Comprehensive classification of ABC ATPases and their functional radiation in nucleoprotein dynamics and biological conflict systems.

ABC ATPases form one of the largest clades of P-loop NTPase fold enzymes that catalyze ATP-hydrolysis and utilize its free energy for a staggering range of functions from transport to nucleoprotein dynamics. Using sensitive sequence and structure analysis with comparative genomics, for the first time we provide a comprehensive classification of the ABC ATPase superfamily. ABC ATPases developed structural hallmarks that unambiguously distinguish them from other P-loop NTPases such as an alternative to arginine-finger-based catalysis. At least five and up to eight distinct clades of ABC ATPases are reconstructed as being present in the last universal common ancestor. They underwent distinct phases of structural innovation with the emergence of inserts constituting conserved binding interfaces for proteins or nucleic acids and the adoption of a unique dimeric toroidal configuration for DNA-threading. Specifically, several clades have also extensively radiated in counter-invader conflict systems where they serve as nodal nucleotide-dependent sensory and energetic components regulating a diversity of effectors (including some previously unrecognized) acting independently or together with restriction-modification systems. We present a unified mechanism for ABC ATPase function across disparate systems like RNA editing, translation, metabolism, DNA repair, and biological conflicts, and some unexpected recruitments, such as MutS ATPases in secondary metabolism.

Mycoplasma promotes malignant transformation in vivo, and its DnaK, a bacterial chaperone protein, has broad oncogenic properties.

We isolated a strain of human mycoplasma that promotes lymphomagenesis in SCID mice, pointing to a p53-dependent mechanism similar to lymphomagenesis in uninfected p53-/- SCID mice. Additionally, mycoplasma infection in vitro reduces p53 activity. Immunoprecipitation of p53 in mycoplasma-infected cells identified several mycoplasma proteins, including DnaK, a member of the Hsp70 chaperon family. We focused on DnaK because of its ability to interact with proteins. We demonstrate that mycoplasma DnaK interacts with and reduces the activities of human proteins involved in critical cellular pathways, including DNA-PK and PARP1, which are required for efficient DNA repair, and binds to USP10 (a key p53 regulator), impairing p53-dependent anticancer functions. This also reduced the efficacy of anticancer drugs that depend on p53 to exert their effect. mycoplasma was detected early in the infected mice, but only low copy numbers of mycoplasma DnaK DNA sequences were found in some primary and secondary tumors, pointing toward a hit-and-run/hide mechanism of transformation. Uninfected bystander cells took up exogenous DnaK, suggesting a possible paracrine function in promoting malignant transformation, over and above cells infected with the mycoplasma. Phylogenetic amino acid analysis shows that other bacteria associated with human cancers have similar DnaKs, consistent with a common mechanism of cellular transformation mediated through disruption of DNA-repair mechanisms, as well as p53 dysregulation, that also results in cancer-drug resistance. This suggests that the oncogenic properties of certain bacteria are DnaK-mediated.

ATPase and GTPase Tangos Drive Intracellular Protein Transport.

The GTPase superfamily of proteins provides molecular switches to regulate numerous cellular processes. The 'GTPase switch' paradigm, in which external regulatory factors control the switch of a GTPase between 'on' and 'off' states, has been used to interpret the regulatory mechanism of many GTPases. However, recent work unveiled a class of nucleotide hydrolases that do not adhere to this classical paradigm. Instead, they use nucleotide-dependent dimerization cycles to regulate key cellular processes. In this review article, recent studies of dimeric GTPases and ATPases involved in intracellular protein targeting are summarized. It is suggested that these proteins can use the conformational plasticity at their dimer interface to generate multiple points of regulation, thereby providing the driving force and spatiotemporal coordination of complex cellular pathways.

[In silico Analyses of Transcriptomes of the Marine Green Microalga Dunaliella tertiolecta: Identification of Sequences Encoding P-type ATPases].

