamilies of the AAAþ family (position 1), two are conserved only in the AAA subfamily (positions 2 and 4), and the remaining one (position 3) is conserved in most subfamilies but not in the AAA subfamily (Fig. 1). As we discuss here, emerging data from studies of various family members indicate that, even though these arginines are strongly conserved, there is significant divergence in their roles in, and contributions to, ATP
binding, ATP hydrolysis and the coupling of these steps to the downstream mechanochemical events.
2. Position 1 arginine The arginine at position 1 is highly conserved in most AAAþ proteins with only minor exceptions. In the AAA subfamily, this is one of the highly conserved residues of the SRH (Fig. 1). Although there are two closely spaced arginines (positions 1 and 2) in the SRH, superposition
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of the p97 (AAA) and HslU (AAAþ ) hexamers suggests that the C-terminal arginine in the SRH (Arg362 in p97) corresponds to Arg325 in HslU (Fig. 2A). In the replication factor C (RFC; deltaÕ and gamma in Fig. 1) and minichromosome maintenance (MCM; Mcm3p in Fig. 1) subfamilies, it corresponds to the conserved arginine in the SRC (Ser–Arg–Cys) or SRF (Ser–Arg–Phe) motif. To avoid confusion it should be noted that the SRH in the AAA subfamily does not refer to a Ser–Arg– His sequence, instead it denotes the second region of homology as described above. The SRH spans 15–20 amino acid residues as shown in Fig. 1. In the crystal structures of AAAþ oligomers, the position 1 arginine is located in the ATP-binding pocket in such a way that it forms interactions with the c-phosphate of ATP bound to the adjacent subunit (Fig. 3). Two crystal structures of the p97 hexamer have been solved (DeLaBarre and Brunger, 2003; Zhang et al., 2000). In these structures, the orientations of the two conserved arginines (positions 1 and 2) in the SRH are significantly different (Fig. 2B). This indicates that the side chains of these arginines are highly flexible, and perhaps that their functions are overlapping and interchangeable as discussed later. To simplify the discussion in respect of comparisons with AAAþ proteins, we adopt the structure in which the position 1 arginine is closer to the c-phosphate. The interaction of the position 1 arginine with the c-phosphate of ATP bound to the adjacent subunit is reminiscent of the interactions of arginine ÔfingerÕ residues in GTPase-activating proteins (GAPs) with the c-phosphate of GTP bound to a small G protein partner. The arginine finger in some sense completes a GTPase active site, contributing to catalysis of GTP hydrolysis by forming intermolecular interactions with the phosphate groups in the transition state of this reaction. Thus, it was hypothesized that this arginine may perform a similar function in ATP hydrolysis mediated by AAAþ ATPases. This hypothesis was examined using site-directed mutagenesis for FtsH and NtrC. In both cases the results clearly established that the position 1 arginine is essential for ATP hydrolysis but not ATP binding (Table 1; Karata et al., 1999; Rombel et al., 1999), leading to the proposal of an intersubunit catalysis model for ATP hydrolysis. OÕDonnell and his colleagues explored the mechanism of ATP hydrolysis in the c complex (bacterial RFC homologs) and MCM proteins by site-directed mutagenesis of the position 1 arginines in the c-subunit (Johnson and OÕDonnell, 2003) and Mcm3p (Davey et al., 2003), respectively, and obtained unambiguous evidence to support inter-subunit catalysis of the ATPase reaction (Table 1). In particular, the mutation in Mcm3p abolished ATP hydrolysis by the partner Mcm7p. In HslU, the position 1 arginine may contribute to stabilization of subunit–subunit interactions in addition to ATP hydrolysis (Table 1; Song et al., 2000). Although the con-
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servation of the position 1 arginine was first noted in the AAAþ family (Lupas et al., 1997), it is a defining feature of all ring-forming ATPases with a RecA fold (Lupas and Martin, 2002). An interesting result was obtained following mutation of this arginine in N -ethylmaleimide-sensitive factor (NSF) (Matveeva et al., 2002). The mutant NSF retained ATPase activity and this activity was stimulated by the presence of soluble NSF attachment proteins (SNAP) to the same extent as that of the wild type protein, implying that the mutation of this arginine to an alanine does not interfere with ATP hydrolysis by NSF. Nevertheless, the mutant NSF was inactive in SNAP receptor (SNARE) disassembly. These results indicate that the position 1 arginine of NSF is not essential for ATP hydrolysis but that it is critical for biological activity, suggesting that it senses nucleotides and, perhaps, contributes to the coupling of the free energy of ATP hydrolysis to the conformational changes necessary to achieve biological function (Table 1).
