Signal Transduction ATPases With Numerous Domains Research Articles

Structural basis for negative regulation of the Escherichia coli maltose system

Proteins from the signal transduction ATPases with numerous domains (STAND) family are known to play an important role in innate immunity. However, it remains less well understood how they function in transcriptional regulation. MalT is a bacterial STAND that controls the Escherichia coli maltose system. Inactive MalT is sequestered by different inhibitory proteins such as MalY. Here, we show that MalY interacts with one oligomerization interface of MalT to form a 2:2 complex. MalY represses MalT activity by blocking its oligomerization and strengthening ADP-mediated MalT autoinhibition. A loop region N-terminal to the nucleotide-binding domain (NBD) of MalT has a dual role in mediating MalT autoinhibition and activation. Structural comparison shows that ligand-binding induced oligomerization is required for stabilizing the C-terminal domains and conferring DNA-binding activity. Together, our study reveals the mechanism whereby a prokaryotic STAND is inhibited by a repressor protein and offers insights into signaling by STAND transcription activators.

Identification and expression profile of novel STAND gene Nwd2 in the mouse central nervous system

In the central nervous system (CNS), neurons need synaptic neurotransmitter release and cellular response for various cellular stress or environmental stimuli. To achieve these highly orchestrated cellular processes, neurons should drive the molecular mechanisms that govern and integrate complex signaling pathways. The signal transduction ATPases with numerous domains (STAND) family of proteins has been shown to play essential roles in diverse signal transduction mechanisms, including apoptosis and innate immunity. However, a comprehensive understanding of STAND genes remains lacking. Previously, we identified the NACHT and WD repeat domain-containing protein 1 (NWD1), a member of STAND family, in the regulation of the assembly of a giant multi-enzyme complex that enables efficient de novo purine biosynthesis during brain development. Here we identified the mouse Nwd2 gene, which is a paralog of Nwd1. A molecular phylogenetic analysis suggested that Nwd1 emerged during the early evolution of the animal kingdom, and that Nwd2 diverged in the process of Nwd1 duplication. RT-PCR and in situ hybridization analyses revealed the unique expression profile of Nwd2 in the developing and adult CNS. Unlike Nwd1, Nwd2 expression was primarily confined to neurons in the medial habenular nucleus, an essential modulating center for diverse psychological states, such as fear, anxiety, and drug addiction. In the adult brain, Nwd2 expression, albeit at a lower level, was also observed in some neuronal populations in the piriform cortex, hippocampus, and substantia nigra pars compacta. NWD2 might play a unique role in the signal transduction required for specific neuronal circuits, especially for cholinergic neurons in the habenula.

Nwd1 Regulates Neuronal Differentiation and Migration through Purinosome Formation in the Developing Cerebral Cortex.

SummaryEngagement of neural stem/progenitor cells (NSPCs) into proper neuronal differentiation requires the spatiotemporally regulated generation of metabolites. Purines are essential building blocks for many signaling molecules. Enzymes that catalyze de novo purine synthesis are assembled as a huge multienzyme complex called “purinosome.” However, there is no evidence of the formation or physiological function of the purinosome in the brain. Here, we showed that a signal transduction ATPases with numerous domains (STAND) protein, NACHT and WD repeat domain-containing 1 (Nwd1), interacted with Paics, a purine-synthesizing enzyme, to regulate purinosome assembly in NSPCs. Altered Nwd1 expression affected purinosome formation and induced the mitotic exit and premature differentiation of NSPCs, repressing neuronal migration and periventricular heterotopia. Overexpression/knockdown of Paics or Fgams, other purinosome enzymes, in the developing brain resulted in a phenocopy of Nwd1 defects. These findings indicate that strict regulation of purinosome assembly/disassembly is crucial for maintaining NSPCs and corticogenesis.

