Phylogenetic analysis of AAA proteins

AAA ATPases form a large protein family with manifold cellular roles. They belong to the AAA+ superfamily of ringshaped P-loop NTPases, which exert their activity through the energy-dependent unfolding of macromolecules. Phylogenetic analyses have suggested the existence of five major clades of AAA domains (proteasome subunits, metalloproteases, domains D1 and D2 of ATPases with two AAA domains, and the MSP1/katanin/spastin group), as well as a number of deeply branching minor clades. These analyses however have been characterized by a lack of consistency in defining the boundaries of the AAA family. We have used cluster analysis to delineate unambiguously the group of AAA sequences within the AAA+ superfamily. Phylogenetic and cluster analysis of this sequence set revealed the existence of a sixth major AAA clade, comprising the mitochondrial, membrane-bound protein BCS1 and its homologues. In addition, we identified several deep branches consisting mainly of hypothetical proteins resulting from genomic projects. Analysis of the AAA N-domains provided direct support for the obtained phylogeny for most branches, but revealed some deep splits that had not been apparent from phylogenetic analysis and some unexpected similarities between distant clades. It also revealed highly degenerate D1 domains in plant MSP1 sequences and in at least one deeply branching group of hypothetical proteins (YC46), showing that AAA proteins with two ATPase domains arose at least three times independently.     

The PS1 Hairpin of Mcm3 Is Essential for Viability and for DNA Unwinding In Vitro

The pre-sensor 1 (PS1) hairpin is found in ring-shaped helicases of the AAA+ family (ATPases associated with a variety of cellular activities) of proteins and is implicated in DNA translocation during DNA unwinding of archaeal mini-chromosome maintenance (MCM) and superfamily 3 viral replicative helicases. To determine whether the PS1 hairpin is required for the function of the eukaryotic replicative helicase, Mcm2-7 (also comprised of AAA+ proteins), we mutated the conserved lysine residue in the putative PS1 hairpin motif in each of the Saccharomyces cerevisiae Mcm2-7 subunits to alanine. Interestingly, only the PS1 hairpin of Mcm3 was essential for viability. While mutation of the PS1 hairpin in the remaining MCM subunits resulted in minimal phenotypes, with the exception of Mcm7 which showed slow growth under all conditions examined, the viable alleles were synthetic lethal with each other. Reconstituted Mcm2-7 containing Mcm3 with the PS1 mutation (Mcm3_K499A) had severely decreased helicase activity. The lack of helicase activity provides a probable explanation for the inviability of the mcm3 _K499A strain. The ATPase activity of Mcm2-7_3K499A was similar to the wild type complex, but its interaction with single-stranded DNA in an electrophoretic mobility shift assay and its associations in cells were subtly altered. Together, these findings indicate that the PS1 hairpins in the Mcm2-7 subunits have important and distinct functions, most evident by the essential nature of the Mcm3 PS1 hairpin in DNA unwinding.     

RecA-like motor ATPases—lessons from structures

A large class of ATPases contains a RecA-like structural domain and uses the energy of nucleotide binding and hydrolysis to perform mechanical work, for example, to move polypeptides or nucleic acids. These ATPases include helicases, ABC transporters, clamp loaders, and proteases. The functional units of the ATPases contain different numbers of RecA-like domains, but the nucleotide is always bound at the interface between two adjacent RecA-like folds and the two domains move relative to one another during the ATPase cycle. The structures determined for different RecA-like motor ATPases begin to reveal how they move macromolecules.     

RecA-like motor ATPases--lessons from structures

A large class of ATPases contains a RecA-like structural domain and uses the energy of nucleotide binding and hydrolysis to perform mechanical work, for example, to move polypeptides or nucleic acids. These ATPases include helicases, ABC transporters, clamp loaders, and proteases. The functional units of the ATPases contain different numbers of RecA-like domains, but the nucleotide is always bound at the interface between two adjacent RecA-like folds and the two domains move relative to one another during the ATPase cycle. The structures determined for different RecA-like motor ATPases begin to reveal how they move macromolecules.     