De novo assembled transcriptomes of the marine microalga Dunaliella tertiolecta (Chlorophyta) were analyzed. Transcriptome assemblies were performed using short-read RNA-seq data deposited in the SRA database (DNA and RNA Sequence Read Archive, NCBI). A merged transcriptome was assembled using a pooled RNA-seq data set. The goal of the study was in silico identification of nucleotide sequences encoding P-type ATPases in D. tertiolecta transcriptomes. P-type ATPases play a considerable role in the adaptation of an organism to a variable environment, and this problem is particularly significant for microalgae inhabiting an environment with an unstable ionic composition. Particular emphasis was given to searching for a sequence coding Na^(+)-ATPase. This enzyme is expected to function in the plasma membrane of D. tertiolecta like in some marine algae, in particular, in the closely related alga Dunaliella maritima. An ensemble of 12 P-type ATPases consisting of members belonging to the five main subfamilies of the P-type ATPase family was revealed in the assembled transcriptomes. The genes of the following P-type ATPases were found: (1) heavy metal ATPases (subfamily PIB); (2) Ca^(2+)-ATPases of SERCA type (subfamily P2A); (3) H^(+)-ATPases (subfamily P3); (4) phospholipid-transporting ATPases (flippases) (subfamily P4); (5) cation-transporting ATPases of uncertain specificities (subfamily P5). The presence of functional Na^(+)-ATPases in marine algae is presently undoubted. However, contrary to expectations, we failed to find a nucleotide sequence encoding a protein that could unequivocally be considered a Na^(+)-ATPase. Further study is necessary to elucidate the roles of in silico revealed D. tertiolecta ATPases in Na^(+) transport.

Distinguishing between biochemical and cellular function: Are there peptide signatures for cellular function of proteins?

The genome annotation and identification of gene function depends on conserved biochemical activity. However, in the cell, proteins with the same biochemical function can participate in different cellular pathways and cannot complement one another. Similarly, two proteins of very different biochemical functions are put in the same class of cellular function; for example, the classification of a gene as an oncogene or a tumour suppressor gene is not related to its biochemical function, but is related to its cellular function. We have taken an approach to identify peptide signatures for cellular function in proteins with known biochemical function. ATPases as a test case, we classified ATPases (2360 proteins) and kinases (517 proteins) from the human genome into different cellular function categories such as transcriptional, replicative, and chromatin remodelling proteins. Using publicly available tool, MEME, we identify peptide signatures shared among the members of a given category but not between cellular functional categories; for example, no motif sharing is seen between chromatin remodelling and transporter ATPases, similarly between receptor Serine/Threonine Kinase and Receptor Tyrosine Kinase. There are motifs shared within each category with significant E value and high occurrence. This concept of signature for cellular function was applied to developmental regulators, the polycomb and trithorax proteins which led to the prediction of the role of INO80, a chromatin remodelling protein, in development. This has been experimentally validated earlier for its role in homeotic gene regulation and its interaction with regulatory complexes like the Polycomb and Trithorax complex. Proteins 2017; 85:682-693. © 2016 Wiley Periodicals, Inc.

Primary endosymbiosis events date to the later Proterozoic with cross-calibrated phylogenetic dating of duplicated ATPase proteins.

Chloroplasts and mitochondria descended from bacterial ancestors, but the dating of these primary endosymbiosis events remains very uncertain, despite their importance for our understanding of the evolution of both bacteria and eukaryotes. All phylogenetic dating in the Proterozoic and before is difficult: Significant debates surround potential fossil calibration points based on the interpretation of the Precambrian microbial fossil record, and strict molecular clock methods cannot be expected to yield accurate dates over such vast timescales because of strong heterogeneity in rates. Even with more sophisticated relaxed-clock analyses, nodes that are distant from fossil calibrations will have a very high uncertainty in dating. However, endosymbiosis events and gene duplications provide some additional information that has never been exploited in dating; namely, that certain nodes on a gene tree must represent the same events, and thus must have the same or very similar dates, even if the exact date is uncertain. We devised techniques to exploit this information: cross-calibration, in which node date calibrations are reused across a phylogeny, and cross-bracing, in which node date calibrations are formally linked in a hierarchical Bayesian model. We apply these methods to proteins with ancient duplications that have remained associated and originated from plastid and mitochondrial endosymbionts: the α and ß subunits of ATP synthase and its relatives, and the elongation factor thermo unstable. The methods yield reductions in dating uncertainty of 14-26% while only using date calibrations derived from phylogenetically unambiguous Phanerozoic fossils of multicellular plants and animals. Our results suggest that primary plastid endosymbiosis occurred ∼900 Mya and mitochondrial endosymbiosis occurred ∼1,200 Mya.