Nwd1 Regulates Neuronal Differentiation and Migration Through Purinosome Formation in the Developing Cerebral Cortex

Engagement of neural stem/progenitor cells (NSPCs) into proper neuronal differentiation requires the spatiotemporally regulated generation of metabolites. Purines are essential building blocks for many signaling molecules. Enzymes that catalyze de novo purine synthesis are assembled as a huge multienzyme complex called “purinosome.” However, there is no evidence of the formation or physiological function of the purinosome in the brain. Here, we showed that a signal transduction ATPases with numerous domains (STAND) protein, NACHT and WD repeat d omain-containing 1 (Nwd1), interacted with Paics, a purine-synthesizing enzyme, to regulate purinosome assembly in NSPCs. Altered Nwd1 expression affected purinosome formation and induced the mitotic exit and premature differentiation of NSPCs, repressing neuronal migration and periventricular heterotopia. Overexpression/knockdown of Paics or Fgams, other purinosome enzymes, in the developing brain resulted in a phenocopy of Nwd1 defects. These findings indicate that strict regulation of purinosome assembly/disassembly is crucial for maintaining NSPCs and corticogenesis.

Double autoinhibition mechanism of signal transduction ATPases with numerous domains (STAND) with a tetratricopeptide repeat sensor.

Upon triggering by their inducer, signal transduction ATPases with numerous domains (STANDs), initially in monomeric resting forms, multimerize into large hubs that activate target macromolecules. This process requires conversion of the STAND conserved core (the NOD) from a closed form encasing an ADP molecule to an ATP-bound open form prone to multimerize. In the absence of inducer, autoinhibitory interactions maintain the NOD closed. In particular, in resting STAND proteins with an LRR- or WD40-type sensor domain, the latter establishes interactions with the NOD that are disrupted in the multimerization-competent forms. Here, we solved the first crystal structure of a STAND with a tetratricopeptide repeat sensor domain, PH0952 from Pyrococcus horikoshii, revealing analogous NOD-sensor contacts. We use this structural information to experimentally demonstrate that similar interactions also exist in a PH0952 homolog, the MalT STAND archetype, and actually contribute to the MalT autoinhibition in vitro and in vivo. We propose that STAND activation occurs by stepwise release of autoinhibitory contacts coupled to the unmasking of inducer-binding determinants. The MalT example suggests that STAND weak autoinhibitory interactions could assist the binding of inhibitory proteins by placing in register inhibitor recognition elements born by two domains.

Expression profile of the STAND protein Nwd1 in the developing and mature mouse central nervous system.

The orchestrated events required during brain development, as well as the maintenance of adult neuronal plasticity, highly depend on the accurate responses of neuronal cells to various cellular stress or environmental stimuli. Recent studies have defined a previously unrecognized, broad class of multidomain proteins, designated as signal transduction ATPases with numerous domains (STAND), which comprises a large number of proteins, including the apoptotic peptidase activating factor 1 (Apaf1) and nucleotide-binding oligomerization domain-like receptors (NLRs), central players in cell death and innate immune responses, respectively. Although the involvement of STANDs in the central nervous system (CNS) has been postulated in terms of neuronal development and function, it remains largely unclear. Here, we identified Nwd1 (NACHT and WD repeat domain-containing protein 1), as a novel STAND protein, expressed in neural stem/progenitor cells (NSPCs). Structurally, Nwd1 was most analogous to the apoptosis regulator Apaf1, also involved in mitosis and axonal outgrowth regulation in the CNS. Using a specific antibody, we show that, during the embryonic and postnatal period, Nwd1 is expressed in nestin-positive NSPCs in vivo and in vitro, while postnatally it is found in terminally differentiated neurons and blood vessels. At the subcellular level, we demonstrate that Nwd1 is preferentially located in the cytosolic compartment of cultured NSPCs, partially overlapping with cytochrome c. These observations imply that Nwd1 might be involved in the neuronal lineage as a new STAND gene, including having a pro-apoptotic or nonapoptotic role, similar to Apaf1.

