AAA-ATPases

A AAA-ATPases J Martin and A N Lupas, Max Planck Institute for Developmental Biology, Tu¨bingen, Germany ã 2013 Elsevier Inc. All rights reserved.

Glossary AAA proteins A protein family of oligomeric ATPases with diverse cellular activities. AAAþ proteins A superfamily that encompasses, among other families, the AAA proteins. Arginine finger A conserved arginine residue in the SRH that points into the nucleotide-binding pocket. Clade A group of organisms or molecules, whose members share homologous features derived from a common ancestor. Cluster analysis A data analysis tool for solving classification problems. Organisms or molecules are sorted into clusters, so that the degree of association is strong between members of the same and weak between members

AAA Proteins Complex cellular events commonly depend on the activity of molecular machines that efficiently couple enzymatic and regulatory functions within a multiprotein assembly. As these functions require energy, adenosine triphosphatases (ATPases) are often found as integral components in these assemblies. All ATPases harness energy gained from breaking the g-phosphate bond of ATP. An essential and expanding subset in the areas of protein remodeling and quality control comprises proteins of the AAA family.

Structure and Mechanism of Action AAA proteins, which belong to the superfamily of P-loop nucleoside triphosphatases (NTPases), share the common feature of a highly conserved, extended nucleotidase domain containing 200–250 residues. This so-called AAA domain is formed by two subdomains, an amino-terminal nucleotidebinding subdomain and a smaller carboxy-terminal helical subdomain. Comparison to the structures of other AAAþ proteins reveals that this subdomain arrangement represents the common structural core of the AAAþ superfamily (see later). The nucleotide-binding subdomain contains two classical Walker A (GX4GKT) and Walker B (HyDE) motifs, which are involved in binding of the triphosphate moiety of the

of different clusters. Each cluster thus describes the class to which its members belong. P-loop NTPases Nucleotidases that are characterized by the presence of a loop element that is associated with the phosphates of bound nucleotides. Second region of homology (SRH) A hallmark of the AAA family. It is a conserved sequence region containing sensor 1 residue and arginine finger. Sensor 1 and 2 Conserved amino acid residues in AAAþ proteins that contact bound nucleotide; sensor 1 is a polar residue, sensor 2 typically arginine. Walker A and B motifs Consensus sequence elements that are characteristic of nucleotide-binding folds.

nucleotide and coordination of a Mg2þ-ion, which is important for subsequent hydrolysis of ATP. It is a RecA-like, threelayered aba structure with a central five-stranded, parallel b sheet. The property that separates this fold from that of all other P-loop NTPases is the insertion of a b strand which results in the addition of a conserved polar residue to the active site. The residue has been named sensor-1 for the fact that it contacts the g-phosphate of the bound nucleotide and is thought to discriminate between ATP and ADP. Indeed, mutagenesis of this residue highlights its critical role in cooperative ATP hydrolysis. The so-called second region of homology (SRH), in which sensor-1 lies, is the hallmark of the AAA family and makes it distinguishable from the larger and more diverse superfamily of AAAþ proteins. The carboxy-terminal subdomain is largely helical and shows substantially greater structural variation, although it invariably occupies a similar position, diagonally above the base of the bound nucleotide. A recurring feature of this subdomain is a residue (typically arginine), called sensor-2 for its ability to interact with the nucleotide. Depending on the subtype of AAA proteins, either one or two AAA domains (D1 and D2) are present. In some proteins with two domains, both are evolutionarily well conserved (like in Cdc48p/p97); in others, either the D2 domain (like in Pex1p and Pex6p) or the D1 domain (in Sec18p/NSF) is better conserved. AAA proteins assemble into oligomers, which form ringshaped structures with a rather narrow central pore. These