Structural Features Reminiscent of ATP-Driven Protein Translocases Are Essential for the Function of a Type III Secretion-Associated ATPase.

UNLABELLED: Many bacterial pathogens and symbionts utilize type III secretion systems to interact with their hosts. These machines have evolved to deliver bacterial effector proteins into eukaryotic target cells to modulate a variety of cellular functions. One of the most conserved components of these systems is an ATPase, which plays an essential role in the recognition and unfolding of proteins destined for secretion by the type III pathway. Here we show that structural features reminiscent of other ATP-driven protein translocases are essential for the function of InvC, the ATPase associated with a Salmonella enterica serovar Typhimurium type III secretion system. Mutational and functional analyses showed that a two-helix-finger motif and a conserved loop located at the entrance of and within the predicted pore formed by the hexameric ATPase are essential for InvC function. These findings provide mechanistic insight into the function of this highly conserved component of type III secretion machines. IMPORTANCE: Type III secretion machines are essential for the virulence or symbiotic relationships of many bacteria. These machines have evolved to deliver bacterial effector proteins into host cells to modulate cellular functions, thus facilitating bacterial colonization and replication. An essential component of these machines is a highly conserved ATPase, which is necessary for the recognition and secretion of proteins destined to be delivered by the type III secretion pathway. Using modeling and structure and function analyses, we have identified structural features of one of these ATPases from Salmonella enterica serovar Typhimurium that help to explain important aspects of its function.

An ENA ATPase, MaENA1, of Metarhizium acridum influences the Na(+)-, thermo- and UV-tolerances of conidia and is involved in multiple mechanisms of stress tolerance.

In fungi, ENA ATPases play key roles in osmotic and alkaline pH tolerance, although their functions in thermo- and UV-tolerances have not been explored. Entomopathogenic fungi are naturally widespread and have considerable potential in pest control. An ENA ATPase gene, MaENA1, from the entomopathogenic fungus Metarhizium acridum was functionally analyzed by deletion. MaENA1-disruption strain (ΔMaENA1) was less tolerant to NaCl, heat, and UV radiation than a wild-type strain (WT). Digital Gene Expression profiling of conidial RNAs resulted in 281 differentially expressed genes (DEGs) between the WT and ΔMaENA1 strains. Eighty-five DEGs, 56 of which were down-regulated in the ΔMaENA1 strain, were shown to be associated with heat/UV tolerance, including six cytochrome P450 superfamily genes, 35 oxidoreductase genes, 24 ion-binding genes, seven DNA repair genes, and five other genes. In addition, eight genes were components of stress responsive pathways, including the Ras-cAMP PKA pathway, the RIM101 pathway, the Ca(2+)/calmodulin pathway, the TOR pathway, and the HOG/Spc1/Sty1/JNK pathway. These results demonstrated that MaENA1 influences fungal tolerances to Na(+), heat, and UV radiation in M. acridum, and is involved in multiple mechanisms of stress tolerance. Therefore, MaENA1 is required for the adaptation and survival of entomopathogenic fungi in stressful conditions in the environment and in their hosts.

Structural basis for the mechanism of ABC transporters.

The ATP-binding cassette (ABC) transporters are primary transporters that couple the energy stored in adenosine triphosphate (ATP) to the movement of molecules across the membrane. ABC transporters can be divided into exporters and importers; importers mediate the uptake of essential nutrients into cells and are found predominantly in prokaryotes whereas exporters transport molecules out of cells or into organelles and are found in all organisms. ABC exporters have been linked with multi-drug resistance in both bacterial and eukaryotic cells. ABC transporters are powered by the hydrolysis of ATP and transport their substrate via the alternating access mechanism, whereby the protein alternates between a conformation in which the substrate-binding site is accessible from the outside of the membrane, outward-facing and one in which it is inward-facing. In this mini-review, the structures of different ABC transporter types in different conformations are presented within the context of the alternating access mechanism and how they have shaped our current understanding of the mechanism of ABC transporters.