An NMR Study of a 300-kDa AAA+ Unfoldase

AAA+ ATPases are ubiquitous hexameric unfoldases acting in cellular protein quality control. In complex with proteases, they form protein degradation machinery (the proteasome) in both archaea and eukaryotes. Here, we use solution-state NMR spectroscopy to determine the symmetry properties of the archaeal PAN AAA+ unfoldase and gain insights into its functional mechanism. PAN consists of three folded domains: the coiled-coil (CC), OB and ATPase domains. We find that full-length PAN assembles into a hexamer with C₂ symmetry, and that this symmetry extends over the CC, OB and ATPase domains. The NMR data, collected in the absence of substrate, are incompatible with the spiral staircase structure observed in electron-microscopy studies of archaeal PAN in the presence of substrate and in electron-microscopy studies of eukaryotic unfoldases both in the presence and in the absence of substrate. Based on the C₂ symmetry revealed by NMR spectroscopy in solution, we propose that archaeal ATPases are flexible enzymes, which can adopt distinct conformations in different conditions. This study reaffirms the importance of studying dynamic systems in solution.     

DNA helicase activity is associated with the replication initiator protein rep of tomato yellow leaf curl geminivirus

The Rep protein of tomato yellow leaf curl Sardinia virus (TYLCSV), a single-stranded DNA virus of plants, is the replication initiator essential for virus replication. TYLCSV Rep has been classified among ATPases associated with various cellular activities (AAA+ ATPases), in superfamily 3 of small DNA and RNA virus replication initiators whose paradigmatic member is simian virus 40 large T antigen. Members of this family are DNA- or RNA-dependent ATPases with helicase activity necessary for viral replication. Another distinctive feature of AAA+ ATPases is their quaternary structure, often composed of hexameric rings. TYLCSV Rep has ATPase activity, but the helicase activity, which is instrumental in further characterization of the mechanism of rolling-circle replication used by geminiviruses, has been a longstanding question. We present results showing that TYLCSV Rep lacking the 121 N-terminal amino acids has helicase activity comparable to that of the other helicases: requirements for a 3' overhang and 3'-to-5' polarity of unwinding, with some distinct features and with a minimal AAA+ ATPase domain. We also show that the helicase activity is dependent on the oligomeric state of the protein.     

The third P-loop domain in cytoplasmic dynein heavy chain is essential for dynein motor function and ATP-sensitive microtubule binding

Sequence comparisons and structural analyses show that the dynein heavy chain motor subunit is related to the AAA family of chaperone-like ATPases. The core structure of the dynein motor unit derives from the assembly of six AAA domains into a hexameric ring. In dynein, the first four AAA domains contain consensus nucleotide triphosphate-binding motifs, or P-loops. The recent structural models of dynein heavy chain have fostered the hypothesis that the energy derived from hydrolysis at P-loop 1 acts through adjacent P-loop domains to effect changes in the attachment state of the microtubule-binding domain. However, to date, the functional significance of the P-loop domains adjacent to the ATP hydrolytic site has not been demonstrated. Our results provide a mutational analysis of P-loop function within the first and third AAA domains of the Drosophila cytoplasmic dynein heavy chain. Here we report the first evidence that P-loop-3 function is essential for dynein function. Significantly, our results further show that P-loop-3 function is required for the ATP-induced release of the dynein complex from microtubules. Mutation of P-loop-3 blocks ATP-mediated release of dynein from microtubules, but does not appear to block ATP binding and hydrolysis at P-loop 1. Combined with the recent recognition that dynein belongs to the family of AAA ATPases, the observations support current models in which the multiple AAA domains of the dynein heavy chain interact to support the translocation of the dynein motor down the microtubule lattice.     

Structure of the ABC ATPase domain of human TAP1, the transporter associated with antigen processing

The transporter associated with antigen processing (TAP) is an ABC transporter formed of two subunits, TAP1 and TAP2, each of which has an N-terminal membrane-spanning domain and a C-terminal ABC ATPase domain. We report the structure of the C-terminal ABC ATPase domain of TAP1 (cTAP1) bound to ADP. cTAP1 forms an L-shaped molecule with two domains, a RecA-like domain and a small alpha-helical domain. The diphosphate group of ADP interacts with the P-loop as expected. Residues thought to be involved in gamma-phosphate binding and hydrolysis show flexibility in the ADP-bound state as evidenced by their high B-factors. Comparisons of cTAP1 with other ABC ATPases from the ABC transporter family as well as ABC ATPases involved in DNA maintenance and repair reveal key regions and residues specific to each family. Three ATPase subfamilies are identified which have distinct adenosine recognition motifs, as well as distinct subdomains that may be specific to the different functions of each subfamily. Differences between TAP1 and TAP2 in the nucleotide-binding site may be related to the observed asymmetry during peptide transport.     