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Protein/Enzyme Structure Function and Degradation | AAA-ATPases

oligomers are typically hexameric, although in a few cases, heptameric forms have also been proposed. Depending on the respective protein, the inter-subunit contacts that mediate assembly can lie either in the N-domain (see later) or the AAA domain or both. Some members with two AAA domains have dedicated one domain for the purpose of maintaining the structural stability and integrity of the oligomeric complex, such as D1 in p97/Cdc48p or D2 in Sec18/NSF. These domains are degenerate and have lost their ability to hydrolyze ATP. In addition to the physiologically active hexameric form, some AAA proteins, like katanin, do exist also as dimers, which assemble into rings in the presence of substrate proteins or regulatory cofactors. Further oligomerization to dodecameric complexes was also noticed for some proteins, but the physiological relevance of dodecamers remains unclear. Several crystal structures of AAA proteins have been determined, among them the complete structure of p97, an ATPase with two canonical AAA domains (Figure 1). These structures have shown that in the hexameric conformation, the ATP-binding site is positioned at the interface between subunits, such that the SRH bridges the space between nucleotide-binding pockets of adjacent subunits. Whereas the sensor-1 residue of one subunit points into its own pocket, the conserved arginine residue (the socalled arginine finger) points into the next pocket in the ring (Figure 2). This observation has suggested a mechanism for concerted nucleotide hydrolysis, in which loss of the g-phosphate in one binding site is conveyed to the next active site via rigid-body motion of the helix connecting the sensor-1 and arginine finger residues. It also provides an explanation for the high degree of sequence conservation in the SRH. Interestingly, the meiotic clade of the AAA family and some minor clades (see later), are characterized by a two-residue deletion in the SRH immediately preceding the arginine finger. Amino-terminally to the AAA domains are non-ATPase domains (N-domains) which are involved in substrate binding and recognition. Divergence in the function of the various family members occurs to a large part from the wide variety of N-domains that are found. These domains act as tool heads interacting with substrates, either directly or through adaptor molecules. Upon ATP binding and hydrolysis, AAA proteins undergo conformational changes in the AAA domains that are registered in the N-domains. The result is a repositioning of the N-domains in the ring relative to each other and to the AAA domains. Motions in the Ndomains can be transmitted as mechanical force to a bound substrate, resulting, for example, in remodeling or dissociation of a protein complex, or in the complete unfolding of a polypeptide chain and its subsequent translocation through the central pore of the AAA protein ring. In a sense, AAA proteins use the AAA module like a chemo-mechanical converter (motor) powered by ATP hydrolysis to energize such a mechanical force. The conserved features of AAA domains imply a common mechanism for the operation of the motor. Yet, small differences in nucleotide-sensing residues, in the domains attached to the motor or in the connecting regions between these parts, may determine the direction and timing of the forces that are exerted. They may also

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Figure 1 Crystal structure of a representative AAA protein with two ATPase domains, mouse p97. The domains are colored in their succession from amino- to carboxy terminus: N-domain blue, D1 P-loop subdomain green, D1 helical subdomain yellow, D2 P-loop subdomain orange, D2 helical subdomain red. The top panel shows a top view and the middle panel a side view of the complex. The bottom panel illustrates schematically the succession of N, D1, and D2 domains in the polypeptide chain, as well as an enlargement of one of the D domains with the salient sequence motifs labeled.

affect substrate specificity of these proteins. Sequence divergence in the AAA domains, acquisition of different Ndomains, followed by gene duplication in case of proteins with two AAA domains, have thus produced an array of biological activities from a common ancestral protein.

Protein/Enzyme Structure Function and Degradation | AAA-ATPases

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Sensor-1 (subunit A) Arg finger (subunit B)

Nucleotide (subunit A)

Nucleotide (subunit C) Sensor-1 (subunit B) Arg finger (subunit A)

Walker B motif (subunit B)

Walker A motif (subunit B) Nucleotide (subunit B)

Figure 2 Enlarged, cut-away view of three consecutive nucleotide-binding sites in the p97 ring. Only the structural elements needed to illustrate the communication between active sites are shown. The residues participating in nucleotide binding and hydrolysis are labeled and color coded as in Figure 1. (Note that most AAA proteins, including p97 depicted here, lack a sensor-2 arginine.)