3. Position 2 arginine In the AAA subfamily, there is a second highly conserved arginine residue within the SRH adjacent to the arginine finger (Fig. 1) as described above. Mutations of this position 2 arginine in FtsH also abolished ATPase activity. A mutation of the position 2 arginine in NSF abolished the SNARE disassembling activity without having any effect on ATP hydrolysis, a phenotype similar to that of the position 1 mutant. Thus, it could function as a second arginine finger as discussed in more detail later. The position 2 arginine is usually separated by two amino acid residues, often Pro–Gly, from the position 1 arginine finger (Fig. 1). In some AAA proteins, such as katanin and Vps4p/ SKD1, the Pro–Gly sequence is absent and the two arginines are adjacent (Fig. 1). Deletion of the Pro–Gly sequence of FtsH did not significantly affect activity (Karata et al., 2001). Modeling studies suggested that deletion of these residues will not significantly alter the conformation of the ATP-binding pocket region or of the two conserved arginines. A temperature-sensitive mutant of Drosophila NSF1 (comtTP7 ), has been found to have a Ser substitution at the Pro residue of the Pro–Gly sequence (Kawasaki et al., 1998), implying that the Pro to Ser substitution may alter the conformation of this region, perhaps affecting the orientation of the position 1 and/or 2 arginines. The position 2 arginine is usually absent in other AAAþ subfamilies (Fig. 1). The functional importance of this arginine in those AAAþ members that do possess it (class 1 members of the Clp/Hsp100 subfamily; Fig. 1) has not been examined.
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Table 1 Functions of the conserved arginine residues in the AAA and AAAþ modules Positions (motifs)
Mutations
Phenotypes
References
Functions
Position 1 (SRH in AAA, SRC/SRF in RFC and MCM)
R315A/L/K of FtsH
Loss of ATP hydrolysis
Karata et al. (1999)
ATP hydrolysis (arginine finger; stabilization of transition state of ATP)
R169A of gamma
Loss of ATP hydrolysis
R542A of Mcm3p
Loss of ATP hydrolysis by Mcm7p Loss of ATP hydrolysis but normal oligomerization Loss of ATP hydrolysis Dominant negative Loss of ATP hydrolysis and hexamerization Loss of SNARE disassembly but not ATP hydrolysis
Johnson and OÕDonnell (2003) Davey et al. (2003)
R294C of NtrC R170A/K of TmRuvB R174H of RuvB R325E of HslU R388A of NSF Position 2 (second Arg in SRH)
Position 3 (sensor 2)
Putnam et al. (2001) Iwasaki et al. (2000) Song et al. (2000) Matveeva et al. (2002)
Loss of ATP hydrolysis
R385A of NSF
Loss of SNARE disassembly Matveeva et al. (2002) but not ATP hydrolysis
R334A/H of DnaA
Loss of ATP hydrolysis, overinitiation Loss of ATP hydrolysis Loss of ATP hydrolysis Loss or decrease of ATP hydrolysis Dominant negative Loss of ATP hydrolysis, ATP-independent nonfunctional multimerization Reduced affinity for ATP and ADP Loss of ATP binding Loss of ATP binding Loss or decrease of ATP binding Inefficient MCM loading
Nishida et al. (2002) and SuÕetsugu et al. (2001) Song et al. (2000) Hansson et al. (2002) Putnam et al. (2001)
ATP hydrolysis or sensing?