Tracking in atomic detail the functional specializations in viral RecA helicases that occur during evolution.

Many complex viruses package their genomes into empty protein shells and bacteriophages of the Cystoviridae family provide some of the simplest models for this. The cystoviral hexameric NTPase, P4, uses chemical energy to translocate single-stranded RNA genomic precursors into the procapsid. We previously dissected the mechanism of RNA translocation for one such phage, 12, and have now investigated three further highly divergent, cystoviral P4 NTPases (from 6, 8 and 13). High-resolution crystal structures of the set of P4s allow a structure-based phylogenetic analysis, which reveals that these proteins form a distinct subfamily of the RecA-type ATPases. Although the proteins share a common catalytic core, they have different specificities and control mechanisms, which we map onto divergent N- and C-terminal domains. Thus, the RNA loading and tight coupling of NTPase activity with RNA translocation in 8 P4 is due to a remarkable C-terminal structure, which wraps right around the outside of the molecule to insert into the central hole where RNA binds to coupled L1 and L2 loops, whereas in 12 P4, a C-terminal residue, serine 282, forms a specific hydrogen bond to the N7 of purines ring to confer purine specificity for the 12 enzyme.

[GABA(A)-Coupled Cl-/HCO3(-)-ATPase: Candidate for an Novel Primary Active Transporter in Neuronal Membranes].

Cl(-)-transport systems in cell membranes from various origins (including neurons) play an important role in different processes of their vital functions. Various transport mechanisms involved in the maintenance of intracellular concentration of Cl- that differs from concentration equilibrium have been considered. This review provides the biochemical properties of the GABA(A)-coupled Cl-/HCO3(-)-ATPase which is a candidate for an novel primary active system in neuronal membranes. Special emphasis has been placed on a review of the prerequisites for the existence of the GABA(A)-coupled ATPase. This work provides data for the benefit not only functional but also the alleged structural coupling of the enzyme with GABA(A)-receptors. It is concluded on the importance of the found ATPase in primary active transport processes across the plasma membrane of neuronal cells with different level of the organization.

A member of the heavy metal P-type ATPase OsHMA5 is involved in xylem loading of copper in rice.

Heavy metal-transporting P-type ATPase (HMA) has been implicated in the transport of heavy metals in plants. Here, we report the function and role of an uncharacterized member of HMA, OsHMA5 in rice (Oryza sativa). Knockout of OsHMA5 resulted in a decreased copper (Cu) concentration in the shoots but an increased Cu concentration in the roots at the vegetative stage. At the reproductive stage, the concentration of Cu in the brown rice was significantly lower in the mutants than in the wild-type rice; however, there was no difference in the concentrations of iron, manganese, and zinc between two independent mutants and the wild type. The Cu concentration of xylem sap was lower in the mutants than in the wild-type rice. OsHMA5 was mainly expressed in the roots at the vegetative stage but also in nodes, peduncle, rachis, and husk at the reproductive stage. The expression was up-regulated by excess Cu but not by the deficiency of Cu and other metals, including zinc, iron, and manganese, at the vegetative stage. Analysis of the transgenic rice carrying the OsHMA5 promoter fused with green fluorescent protein revealed that it was localized at the root pericycle cells and xylem region of diffuse vascular bundles in node I, vascular tissues of peduncle, rachis, and husk. Furthermore, immunostaining with an antibody against OsHMA5 revealed that it was localized to the plasma membrane. Expression of OsHMA5 in a Cu transport-defective mutant yeast (Saccharomyces cerevisiae) strain restored the growth. Taken together, OsHMA5 is involved in loading Cu to the xylem of the roots and other organs.

Diversity of the metal-transporting P1B-type ATPases.