Table 1

Examples of AAA proteins (upper part) and AAAþ proteins (lower part) involved in cell biological processes

Protein

Function

Cellular location

PAN, ARC, Rpt1-Rpt6

Accessory factors to the proteasome in archaea, bacteria, and eukaryotes; unfolding and translocation of proteins Dislocation of PTS receptor from the peroxisomal membrane for recycling in the cytosol Disassembly of SNARE complexes for vesicular transport Disassembly of ESCRT-III complex during budding into multivesicular bodies; related function in archaea? Retranslocation of misfolded proteins during ERAD, nuclear envelope, and Golgi reassembly after mitosis; archaeal VAT homologs with possible function in protein quality control Metalloprotease; degradation of misfolded membrane proteins

Cytosol

Pex1 and Pex6 NSF Vps4 Cdc48p/p97

FtsH i-AAA and m-AAA proteases Katanin BCS1 Dynein Lon Clp/Hsp100 DnaA RuvB clamp loader BchI

Maturation of mitochondrial proteins and mitochondrial protein quality control Microtubule severing Formation of Rieske iron–sulfur cluster proteins Minus-end-directed microtubule-based motor protein Protease; degradation of short-lived and misfolded proteins Protein quality control in conjuction with proteases, disaggregation, and unfolding of proteins Initiation of chromosomal replication in bacteria, promotion of local DNA unwinding within the replication origin Branch migration helicase in DNA recombination Component of magnesium chelatase complex involved in chlorophyll synthesis

Classification and Evolution: AAA and AAAþ (Super)Families The classical AAA family is part of the AAAþ superfamily of ATPases that includes also a number of more distantly related cellular regulators. This superfamily has an expanded activity spectrum compared to the core AAA family (Table 1). AAAþ

Peroxisome and cytosol Cytosol, vesicles, Golgi, and endosomes Multvesicular body and endosomes in eukaryotes; cytosol in archaea Cytosol, endoplasmic reticulum, Golgi

Inner membrane of bacteria and endosymbiontic organelles Mitochondrial inner membrane Centrosome and spindle Mitochondrial inner membrane Microtubules, spindle, vesicles Organelles and bacterial cytosol Cytosol Bacterial cytosol Bacterial cytosol Plastids and bacterial cytosol

proteins are involved in protein degradation, membrane fusion, DNA replication, microtubule dynamics, intracellular transport, flagellar and ciliary beating, and disassembly of protein complexes and protein aggregates. Notably, some proteins like DnaA and RuvB interact with DNA. In their case, mechanical force results in the unwinding of the nucleic acid rather than unfolding of a protein.

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Protein/Enzyme Structure Function and Degradation | AAA-ATPases

The classification of AAA proteins is based on the analysis of their AAA domains. Several approaches have been used to compute phylogenies for AAA proteins, differing in the data available at the time (i.e., sequenced genomes) and in the inclusion of degenerate, inactive, and/or rapidly evolving sequences. More recently, cluster analysis has been applied toward the same goal. A cluster map of the AAAþ superfamily yields a well defined and compact group for AAA proteins, whose substructure can be further analyzed either by clustering at more stringent cutoffs or by classical phylogenetic methods. This approach outlines six major clades of AAA domains, namely proteasome subunits, metalloproteases, domains D1 and D2 of ATPases with two AAA domains, the meiotic group with MSP1/katanin/spastin, and BCS1 and it homologs, as well as a number of minor clades (Figure 3). Although deep branching, two of these minor clades are located next to major clades: ARC (AAA ATPase forming ring-shaped complexes)