Structural mapping of the ClpB ATPases of Plasmodium falciparum: Targeting protein folding and secretion for antimalarial drug design

Caseinolytic chaperones and proteases (Clp) belong to the AAA+ protein superfamily and are part of the protein quality control machinery in cells. The eukaryotic parasite Plasmodium falciparum, the causative agent of malaria, has evolved an elaborate network of Clp proteins including two distinct ClpB ATPases. ClpB1 and ClpB2 are involved in different aspects of parasitic proteostasis. ClpB1 is present in the apicoplast, a parasite-specific and plastid-like organelle hosting various metabolic pathways necessary for parasite growth. ClpB2 localizes to the parasitophorous vacuole membrane where it drives protein export as core subunit of a parasite-derived protein secretion complex, the Plasmodium Translocon of Exported proteins (PTEX); this process is central to parasite virulence and survival in the human host. The functional associations of these two chaperones with parasite-specific metabolism and protein secretion make them prime drug targets. ClpB proteins function as unfoldases and disaggregases and share a common architecture consisting of four domains—a variable N-terminal domain that binds different protein substrates, followed by two highly conserved catalytic ATPase domains, and a C-terminal domain. Here, we report and compare the first crystal structures of the N terminal domains of ClpB1 and ClpB2 from Plasmodium and analyze their molecular surfaces. Solution scattering analysis of the N domain of ClpB2 shows that the average solution conformation is similar to the crystalline structure. These structures represent the first step towards the characterization of these two malarial chaperones and the reconstitution of the entire PTEX to aid structure-based design of novel anti-malarial drugs.     

Crystal structure of the AAA+ alpha domain of E. coli Lon protease at 1.9A resolution

The crystal structure of the small, mostly helical alpha domain of the AAA+ module of the Escherichia coli ATP-dependent protease Lon has been solved by single isomorphous replacement combined with anomalous scattering and refined at 1.9A resolution to a crystallographic R factor of 17.9%. This domain, comprising residues 491-584, was obtained by chymotrypsin digestion of the recombinant full-length protease. The alpha domain of Lon contains four alpha helices and two parallel strands and resembles similar domains found in a variety of ATPases and helicases, including the oligomeric proteases HslVU and ClpAP. The highly conserved "sensor-2" Arg residue is located at the beginning of the third helix. Detailed comparison with the structures of 11 similar domains established the putative location of the nucleotide-binding site in this first fragment of Lon for which a crystal structure has become available.     

Stairway to translocation: AAA+ motor structures reveal the mechanisms of ATP‐dependent substrate translocation

Translocases of the AAA+ (ATPases Associated with various cellular Activities) family are powerful molecular machines that use the mechano‐chemical coupling of ATP hydrolysis and conformational changes to thread DNA or protein substrates through their central channel for many important biological processes. These motors comprise hexameric rings of ATPase subunits, in which highly conserved nucleotide‐binding domains form active‐site pockets near the subunit interfaces and aromatic pore‐loop residues extend into the central channel for substrate binding and mechanical pulling. Over the past 2 years, 41 cryo‐EM structures have been solved for substrate‐bound AAA+ translocases that revealed spiral‐staircase arrangements of pore‐loop residues surrounding substrate polypeptides and indicating a conserved hand‐over‐hand mechanism for translocation. The subunits' vertical positions within the spiral arrangements appear to be correlated with their nucleotide states, progressing from ATP‐bound at the top to ADP or apo states at the bottom. Studies describing multiple conformations for a particular motor illustrate the potential coupling between ATP‐hydrolysis steps and subunit movements to propel the substrate. Experiments with double‐ring, Type II AAA+ motors revealed an offset of hydrolysis steps between the two ATPase domains of individual subunits, and the upper ATPase domains lacking aromatic pore loops frequently form planar rings. This review summarizes the critical advances provided by recent studies to our structural and functional understanding of hexameric AAA+ translocases, as well as the important outstanding questions regarding the underlying mechanisms for coordinated ATP‐hydrolysis and mechano‐chemical coupling.     