The P1B-ATPases are integral membrane proteins that couple ATP hydrolysis to metal cation transport. Widely distributed across all domains of life, these enzymes have been previously shown to transport copper, zinc, cobalt, and other thiophilic heavy metals. Recent data suggest that these enzymes may also be involved in nickel and/or iron transport. Here we have exploited large amounts of genomic data to examine and classify the various P1B-ATPase subfamilies. Specifically, we have combined new methods of data partitioning and network visualization known as Transitivity Clustering and Protein Similarity Networks with existing biochemical data to examine properties such as length, speciation, and metal-binding motifs of the P1B-ATPase subfamily sequences. These data reveal interesting relationships among the enzyme sequences of previously established subfamilies, indicate the presence of two new subfamilies, and suggest the existence of new regulatory elements in certain subfamilies. Taken together, these findings underscore the importance of P1B-ATPases in homeostasis of nearly every biologically relevant transition metal and provide an updated framework for future studies.

Mycobacterium smegmatis SftH exemplifies a distinctive clade of superfamily II DNA-dependent ATPases with 3' to 5' translocase and helicase activities.

Bacterial DNA helicases are nucleic acid-dependent NTPases that play important roles in DNA replication, recombination and repair. We are interested in the DNA helicases of Mycobacteria, a genus of the phylum Actinobacteria, which includes the human pathogen Mycobacterium tuberculosis and its avirulent relative Mycobacterium smegmatis. Here, we identify and characterize M. smegmatis SftH, a superfamily II helicase with a distinctive domain structure, comprising an N-terminal NTPase domain and a C-terminal DUF1998 domain (containing a putative tetracysteine metal-binding motif). We show that SftH is a monomeric DNA-dependent ATPase/dATPase that translocates 3' to 5' on single-stranded DNA and has 3' to 5' helicase activity. SftH homologs are found in bacteria representing 12 different phyla, being especially prevalent in Actinobacteria (including M. tuberculosis). SftH homologs are evident in more than 30 genera of Archaea. Among eukarya, SftH homologs are present in plants and fungi.

Co-evolution of primordial membranes and membrane proteins.

Studies of the past several decades have provided major insights into the structural organization of biological membranes and mechanisms of many membrane molecular machines. However, the origin(s) of the membrane(s) and membrane proteins remains enigmatic. We discuss different concepts of the origin and early evolution of membranes with a focus on the evolution of the (im)permeability to charged molecules such as proteins, nucleic acids and small ions. Reconstruction of the evolution of F-type and A/V-type membrane ATPases (ATP synthases), which are either proton- or sodium-dependent, might help us to understand not only the origin of membrane bioenergetics but also of membranes themselves. We argue that evolution of biological membranes occurred as a process of co-evolution of lipid bilayers, membrane proteins and membrane bioenergetics.

Structure and function of the AAA+ nucleotide binding pocket.

Members of the diverse superfamily of AAA+ proteins are molecular machines responsible for a wide range of essential cellular processes. In this review we summarise structural and functional data surrounding the nucleotide binding pocket of these versatile complexes. Protein Data Bank (PDB) structures of closely related AAA+ ATPase are overlaid and biologically relevant motifs are displayed. Interactions between protomers are illustrated on the basis of oligomeric structures of each AAA+ subgroup. The possible role of conserved motifs in the nucleotide binding pocket is assessed with regard to ATP binding and hydrolysis, oligomerisation and inter-subunit communication. Our comparison indicates that in particular the roles of the arginine finger and sensor 2 residues differ subtly between AAA+ subgroups, potentially providing a means for functional diversification.

A non-mitotic CENP-E homolog in Dictyostelium discoideum with slow motor activity.

Kinesins are ATP-dependent molecular motors that mediate unidirectional intracellular transport along microtubules. Dictyostelium discoideum has 13 different kinesin isoforms including two members of the kinesin-7 family, Kif4 and Kif11. While Kif4 is structurally and functionally related to centromere-associated CENP-E proteins involved in the transport of chromosomes to the poles during mitosis, the function of the unusually short CENP-E variant Kif11 is unclear. Here we show that orthologs of short CENP-E variants are present in plants and fungi, and analyze functional properties of the Dictyostelium CENP-E version, Kif11. Gene knockout mutants reveal that Kif11 is not required for mitosis or development. Imaging of GFP-labeled Kif11 expressing Dictyostelium cells indicates that Kif11 is a plus-end directed motor that accumulates at microtubule plus ends. By multiple motor gliding assays, we show that Kif11 moves with an average velocity of 38nm/s, thus defining Kif11 as a very slow motor. The activity of the Kif11 motor appears to be modulated via interactions with the non-catalytic tail region. Our work highlights a subclass of kinesin-7-like motors that function outside of a role in mitosis.