next to the D1 and D2 clade, and a group of methanogenic sequences next to the metalloproteases, named AMA for its occurrence in Archaeoglobus fulgidus and methanogenic archaea. Most of the minor clades close to the root have gapped SRH regions and are similar to the AAAþ proteins in the positions of the conserved arginine finger residues, which may indicate an ancestral trait of these sequences. Clear clustering of the eukaryotic clades and predominance of prokaryotic sequences in deep-branching clades suggest that the AAA family had already reached most of its diversity before the three domains of life separated. As mentioned above, the N-domains of the AAA proteins are crucial for substrate specificity and consequently for the cellular function of the respective AAA protein. A cluster analysis of N-domains resolves these sequences into 20 groups. This implies that the wide variety of biological functions of these proteins originates primarily from divergence in their

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Protein/Enzyme Structure Function and Degradation | AAA-ATPases

N-domains. Some of these differences were acquired after the division into plants and other eukaryotes (fungi and animals) as observed in the BCS1 clade. Other family members show high divergence in their N-domains although their AAA domains are of obvious monophyletic origin, suggesting an evolutionary exchange or recruitment of N-domains (like smallminded and Yta7 in the D1 clade). Interestingly, there are also N-domain homologies between the CDC48p/p97 group and the deep-branching AMA group as well as between ARC and proteosomal ATPases. Sequence analysis of N-domains of AMA proteins using PSI-Blast led to the assumption that these proteins adopt a structure similar to the b-clam fold found in N-domains of the CDC48p/p97 group. The homology between ARC and PAN proteins was proposed earlier mainly because of the presence of a coiled-coil region at the very end of the N-domains and of a similar genomic context (proteasome loci). Subsequent crystallographic and biochemical characterization of ARC indicated that it possesses indeed structural similarity to PAN. This similarity extends to function; ARC interacts with bacterial proteasomes and is thought to funnel unfolded substrates into the proteasomal ring chamber for proteolysis (see later for more details). This example demonstrates the usefulness of phylogenetic trees for gaining insight into possible biological functions of as yet uncharacterized members of the AAA protein family.

Cellular Functions of AAA Proteins Members of the AAA family are found in all three organismal kingdoms and they are essential for many cellular processes (see Table 1). To showcase the functional diversity of this protein family, the following sections provide three important examples of well-characterized AAA proteins.

Mediating-Protein Degradation by the Proteasome The proteasome forms the core of the protein quality control system in archaea and eukaryotes and also occurs in one bacterial lineage, the Actinobacteria. AAA proteins are destined as accessory factors of proteasomes, as they can use the energy from ATP hydrolysis to unfold potential protease substrates and transfer them into the interior of the associated proteasomal cylinder for degradation. The AAA proteins in the proteasome subunit clade form homooligomeric complexes in archaea and heterooligomeric complexes in eukaryotes. In eukaryotes, six distinct monomers form a hexameric ring that acts as an unfoldase and de-ubiquitinates tagged proteins prior to threading them through the proteasome for digestion. It appears that the different AAA proteins within a proteasome aid in the recognition and degradation of different subsets of proteins. The N-domains of the archaeal proteasomal ATPase proteasomeactivating nucleotidase (PAN) and of its actinobacterial homolog, ARC or Mpa, form hexameric rings, whose identical subunits consist of an amino-terminal coiled coil and a carboxy-terminal OB domain. In ARC-N, the OB domains are duplicated and form separate rings. Detached from the ATPase domains, PAN-N and ARC-N can act as chaperones, demonstrating their general ability to interact with denatured polypeptides. It has been suggested that, in the context of the entire AAA protein, concerted radial