Model for the motor component of dynein heavy chain based on homology to the AAA family of oligomeric ATPases

BACKGROUND: Recent iterative methods for sequence alignment have indicated that the 380 kDa motor unit of dynein belongs to the AAA class of chaperone-like ATPases. These alignments indicate that the core of the 380 kDa motor unit contains a concatenated chain of six AAA modules, of which four correspond to the ATP binding sites with P-loop signatures described previously, and two are modules in which the P loop has been lost in evolution. RESULTS: We report predicted structures for the six AAA modules in the beta heavy chain of axonemal dynein, based upon their homology to a template of structurally conserved regions derived from three AAA proteins with experimentally determined structures (pdb:1A5T, pdb:1DOO, and pdb:1NSF). The secondary structural elements of the AAA modules in dynein correspond to regions of sequence that are relatively well conserved in different dynein isoforms. The tertiary structure of each AAA module comprises a major alpha/beta N domain from which a smaller all-alpha C domain protrudes at an angle, as part of the putative nucleotide binding cavity. The structures of the six modules are assembled into a ring, approximately 125 A in diameter, that resembles the structure of the dynein motor unit observed by electron microscopy. CONCLUSION: The predicted structures are supported by procedures that assess global, regional, and local quality, with the module containing the hydrolytic ATP binding site being supported the most strongly. The structural resemblance of the dynein motor to the hexameric assembly of AAA modules in the hsp100 family of chaperones suggests that the basic mechanism underlying the ATP-dependent translocation of dynein along a microtubule may have aspects in common with the ATP-dependent translocation of polypeptides into the interior compartment of chaperones.     

Foot-and-Mouth Disease Virus 2C Is a Hexameric AAA+ Protein with a Coordinated ATP Hydrolysis Mechanism

Foot-and-mouth disease virus (FMDV), a positive sense, single-stranded RNA virus, causes a highly contagious disease in cloven-hoofed livestock. Like other picornaviruses, FMDV has a conserved 2C protein assigned to the superfamily 3 helicases a group of AAA+ ATPases that has a predicted N-terminal membrane-binding amphipathic helix attached to the main ATPase domain. In infected cells, 2C is involved in the formation of membrane vesicles, where it co-localizes with viral RNA replication complexes, but its precise role in virus replication has not been elucidated. We show here that deletion of the predicted N-terminal amphipathic helix enables overexpression in Escherichia coli of a highly soluble truncated protein, 2C(34–318), that has ATPase and RNA binding activity. ATPase activity was abrogated by point mutations in the Walker A (K116A) and B (D160A) motifs and Motif C (N207A) in the active site. Unliganded 2C(34–318) exhibits concentration-dependent self-association to yield oligomeric forms, the largest of which is tetrameric. Strikingly, in the presence of ATP and RNA, FMDV 2C(34–318) containing the N207A mutation, which binds but does not hydrolyze ATP, was found to oligomerize specifically into hexamers. Visualization of FMDV 2C-ATP-RNA complexes by negative stain electron microscopy revealed hexameric ring structures with 6-fold symmetry that are characteristic of AAA+ ATPases. ATPase assays performed by mixing purified active and inactive 2C(34–318) subunits revealed a coordinated mechanism of ATP hydrolysis. Our results provide new insights into the structure and mechanism of picornavirus 2C proteins that will facilitate new investigations of their roles in infection.     

Classification and evolution of P-loop GTPases and related ATPases

Sequences and available structures were compared for all the widely distributed representatives of the P-loop GTPases and GTPase-related proteins with the aim of constructing an evolutionary classification for this superclass of proteins and reconstructing the principal events in their evolution. The GTPase superclass can be divided into two large classes, each of which has a unique set of sequence and structural signatures (synapomorphies). The first class, designated TRAFAC (after translation factors) includes enzymes involved in translation (initiation, elongation, and release factors), signal transduction (in particular, the extended Ras-like family), cell motility, and intracellular transport. The second class, designated SIMIBI (after signal recognition particle, MinD, and BioD), consists of signal recognition particle (SRP) GTPases, the assemblage of MinD-like ATPases, which are involved in protein localization, chromosome partitioning, and membrane transport, and a group of metabolic enzymes with kinase or related phosphate transferase activity. These two classes together contain over 20 distinct families that are further subdivided into 57 subfamilies (ancient lineages) on the basis of conserved sequence motifs, shared structural features, and domain architectures. Ten subfamilies show a universal phyletic distribution compatible with presence in the last universal common ancestor of the extant life forms (LUCA). These include four translation factors, two OBG-like GTPases, the YawG/YlqF-like GTPases (these two subfamilies also consist of predicted translation factors), the two signal-recognition-associated GTPases, and the MRP subfamily of MinD-like ATPases. The distribution of nucleotide specificity among the proteins of the GTPase superclass indicates that the common ancestor of the entire superclass was a GTPase and that a secondary switch to ATPase activity has occurred on several independent occasions during evolution. The functions of most GTPases that are traceable to LUCA are associated with translation. However, in contrast to other superclasses of P-loop NTPases (RecA-F1/F0, AAA+, helicases, ABC), GTPases do not participate in NTP-dependent nucleic acid unwinding and reorganizing activities. Hence, we hypothesize that the ancestral GTPase was an enzyme with a generic regulatory role in translation, with subsequent diversification resulting in acquisition of diverse functions in transport, protein trafficking, and signaling. In addition to the classification of previously known families of GTPases and related ATPases, we introduce several previously undetected families and describe new functional predictions.     