The NBS-LRR architectures of plant R-proteins and metazoan NLRs evolved in independent events

There are intriguing parallels between plants and animals, with respect to the structures of their innate immune receptors, that suggest universal principles of innate immunity. The cytosolic nucleotide binding site-leucine rich repeat (NBS-LRR) resistance proteins of plants (R-proteins) and the so-called NOD-like receptors of animals (NLRs) share a domain architecture that includes a STAND (signal transduction ATPases with numerous domains) family NTPase followed by a series of LRRs, suggesting inheritance from a common ancestor with that architecture. Focusing on the STAND NTPases of plant R-proteins, animal NLRs, and their homologs that represent the NB-ARC (nucleotide-binding adaptor shared by APAF-1, certain R gene products and CED-4) and NACHT (named for NAIP, CIIA, HET-E, and TEP1) subfamilies of the STAND NTPases, we analyzed the phylogenetic distribution of the NBS-LRR domain architecture, used maximum-likelihood methods to infer a phylogeny of the NTPase domains of R-proteins, and reconstructed the domain structure of the protein containing the common ancestor of the STAND NTPase domain of R-proteins and NLRs. Our analyses reject monophyly of plant R-proteins and NLRs and suggest that the protein containing the last common ancestor of the STAND NTPases of plant R-proteins and animal NLRs (and, by extension, all NB-ARC and NACHT domains) possessed a domain structure that included a STAND NTPase paired with a series of tetratricopeptide repeats. These analyses reject the hypothesis that the domain architecture of R-proteins and NLRs was inherited from a common ancestor and instead suggest the domain architecture evolved at least twice. It remains unclear whether the NBS-LRR architectures were innovations of plants and animals themselves or were acquired by one or both lineages through horizontal gene transfer.

Prion folding sends a death signal in fungus.

Prions are kind of like Batman—they have a bad reputation, but they can also serve a number of good purposes. While prion domains—folded regions of proteins that can induce similar folding in other susceptible proteins—are responsible for the human prion diseases and may be involved in other neurodegenerative diseases as well, they have also been linked to normal processes of memory and innate immunity. In the fungus Podospora anserina, the protein HET-S includes a prion-forming domain, which, when it folds into the so-called beta-solenoid conformation, causes the protein to embed in the plasma membrane, where it forms a pore that ultimately results in cell death. One trigger for this folding is interaction with a very similar protein (called HET-s) from a different strain of the same fungus. When the two strains meet, their prion-mediated interaction prevents them from fusing and limits the transmission of pathogens between strains. But HET-s isn’t the only protein that induces prion folding in HET-S, according to a new study by Asen Daskalov, Sven Saupe, and colleagues in this issue of PLOS Biology. They show that the HET-S conformation change can be triggered through interaction with a member of a widely distributed protein family, suggesting that prion-based signal transduction may be more common than currently appreciated (Fig. 1). Figure 1 The fungal NOD-like receptor NWD2 controls activation of the HET-S pore-forming protein. Several lines of evidence suggested that the protein, NWD2, might be a HET-S prion partner. The gene sits right next to the gene for HET-S, and the NWD2 protein contains a short beta-solenoid motif of its own. It is a member of the signal transduction ATPases with numerous domains (STAND) family of proteins, which function in innate immunity systems in a wide range of organisms, including humans, in whom APAF-1 induces apoptosis. NWD2 is a receptor, but its ligand is unknown, so to test whether NWD2 and HET-S interact, the authors constructed chimeric proteins, in which the ligand-binding domain was replaced with homologous regions from related proteins whose ligands are known. Introduction of those ligands induced folding in the prion-forming domain of a HET-S/s protein. Mutation of NWD2 residues that altered the beta-solenoid domain of NWD2 prevented this induction. NWD2’s prion-forming domain alone could induce prion formation in HET-S/s proteins, even without ligands, suggesting an inhibitory role for some part of the protein that was removed. When portions of NWD2 and HET-S were transfected into yeast, which doesn’t contain its own version of these proteins, prion formation was nonetheless induced, indicating that other cellular partners are likely not needed to trigger prion folding in HET-S, as long as NWD2 is present. The authors note that the HET-S/NWD2 system is found widely in the fungal kingdom, while the HET-S/HET-s incompatibility system is limited to P. anserina, suggesting that the latter system is an evolutionarily modified version of the former one. Whether the same mechanism is at work in related prion-forming proteins in mammals remains to be seen, but several structural features, including its compactness and its ability to tightly regulate folding, may give this system advantages for signal transduction, including in, but perhaps not limited to, cell defense systems of the immune system.