A helminth cathelicidin-like protein suppresses antigen processing and presentation in macrophages via inhibition of lysosomal vATPase.

We previously reported the identification of a novel family of immunomodulatory proteins, termed helminth defense molecules (HDMs), that are secreted by medically important trematode parasites. Since HDMs share biochemical, structural, and functional characteristics with mammalian cathelicidin-like host defense peptides (HDPs), we proposed that HDMs modulate the immune response via molecular mimicry of host molecules. In the present study, we report the mechanism by which HDMs influence the function of macrophages. We show that the HDM secreted by Fasciola hepatica (FhHDM-1) binds to macrophage plasma membrane lipid rafts via selective interaction with phospholipids and/or cholesterol before being internalized by endocytosis. Following internalization, FhHDM-1 is rapidly processed by lysosomal cathepsin L to release a short C-terminal peptide (containing a conserved amphipathic helix that is a key to HDM function), which then prevents the acidification of the endolysosomal compartments by inhibiting vacuolar ATPase activity. The resulting endolysosomal alkalization impedes macrophage antigen processing and prevents the transport of peptides to the cell surface in conjunction with MHC class II for presentation to CD4(+) T cells. Thus, we have elucidated a novel mechanism by which helminth pathogens alter innate immune cell function to assist their survival in the host.

Characterization of a cobalt-specific P(1B)-ATPase.

The P(1B)-type ATPases are a ubiquitous family of P-type ATPases involved in the transport of transition metal ions. Divided into subclasses based on sequence characteristics and substrate specificity, these integral membrane transporters play key roles in metal homeostasis, metal tolerance, and the biosynthesis of metalloproteins. The P(1B-4)-ATPases have the simplest architecture of the five P(1B)-ATPase families and have been suggested to play a role in Co(2+) transport. A P(1B-4)-ATPase from Sulfitobacter sp. NAS-14.1, designated sCoaT, has been cloned, expressed, and purified. Activity assays indicate that sCoaT is specific for Co(2+). A single Co(2+) binding site is present, and optical, electron paramagnetic resonance, and X-ray absorption spectroscopic data are consistent with tetrahedral coordination by oxygen and nitrogen ligands, including a histidine and likely a water. Surprisingly, there is no evidence for coordination by sulfur. Mutation of a conserved cysteine residue, Cys 327, in the signature transmembrane Ser-Pro-Cys metal binding motif does not abolish the ATP hydrolysis activity or affect the spectroscopic analysis, establishing that this residue is not involved in the initial Co(2+) binding by sCoaT. In contrast, replacements of conserved transmembrane residues Ser 325, His 657, Glu 658, and Thr 661 with alanine abolish ATP hydrolysis activity and Co(2+) binding, indicating that these residues are necessary for Co(2+) transport. These data represent the first in vitro characterization of a P(1B-4)-ATPase and its Co(2+) binding site.

Structural divergence between the two subgroups of P5 ATPases.

Evolution of P5 type ATPases marks the origin of eukaryotes but still they remain the least characterized pumps in the superfamily of P-type ATPases. Phylogenetic analysis of available sequences suggests that P5 ATPases should be divided into at least two subgroups, P5A and P5B. P5A ATPases have been identified in the endoplasmic reticulum and seem to have basic functions in protein maturation and secretion. P5B ATPases localize to vacuolar/lysosomal or apical membranes and in animals play a role in hereditary neuronal diseases. Here we have used a bioinformatical approach to identify differences in the primary sequences between the two subgroups. P5A and P5B ATPases appear have a very different membrane topology from other P-type ATPases with two and one, respectively, additional transmembrane segments inserted in the N-terminal end. Based on conservation of residues in the transmembrane region, the two P5 subgroups most likely have different substrate specificities although these cannot be predicted from their sequences. Furthermore, sequence differences between P5A and P5B ATPases are identified in the catalytic domains that could influence key kinetic properties differentially. Together these findings indicate that P5A and P5B ATPases are structurally and functionally different.