Iwasaki et al. (2000) Perez-Martin and de Lorenzo (1996)
ATP hydrolysis, sensing? Inter-subunit interaction?
Loss or decrease of protease activity
Karata et al. (2001)
R221A of RuvB R453H of XylR
R826M of Hsp104 R358C/H of NtrC R453C/H of DmpR R453C/H of PspF R332A/E of Cdc6p R263D/E/K of FtsH
Karata et al. (1999)
ATP hydrolysis, inter-subunit interaction Nucleotide sensing, energy coupling
R312A/L/K of FtsH
R393A of HslU R289K of BchI R217A/K of TmRuvB
Position 4 (downstream of Walker B)
Rombel et al. (1999)
Hattendorf and Lindquist (2002) Rombel et al. (1999) Wikstr€ om et al. (2001) Lew and Gralla (2002)
ATP hydrolysis (second arginine finger) Nucleotide sensing, energy coupling ATP hydrolysis
Nucleotide binding, sensing
Schepers and Diffley (2001) and Takahashi et al. (2002) Structural stability, inter-subunit interaction?
For origins (species) of AAAþ proteins listed in this table, refer to references indicated in the table. Since RuvB proteins from two different species are listed in this table, RuvB from E. coli and Thermotoga maritima are indicated as RuvB and TmRuvB to avoid confusion.
4. Position 3 arginine There is a conserved arginine in the C-terminal a-helical domain of most AAAþ members that is generally not present in the AAA subfamily members (Fig. 1). The arginine at this position is referred to as Ôsensor 2Õ (Guenther et al., 1997) by analogy with an arginine in adenylate kinase which mediates conformational changes upon ATP binding (M€ uller and Schulz, 1992). Many mutants of this arginine have been isolated for AAAþ proteins which function as transcriptional activators such as NtrC, PspF, DmpR, and XylR, which remodel r54 and are thus referred to as r54 activators (Table 1). Zhang et al. (2002) have discussed the function of the sensor 2 arginines in r54 activators. Briefly, it
has been shown that mutations in the sensor 2 arginine result in defective ATP hydrolysis, nucleotide-sensing, and/or inter-subunit interaction. Two points here are worthy of note. First, in most cases, mutations at this position diminish ATP binding (Table 1; Lew and Gralla, 2002; Rombel et al., 1999; Wikstr€ om et al., 2001), implying that this arginine is an important part of the ATP-binding pocket. A similar result has also been obtained with a corresponding mutant of Hsp104, in which the affinity for both ATP and ADP was greatly reduced (Hattendorf and Lindquist, 2002). Second, an R453H mutant of XylR oligomerizes in the absence of ATP, though wild type XylR requires ATP for oligomerization (Perez-Martin and de Lorenzo, 1996). However, this oligomer is unable to activate r54 and the
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mutant can bind ATP but not hydrolyze it (Table 1). Results from mutants of the replication initiator protein, DnaA, are interesting. The ATP-bound form of DnaA is active in the initiation of DNA replication, while the ADP-bound form is not. Sensor 2 arginine mutants of DnaA cause overinitiation of replication due to defects in ATP hydrolysis but not ATP binding (Table 1; Nishida et al., 2002; SuÕetsugu et al., 2001). Taken together, these results suggest that the function of the sensor 2 arginine may be rather divergent and that it may play multiple roles including ATP binding, hydrolysis, and inter-subunit interaction as well as nucleotide sensing. Positions 2 and 3 arginines are mutually exclusive in AAAþ proteins (Fig. 1). This is vividly illustrated in class 1 members of the Clp/Hsp100 subfamily, which contain tandem AAAþ modules; the first D1, has a position 2 arginine in common with the AAA subfamily while the second D2, has a position 3 arginine akin to other AAAþ subfamilies (Lupas et al., 1997; Schirmer et al., 1996; http://finley.med.harvard.edu/ubiquitin/). These data point to an interesting mechanical divergence of the two AAAþ modules in the class 1 members of the Clp/Hsp100 subfamily. The above considerations emphasize that AAAþ ATPases usually have a pair of conserved arginines near the ATP-binding pocket, both of which may interact with the c-phosphate of ATP. In a recently