AAA+ ATPases: achieving diversity of function with conserved machinery

AAA+ adenosine triphosphatases (ATPases) are molecular machines that perform a wide variety of cellular functions. For instance, they can act in vesicle transport, organelle assembly, membrane dynamics and protein unfolding. In most cases, the ATPase domains of these proteins assemble into active ring-shaped hexamers. As AAA+ proteins have a common structure, a central issue is determining how they use conserved mechanistic principles to accomplish specific biological actions. Here, we review the features and motifs that partially define AAA+ domains, describe the cellular activities mediated by selected AAA+ proteins and discuss the recent work, suggesting that various AAA+ machines with very different activities employ a common core mechanism. The importance of this mechanism to human health is demonstrated by the number of genetic diseases caused by mutant AAA+ proteins.     

Evolutionary links between FliH/YscL-like proteins from bacterial type III secretion systems and second-stalk components of the FoF1 and vacuolar ATPases

Bacterial type III secretion drives flagellar biosynthesis and mediates bacterial-eukaryotic interactions. Type III secretion is driven by an ATPase that is homologous to the catalytic subunits of proton-translocating ATPases, such as the F(o)F(1) ATPase. Here we use PSI-BLAST searches to show that some noncalatytic components are also conserved between type III secretion systems and proton-translocating ATPases. In particular, we show that the FliH/YscL-like proteins and the E subunits of vacuolar ATPases represent fusions of domains homologous to second-stalk components of the F(o)F(1) ATPase (the b and delta subunits).     

Structural Insights into the Unusually Strong ATPase Activity of the AAA Domain of the Caenorhabditis elegans Fidgetin-like 1 (FIGL-1) Protein

The FIGL-1 (fidgetin like-1) protein is a homolog of fidgetin, a protein whose mutation leads to multiple developmental defects. The FIGL-1 protein contains an AAA (ATPase associated with various activities) domain and belongs to the AAA superfamily. However, the biological functions and developmental implications of this protein remain unknown. Here, we show that the AAA domain of the Caenorhabditis elegans FIGL-1 protein (CeFIGL-1-AAA), in clear contrast to homologous AAA domains, has an unusually high ATPase activity and forms a hexamer in solution. By determining the crystal structure of CeFIGL-1-AAA, we found that the loop linking helices α9 and α10 folds into the short helix α9a, which has an acidic surface and interacts with a positively charged surface of the neighboring subunit. Disruption of this charge interaction by mutagenesis diminishes both the ATPase activity and oligomerization capacity of the protein. Interestingly, the acidic residues in helix α9a of CeFIGL-1-AAA are not conserved in other homologous AAA domains that have relatively low ATPase activities. These results demonstrate that the sequence of CeFIGL-1-AAA has adapted to establish an intersubunit charge interaction, which contributes to its strong oligomerization and ATPase activity. These unique properties of CeFIGL-1-AAA distinguish it from other homologous proteins, suggesting that CeFIGL-1 may have a distinct biological function.     

ClpP: a distinctive family of cylindrical energy-dependent serine proteases

Processes maintaining protein homeostasis in the cell are governed by the activities of molecular chaperones that mainly assist in the folding of polypeptide chains and by a large class of proteases that regulate protein levels through degradation. ClpP proteases define a distinctive family of cylindrical, energy-dependent serine proteases that are highly conserved throughout bacteria and eukaryota. They typically interact with ATP-dependent AAA+ chaperones that bind and unfold target substrates and then translocate them into ClpP for degradation. Structural and functional studies have provided a detailed view of the mechanism of function of this class of proteases.