A novel family of P-loop NTPases with an unusual phyletic distribution and transmembrane segments inserted within the NTPase domain

Abstract

Background: Recent sequence-structure studies on P-loop-fold NTPases have substantially advanced the existing understanding of their evolution and functional diversity. These studies provide a framework for characterization of novel lineages within this fold and prediction of their functional properties.

Results: Using sequence profile searches and homology-based structure prediction, we have identified a previously uncharacterized family of P-loop NTPases, which includes the neuronal membrane protein and receptor tyrosine kinase substrate Kidins220/ARMS, which is conserved in animals, the F-plasmid PifA protein involved in phage T7 exclusion, and several uncharacterized bacterial proteins. We refer to these (predicted) NTPases as the KAP family, after Kidins220/ARMS and PifA. The KAP family NTPases are sporadically distributed across a wide phylogenetic range in bacteria but among the eukaryotes are represented only in animals. Many of the prokaryotic KAP NTPases are encoded in plasmids and tend to undergo disruption to form pseudogenes. A unique feature of all eukaryotic and certain bacterial KAP NTPases is the presence of two or four transmembrane helices inserted into the P-loop NTPase domain. These transmembrane helices anchor KAP NTPases in the membrane such that the P-loop domain is located on the intracellular side. We show that the KAP family belongs to the same major division of the P-loop NTPase fold with the AAA+, ABC, RecA-like, VirD4-like, PilT-like, and AP/NACHT-like NTPase classes. In addition to the KAP family, we identified another small family of predicted bacterial NTPases, with two transmembrane helices inserted into the P-loop domain. This family is not specifically related to the KAP NTPases, suggesting independent acquisition of the transmembrane helices.

Conclusions: We predict that KAP family NTPases function principally in the NTP-dependent dynamics of protein complexes, especially those associated with the intracellular surface of cell membranes. Animal KAP NTPases, including Kidins220/ARMS, are likely to function as NTP-dependent regulators of the assembly of membrane-associated signaling complexes involved in neurite growth and development. One possible function of the prokaryotic KAP NTPases might be in the exclusion of selfish replicons, such as viruses, from the host cells. Phylogenetic analysis and phyletic patterns suggest that the common ancestor of the animals acquired a KAP NTPase via lateral transfer from bacteria. However, an earlier transfer into eukaryotes followed by multiple losses in several eukaryotic lineages cannot be ruled out.

Figure 1

Multiple alignment of the KAP family NTPases. The secondary structure predicted by the PHD program is displayed above the alignment, where E designates a β-strand and H designates α-helix. The helix and strand numbering is given for the secondary structural elements of the conserved P-loop fold. The 80% consensus coloring reflects the following amino acid classes: h (hydrophobic residues: ACFILMVWY), a (aromatic residues: FHWY), and l (aliphatic residues: VIL) are shaded yellow; b (big residues: LIYERFQKMW) are shaded gray; p (polar residues: CDEHKNQRST), - (acidic residues: DE), + (basic residues: HKR) and c (charged residues:HRKDE) are colored magenta; o (alcohol-group-containing residues: ST) are colored blue; s (small: GASCVDNPT) and u (tiny: GAS) residues are colored green. The protein identifiers in the alignment include the name of the protein/gene, species abbreviation and the GenBank gi separated by underscores. The groups discussed in the text are indicated to the right in the last block of the alignment. The asterisk next to the rat sequence indicates a Kidins paralog with a potentially inactive NTPase domain. Species abbreviations are as follows: Atu: Agrobacterium tumefaciens, Ana: Anabaena sp pcc 7120, Ce: Caenorhabditis elegans, Cpe: Clostridium perfringens, Cgl: Corynebacterium glutamicum, Ceff: Corynebacterium efficiens, Dr: Deinococcus radiodurans, Dm: Drosophila melanogaster, Ec: Escherichia coli, Plaf: F plasmid, Gsu: Geobacter sulfurreducens, Hs: Homo sapiens, Kpne: Klebsiella pneumoniae, Lme: Leuconostoc mesenteroides, Mcsp: Magnetococcus sp mc-1, Mde: Microbulbifer degradans, Npu: Nostoc punctiforme, Pput: Pseudomonas putida, Pfl: Pseudomonas fluorescens, Psy: Pseudomonas syringae, Rme: Ralstonia metallidurans, Rn: Rattus norvegicus, Step: Staphylococcus epidermidis, Ssp: Synechocystis sp, Tm: Thermotoga maritima, Vpar: Vibrio parahaemolyticus, Vvul: Vibrio vulnificus.

Figure 2

Predicted topology of the KAP P-loop NTPases and comparison with other P-loop NTPases. The core conserved strands that are shared by all ASCE division NTPases are numbered 1-5, and X indicates additional strands that are observed only in certain NTPases.

Figure 3

Phylogenetic tree and domain architectures of KAP NTPases. Proteins are denoted by their gene names and species abbreviations. Plasmid-borne genes are denoted by red asterisks, and phage genes are denoted by a red +; the eukaryotic branches are colored green. Species abbreviations are as in Figure 1. Filled yellow circles indicate nodes with bootstrap support of greater than 75% in the full maximum-likelihood analysis. The bootstrap values obtained through different methods (Full maximum likelihood, Rell bootstrap with Protml/Rell BP, Puzzle bootstrap/Puzzle-B, Neighbor Joining, Minimum evolution) are specifically shown for the clade that includes animal and bacterial proteins. In the schematics of protein and gene structure, conserved operons are shown as boxed arrow, and transmembrane regions inserted into the KAP domain are shown in blue. DRC0009-C and PifA-C refer to carboxy-terminal globular regions shared by the DRC0009-C and PifA subfamily KAP ATPases. Note that CPE1287 and Lmes0002 do not have the PifA-C domain.

Figure 4

Multiple alignment of the YobI family NTPases. The coloring scheme and labeling conventions are as in Figure 1. Species abbreviations are as follows: Bs: Bacillus subtilis, Bat: Bacteroides thetaiotaomicron, Cpe: Clostridium perfringens, Smu: Streptococcus mutans.