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motions of the coiled coils relative to the OB rings are crucial for unfolding and/or translocation of the bound polypeptide chain through the pore of the ring. The process that determines which proteins eventually become substrates for the unfoldases and subsequently for the proteasome is still unknown. At least for a range of proteins, however, dedicated coupling factors do exist. Similar to eukaryotic ubiquitin, which tags proteins for degradation, small tagging factors have been found in archaea (SAMP) and actinomycetes (Pup). Pup has been shown to interact with the coiled coils of ARC, thereby recruiting any conjugated protein to the unfoldase. Cdc48p/p97 is perhaps the best-studied AAA protein. It has also been implicated in proteasome-dependent protein degradation, but belongs to a different clade than the abovedescribed proteins (see Figure 3). In eukaryotes, misfolded secretory proteins are exported from the endoplasmic reticulum (ER) and degraded by the ER-associated degradation pathway (ERAD). Nonfunctional membrane and luminal proteins are extracted from the ER and degraded in the cytosol by proteasomes. Substrate retrotranslocation and extraction is assisted by Cdc48p in complex with its adapter proteins Ufd1p and Npl4p on the cytosolic side of the membrane. Once it emerges on the cytosolic side, the substrate is ubiquitinated by ER-based E2 and E3 conjugating enzymes before degradation by the proteasome. The ancestral function of Cdc48p must lie somewhere else, though. Highly conserved homologs (named VAT) are found in archaea, in some organisms even as multiple variants. VATs may play a role in archaeal protein quality control, possibly in conjunction with the proteasome. Their ability to handle misfolded proteins could have made them well suited to adapt to new functions during the evolution of the eukaryotic cell.

Membrane Fusion and Vesicle Transport Several AAA proteins seem to have a central role in reshaping cellular membranes. These include the originally defined AAA proteins with two ATPase domains in the D1 and D2 clade, namely Sec18/NSF, Pex1 and Pex6, YTA7, SPAF, and smallminded. Vesicular transport requires cargo selection and vesicle production through a budding process. The subsequent vesicle transport and targeting process concludes with specific membrane fusion of the vesicle to its target membrane. Early molecular analysis of vesicular trafficking between Golgi cisternae identified an essential N-ethyl-maleimide-sensitive factor (NSF). It functions as a soluble NSF attachment protein (SNAP) receptor (SNARE) dissociation factor. Aided by SNAPs, it binds to SNARE complexes and utilizes the energy of ATP hydrolysis to disassemble them, thus facilitating SNARE recycling. A related process is mediated by vacuolar protein sorting 4 (Vps4), a protein involved in endosomal transport through multivesicular bodies. These bodies are endosomal compartments in eukaryotes where ubiquitinated membrane proteins are sorted into specific vesicles. This process involves the sequential action of three multiprotein complexes, ESCRT I–III (ESCRT standing for endosomal sorting complexes required for transport). Vps4p is anchored to the endosomal membrane and has been shown to catalyze the dissociation of ESCRT complexes. Assembly of Vps4p itself is assisted by the conserved Vta1p protein, which regulates its oligomerization

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Protein/Enzyme Structure Function and Degradation | AAA-ATPases

status and ATPase activity. Bioinformatic analysis has shown Vps4p and ESCRT homologs in archaea, pointing to the existence of a rudimentary mechanism for vesicle transport and membrane invagination. As archaea do not have elaborate intracellular membrane systems, the exact cellular role of these membrane-sculpting processes remains to be discovered. Vps4 belongs to the meiotic clade of the AAA family, which derives its name from its member katanin, a microtubule severing protein implicated in mitosis and meiosis. Spastin, another member of this group, is involved in microtubule disassembly.

AAA Proteins As Membrane-Bound Metalloproteases Proteins in the metalloprotease group are distinguished by a unique domain structure, with an extracellular N-domain, connected by a transmembrane helix to an AAA domain and a carboxy-terminal metalloprotease domain. They are found in the inner membranes of bacteria, where they degrade both soluble and membrane proteins, and in endosymbiont-derived organelles, where they primarily degrade unassembled subunits of membrane complexes. In the latter, they occur as two distinct complexes, one of which has an inverted orientation in the membrane. The paradigm for the metalloprotease group is FtsH, the only essential protease of Escherichia coli. It forms a homohexamer, which, at least in some species, is further complexed with an oligomer of the periplasmic, membrane-bound modulating factor HflKC. FtsH is a processive endopeptidase and degrades certain misassembled membrane proteins, as well as a set of short-lived proteins, thereby enabling cellular regulation at the level of protein stability. Although the details of substrate recognition are largely unknown, the ATPase module is thought to be crucial for the ability of FtsH to dislocate membrane protein substrates out of the membrane and to translocate them to the metalloprotease domain. FtsH, thus, turns out to be an intriguing case of an AAA protein, where translocase activity and proteolytic function are integrated as successive modules into one polypeptide chain.