A dual role for the inducer in signalling by MalT, a signal transduction ATPase with numerous domains (STAND)

Signal transduction ATPases with numerous domains (STAND) are widespread proteins, whose activation involves inducer-dependent conversion of resting ADP-bound monomers into active ATP-bound multimers. This process notably comprises opening of the nucleotide-binding oligomerization domain (NOD), nucleotide exchange and NOD-mediated multimerization. How inducer binding to the sensor domain, whose structure is not conserved throughout the STAND family, causes protein activation remains unclear. We used MalT, an Escherichia coli transcription factor, as a STAND model system, to address this question by dissecting the signalling pathway in vitro. We have found that inducer binding to the sensor is the first step of the activation pathway. It both triggers opening of the NOD and makes the MalT multimer competent for binding promoter MalT sites via its DNA-binding domains. Based on available data, we proposed that inducer trigger of NOD opening is a conserved STAND feature, irrespective of the sensor structure. As discussed, an additional role for the inducer, as found for MalT, might pertain to other types of STANDs.

Conserved Motifs Involved in ATP Hydrolysis by MalT, a Signal Transduction ATPase with Numerous Domains fromEscherichia coli

The signal transduction ATPases with numerous domains (STAND) are sophisticated signaling proteins that are related to AAA+ proteins and control various biological processes, including apoptosis, gene expression, and innate immunity. They function as tightly regulated switches, with the off and on positions corresponding to an ADP-bound, monomeric form and an ATP-bound, multimeric form, respectively. Protein activation is triggered by inducer binding to the sensor domain. ATP hydrolysis by the nucleotide-binding oligomerization domain (NOD) ensures the generation of the ADP-bound form. Here, we use MalT, an Escherichia coli transcription activator, as a model system to identify STAND conserved motifs involved in ATP hydrolysis besides the catalytic acidic residue. Alanine substitution of the conserved polar residue (H131) that is located two residues downstream from the catalytic residue (D129) blocks ATP hydrolysis and traps MalT in an active, ATP-bound, multimeric form. This polar residue is also conserved in AAA+. Based on AAA+ X-ray structures, we proposed that it is responsible for the proper positioning of the catalytic and the sensor I residues for the hydrolytic attack. Alanine substitution of the putative STAND sensor I (R160) abolished MalT activity. Substitutions of R171 impaired both ATP hydrolysis and multimerization, which is consistent with an arginine finger function and provides further evidence that ATP hydrolysis is primarily catalyzed by MalT multimers.

The inducer maltotriose binds in the central cavity of the tetratricopeptide‐like sensor domain of MalT, a bacterial STAND transcription factor

Signal transduction ATPases with numerous domains (STAND) are sophisticated proteins that integrate several signals and respond by building multimeric platforms allowing signalling in various processes: apoptosis, innate immunity, bacterial metabolism. They comprise a conserved nucleotide oligomerization domain (NOD), which functions as a binary switch that oscillates between the OFF (ADP-bound) and the ON (ATP-bound) conformation, and non conserved sensor and effector domains. Transition from the OFF form to the ON form strictly depends on the binding of an inducer to the sensor domain. The interaction of the inducer with this domain was studied in MalT, a model STAND protein. MalT sensor domain has a SUPR (superhelical repeats) fold resembling a cylinder with a central cavity. The cavity was subjected to an alanine-scanning approach, and the effects of the alanine substitutions on inducer binding and transcription activation were analyzed. This work unambiguously showed that the inducer maltotriose binds inside the cavity, and a patch on the inner surface was proposed to be the primary maltotriose binding-site. Furthermore, limited proteolysis suggested that maltotriose binding changes the conformation of the sensor domain.