proposed mechanism of action for F1 -ATPase, a typical RecA fold ATPase as well as an AAAþ ATPase (Weber and Senior, 2003), it has been suggested that a pair of conserved arginines, one from the b-subunit and the other from the neighboring a-subunit, are involved in ATP hydrolysis by stabilizing the catalytic transition state cooperatively. Thus, the arginine from the a-subunit functions as an arginine finger. It seems reasonable to assume that other AAAþ ATPases use a similar mechanism of ATP hydrolysis. As discussed above, both of these arginines (positions 1 and 2) in the AAA subfamily are located in the SRH of the neighboring subunit. Therefore, both these arginines may function as arginine fingers and their relative positions may be interchangeable as suggested by the two crystal structures of the p97 hexamer as already discussed. Perhaps, in some AAAþ ATPases only one of these two arginines is required for ATP hydrolysis, allowing the other to ÔdivergeÕ in a mechanistic sense and evolve other contributions to function.
All of the four conserved arginines in AAA and AAAþ modules have crucial functions in these ATPases, as expected from their degree of conservation. Their importance can be rationalized by examination of the recently determined crystal structures of several AAA/ AAAþ ATPases. Site-directed mutagenesis studies of the various AAA/AAAþ ATPases discussed above lead to the somewhat surprising conclusion that the catalytic functions of these conserved arginines may not always be identical. The data discussed suggest that these critical residues play divergent, context-specific roles in the mechanochemical cycle of this family of ATPases. Further structural and biochemical studies are necessary. In particular, high resolution crystal structures of different nucleotide states including complexes with transition state analogs and comparative biochemical analysis are needed to elucidate each arginineÕs function. Therefore, it is wise to be cautious in assigning generic functions based on the homology alone even for residues which are highly conserved.
5. Position 4 arginine
Acknowledgments
In the AAA subfamily but not in other AAAþ subfamilies, there is an additional conserved arginine downstream of the Walker B motif (Fig. 1). Although this arginine is remote from the ATP-binding pocket (Fig. 3), mutation of this residue to alanine in FtsH
We thank one of the reviewers for particularly helpful input. This work was supported in part by grants from the Ministry of Education, Culture, Science, Sports and Technology, Japan to T.O., from the Japan Society for the Promotion of Science to T.O., from the National
abolishes ATPase activity (Table 1; Karata et al., 2001). From the modeled hexamer (Fig. 3), it seems likely that this arginine makes a salt-bridge with a conserved aspartate (Asp307 in FtsH) in the SRH of the neighboring subunit. Mutation of Asp307 to alanine also leads to loss of activity. Thus, we propose that the position 4 arginine of FtsH stabilizes the FtsH hexamer and facilitates efficient ATP hydrolysis. In support of this possibility, two mutations in which charge is retained, R263K and D307E, exhibit residual activity (Karata et al., 1999, 2001). In the crystal structure of the ADPbound p97 hexamer (DeLaBarre and Brunger, 2003), the corresponding Arg and Asp residues are not adjacent and thus the proposed Arg–Asp salt-bridge is not formed. However, cryo-EM studies have revealed dynamic conformational changes of p97 during the cycle of ATP hydrolysis (Beuron et al., 2003; Rouiller et al., 2002). At a certain stage of the cycle, they may form a salt-bridge and this interaction may be important for progression of the cycle. Therefore, although both biochemical and modeling data for FtsH support a hexamer stabilization role for the position 4 arginine, this assertion remains speculative.
6. Conclusions
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Institutes of Health (HL56652) to S.W.W., and from the BBSRC, UK (Grant 87/B13998) to A.J.W.
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