Conclusions The function of several AAA family members has been well delineated by now and protein unfolding and disassembly emerges as a common mechanistic theme. An increasing number of high-resolution structural studies have provided a detailed picture of the conserved structure of the AAA domain. How these domains assemble into functional oligomers and change their conformation during ATP hydrolysis to generate movement and mechanical force has been studied less extensively. Which are the approaches likely to advance our knowledge in this area? High-resolution structures of oligomeric complexes in different nucleotide-bound states, and eventually

bound to substrates and/or cofactors, will enable a better molecular understanding of the inner workings of AAA proteins. To determine the precise function and mechanism of action of a particular ATPase, reconstitution of its reaction in vitro with purified components remains an essential step. Results from such studies would also shed more light on the fact that there is no strict correlation between AAA protein subtypes and clades on the one side, and a specific function and activity on the other side. This suggests that early in the evolution of AAA, there was a small number of subtypes that subsequently expanded and adapted to allow the processing of a wide variety of targets. Another aspect gaining importance concerns the nature of disease mutations in AAA proteins, of which there is a growing number. Interestingly, some autosomal dominant diseases, such as hereditary spastic paraplegia (spastin defect), and some autosomal recessive diseases, such as mitochondrial complex III deficiency (BCS1 defect), are both caused by mutations in the AAA domain that are thought to affect ATP binding and hydrolysis; it is not clear whether for autosomal dominant diseases, the mutations act as dominant negatives. A thorough dissection of the generic AAA ATPase reaction cycle would, therefore, be beneficial for this research area as well.

See also: Protein/Enzyme Structure Function and Degradation: Chaperonins; Protein Degradation; Protein Folding and Assembly.

Further Reading DeLaBarre B and Brunger AT (2003) Complete structure of p97/valosin containing protein reveals communication between nucleotide domains. Nature Structural Biology 10: 856–863. Erzberger JP and Berger JM (2006) Evolutionary relationships and structural mechanisms of AAAþ proteins. Annual Review of Biophysics and Biomolecular Structure 35: 93–114. Frickey T and Lupas AN (2004) Phylogenetic analysis of AAA proteins. Journal of Structural Biology 146: 2–10. Halawani D and Latterich M (2007) p97: The cell’s molecular purgatory? Molecular Cell 22: 713–717. Hanson PI and Whiteheart SW (2005) AAAþ proteins: Have engine, will work. Nature Reviews Molecular Cell Biology 6: 519–529. Lupas AN and Martin J (2002) AAA proteins. Current Opinion in Structural Biology 12: 746–753. Pye VE, Dreveny I, Briggs LC, et al. (2006) Going through the motions: The ATPase cycle of p97. Special Issue: AAAþ ATPases. Journal of Structural Biology 156: 12–28. Smith DM, Benaroudj N, and Goldberg A (2006) Proteasomes and their associated ATPases: A destructive combination. Special Issue: AAAþ ATPases. Journal of Structural Biology 156: 72–83. Suno R, Niwa H, Tsuchiya D, et al. (2008) Structure of the whole cytosolic region of ATP-dependent protease FtsH. Molecular Cell 22: 575–585. Tucker PA and Sallai L (2007) The AAAþ superfamily – a myriad of motions. Current Opinion in Structural Biology 17: 641–652. White SR and Lauring B (2007) AAAþ ATPases: Achieving diversity of function with conserved machinery. Traffic 8: 1657–1667.