STANDing strong, resistance proteins instigators of plant defence

Resistance (R) proteins are involved in specific pathogen recognition and subsequent initiation of host defence. Most R proteins are nucleotide binding - leucine rich repeat (NB-LRR) proteins, which form a subgroup within the STAND (signal transduction ATPases with numerous domains) family. Activity of these multi-domain proteins depends on their ability to bind and hydrolyse nucleotides. Since R protein activation often triggers cell-death tight regulation of activation is essential. Autoinhibition, which seems to be accomplished by intramolecular interactions between the various domains, is important to retain R proteins inactive. This review summarizes recent data on intra- and intermolecular interactions that support a model in which pathogen perception triggers a series of conformational changes, allowing the newly exposed NB domain to interact with downstream signalling partners and activate defence signalling.

Wheel of Life, Wheel of Death: A Mechanistic Insight into Signaling by STAND Proteins

The signal transduction ATPases with numerous domains (STAND) represent a newly recognized class of widespread, sophisticated ATPases that are related to the AAA+ proteins and that function as signaling hubs. These proteins control diverse biological processes in bacteria and eukaryotes, including gene expression, apoptosis, and innate immunity responses. They function as tightly regulated switches, with the off and on positions corresponding to a long-lived monomeric, ADP-bound form and a multimeric, ATP-bound form, respectively. Inducer binding to the sensor domain activates the protein by promoting ADP for ATP exchange, probably through removal of an intramolecular inhibitory interaction, whereas ATP hydrolysis turns off the protein. One key component of the switch is a three-domain module carrying the ATPase activity (nucleotide-binding oligomerization domain [NOD]). Analysis of the atomic structures of four crystallized nucleotide-bound NOD modules provides an unprecedented insight into the NOD conformational changes underlying the activation process.

Crystal structure of a novel archaeal AAA+ ATPase SSO1545 from Sulfolobus solfataricus

Signal transduction ATPases with numerous domains (STAND), a large class of P-loop NTPases, belong to AAA+ ATPases. They include AP(apoptotic)-ATPases (e.g., animal apoptosis regulators CED4/Apaf-1, plant disease resistance proteins, and bacterial AfsR-like transcription regulators), NACHT NTPases (e.g. CARD4, NAIP, Het-E-1, TLP1), and several other less well-characterized families. STAND differ from other P-loop NTPases by their unique sequence motifs, which include an hhGRExE (h, hydrophobic; x, any residue) motif at the N-terminal region, a GxP/GxxP motif at the C-terminal region of the NTPase domain, in addition to a C-terminal helical domain and additional domains such as WD40, TPR, LRR or catalytic modules. Despite significant biological interests, structural coverage of STAND proteins is very limited and only two other structures are currently known: the cell death regulators Apaf-1 and CED-4. Here, we report the crystal structure of SSO1545 from Sulfolobus solfataricus, which was determined using the semi-automated, high-throughput pipeline of the Joint Center for Structural Genomics (JCSG; http://www.jcsg.org), as part of the National Institute of General Medical Sciences' Protein Structure Initiative (PSI). SSO1545 (NP-342973.1), a representative of the archaeal STANDs, is a member of Pfam PF01637 and encodes a protein of 356 residues with calculated molecular weight and isoelectric point of 41.7more » kD and 8.2, respectively.« less

The Nod-Like Receptor (NLR) Family: A Tale of Similarities and Differences

Innate immunity represents an important system with a variety of vital processes at the core of many diseases. In recent years, the central role of the Nod-like receptor (NLR) protein family became increasingly appreciated in innate immune responses. NLRs are classified as part of the signal transduction ATPases with numerous domains (STAND) clade within the AAA+ ATPase family. They typically feature an N-terminal effector domain, a central nucleotide-binding domain (NACHT) and a C-terminal ligand-binding region that is composed of several leucine-rich repeats (LRRs). NLRs are believed to initiate or regulate host defense pathways through formation of signaling platforms that subsequently trigger the activation of inflammatory caspases and NF-kB. Despite their fundamental role in orchestrating key pathways in innate immunity, their mode of action in molecular terms remains largely unknown. Here we present the first comprehensive sequence and structure modeling analysis of NLR proteins, revealing that NLRs posses a domain architecture similar to the apoptotic initiator protein Apaf-1. Apaf-1 performs its cellular function by the formation of a heptameric platform, dubbed apoptosome, ultimately triggering the controlled demise of the affected cell. The mechanism of apoptosome formation by Apaf-1 potentially offers insight into the activation mechanisms of NLR proteins. Multiple sequence alignment analysis and homology modeling revealed Apaf-1-like structural features in most members of the NLR family, suggesting a similar biochemical behaviour in catalytic activity and oligomerization. Evolutionary tree comparisons substantiate the conservation of characteristic functional regions within the NLR family and are in good agreement with domain distributions found in distinct NLRs. Importantly, the analysis of LRR domains reveals surprisingly low conservation levels among putative ligand-binding motifs. The same is true for the effector domains exhibiting distinct interfaces ensuring specific interactions with downstream target proteins. All together these factors suggest specific biological functions for individual NLRs.

ADP for ATP Exchange Mechanism

The signal transduction ATPases with numerous domains (STAND) family of proteins can be subdivided, according to the characteristics of the nucleotide-binding oligomerization domain (NOD), into three groups: the AP subfamily that includes APAF-1, an apoptotic mediator; the NACHT subfamily members that are involved in mammalian innate immunity; and the MalT subfamily of bacterial transcription factors. Although the presence of ATP is critical for the activation of these proteins, which are generally monomeric in the inactive state and oligomerize upon activation, it has been unclear what role ATP plays in this process and whether ATP hydrolysis was involved. Marquenet and Richet determined that the Escherichia coli MalT transcription factor cycles between an ADP-bound inactive form and an ATP-bound active form and that intrinsic ATP hydrolysis was required to return the protein to the inactive state. This is quite similar to the mechanism by which heterotrimeric guanine nucleotide-binding proteins (G proteins) and small guanosine triphosphatases (GTPases) function, except that the latter bind guanine nucleotides instead of adenosine nucleotides. Endogenous inhibitors of MalT--MalY, Aes, and MalK--bind and stabilize the monomeric form and, in the presence of ATP, maltotriose stimulates the oligomerization and formation of active MalT. The authors compared the activities of mutant MalT proteins that bound, but could not hydrolyze ATP, with the activity of wild-type MalT in vivo. MalT-D129A was most extensively studied. In vivo, MalT-D129A was hyperactive, stimulating transcription of a reporter to higher levels than the wild-type protein, and the activity of the ATP hydrolysis mutant was also not inhibited by MalK or MalY. Gel filtration assays showed that MalT-D129A did not bind effectively to MalY and that MalT-D129A existed in an oligomeric state with no detectable monomers in the presence or absence of added maltotriose or ATP. The wild-type protein bound MalY and existed as only monomers in the absence of maltotriose and ATP and as both oligomeric and monomeric forms in the presence of maltotriose and ATP. Assays to detect the bound nucleotide showed that MalT-D129A was bound to ATP in a 1:1 stoichometry, suggesting that it purified in an ATP-bound state. In contrast, wild-type MalT in the absence of ATP and maltotriose, either alone or when bound to MalY, was bound to ADP. Maltotriose stimulated the exchange of ADP for ATP on wild-type MalT, which was detected either using nonhydrolyzable analogs of ATP or with radiolabeled ATP. Thus, maltotriose appears to switch MalT from an inactive ADP-bound form to an activated ATP-bound form, and hydrolysis of ATP is necessary for MalT cycling and regulation. It will be interesting to see if other NOD proteins show similar adenine nucleotide exchange and hydrolysis as part of their signaling mechanism. E. Marquenet, E. Richet, How integration pf positive and negative regulatory signals by a STAND signaling protein depends on ATP hydrolysis. Mol. Cell 28 , 187-199 (2007). [PubMed]

How Integration of Positive and Negative Regulatory Signals by a STAND Signaling Protein Depends on ATP Hydrolysis

The role of nucleotide hydrolysis in signaling by signal transduction ATPases with numerous domains (STAND) is poorly understood. Here we use MalT, the transcription activator of the Escherichia coli maltose regulon, as a model system to address this question. We have constructed the MalT-D129A variant that binds ATP but does not hydrolyze it and have characterized it in vivo and in vitro. ATP hydrolysis is not essential for transcription activation but is crucial in controlling MalT activity. MalT cycles between an ADP-bound, resting form that is the target of negative effectors and an ATP-bound, active form, which oligomerizes. Conversion to the active form involves nucleotide exchange and depends on maltotriose binding, whereas resetting to the inactive state relies on ATP hydrolysis, which ensues MalT multimerization. Such a controlled binary switch most likely applies to the other STAND NTPases, including Apaf-1 and the human innate immunity proteins NOD2, and CIAS1.

STAND, a Class of P-Loop NTPases Including Animal and Plant Regulators of Programmed Cell Death: Multiple, Complex Domain Architectures, Unusual Phyletic Patterns, and Evolution by Horizontal Gene Transfer

Using sequence profile analysis and sequence-based structure predictions, we define a previously unrecognized, widespread class of P-loop NTPases. The signal transduction ATPases with numerous domains (STAND) class includes the AP-ATPases (animal apoptosis regulators CED4/Apaf-1, plant disease resistance proteins, and bacterial AfsR-like transcription regulators) and NACHT NTPases (e.g. NAIP, TLP1, Het-E-1) that have been studied extensively in the context of apoptosis, pathogen response in animals and plants, and transcriptional regulation in bacteria. We show that, in addition to these well-characterized protein families, the STAND class includes several other groups of (predicted) NTPase domains from diverse signaling and transcription regulatory proteins from bacteria and eukaryotes, and three Archaea-specific families. We identified the STAND domain in several biologically well-characterized proteins that have not been suspected to have NTPase activity, including soluble adenylyl cyclases, nephrocystin 3 (implicated in polycystic kidney disease), and Rolling pebble (a regulator of muscle development); these findings are expected to facilitate elucidation of the functions of these proteins. The STAND class belongs to the additional strand, catalytic E division of P-loop NTPases together with the AAA+ ATPases, RecA/helicase-related ATPases, ABC-ATPases, and VirD4/PilT-like ATPases. The STAND proteins are distinguished from other P-loop NTPases by the presence of unique sequence motifs associated with the N-terminal helix and the core strand-4, as well as a C-terminal helical bundle that is fused to the NTPase domain. This helical module contains a signature GxP motif in the loop between the two distal helices. With the exception of the archaeal families, almost all STAND NTPases are multidomain proteins containing three or more domains. In addition to the NTPase domain, these proteins typically contain DNA-binding or protein-binding domains, superstructure-forming repeats, such as WD40 and TPR, and enzymatic domains involved in signal transduction, including adenylate cyclases and kinases. By analogy to the AAA+ ATPases, it can be predicted that STAND NTPases use the C-terminal helical bundle as a "lever" to transmit the conformational changes brought about by NTP hydrolysis to effector domains. STAND NTPases represent a novel paradigm in signal transduction, whereby adaptor, regulatory switch, scaffolding, and, in some cases, signal-generating moieties are combined into a single polypeptide. The STAND class consists of 14 distinct families, and the evolutionary history of most of these families is riddled with dramatic instances of lineage-specific expansion and apparent horizontal gene transfer. The STAND NTPases are most abundant in developmentally and organizationally complex prokaryotes and eukaryotes. Transfer of genes for STAND NTPases from bacteria to eukaryotes on several occasions might have played a significant role in the evolution of eukaryotic signaling systems.