RESUMEN
Transition metals, such as zinc, are essential micronutrients in all organisms, but also highly toxic in excessive amounts. Heavy-metal transporting P-type (PIB) ATPases are crucial for homeostasis, conferring cellular detoxification and redistribution through transport of these ions across cellular membranes. No structural information is available for the PIB-4-ATPases, the subclass with the broadest cargo scope, and hence even their topology remains elusive. Here, we present structures and complementary functional analyses of an archetypal PIB-4-ATPase, sCoaT from Sulfitobacter sp. NAS14-1. The data disclose the architecture, devoid of classical so-called heavy-metal-binding domains (HMBDs), and provide fundamentally new insights into the mechanism and diversity of heavy-metal transporters. We reveal several novel P-type ATPase features, including a dual role in heavy-metal release and as an internal counter ion of an invariant histidine. We also establish that the turnover of PIB-ATPases is potassium independent, contrasting to many other P-type ATPases. Combined with new inhibitory compounds, our results open up for efforts in for example drug discovery, since PIB-4-ATPases function as virulence factors in many pathogens.
Heavy metals such as zinc and cobalt are toxic at high levels, yet most organisms need tiny amounts for their cells to work properly. As a result, proteins studded through the cell membrane act as gatekeepers to finetune import and export. These proteins are central to health and disease; their defect can lead to fatal illnesses in humans, and they also help bacteria infect other organisms. Despite their importance, little is known about some of these metal-export proteins. This is particularly the case for PIB-4-ATPases, a subclass found in plants and bacteria and which includes, for example, a metal transporter required for bacteria to cause tuberculosis. Intricate knowledge of the three-dimensional structure of these proteins would help to understand how they select metals, shuttle the compounds in and out of cells, and are controlled by other cellular processes. To reveal this three-dimensional organisation, Grønberg et al. used X-ray diffraction, where high-energy radiation is passed through crystals of protein to reveal the positions of atoms. They focused on a type of PIB-4-ATPases found in bacteria as an example. The work showed that the protein does not contain the metal-binding regions seen in other classes of metal exporters; however, it sports unique features that are crucial for metal transport such as an adapted pathway for the transport of zinc and cobalt across the membrane. In addition, Grønberg et al. tested thousands of compounds to see if they could block the activity of the protein, identifying two that could kill bacteria. This better understanding of how PIB-4-ATPases work could help to engineer plants capable of removing heavy metals from contaminated soils, as well as uncover new compounds to be used as antibiotics.
Asunto(s)
Iones/metabolismo , Metales Pesados/metabolismo , ATPasas Tipo P/química , ATPasas Tipo P/metabolismo , Rhodobacteraceae/enzimología , Sitios de Unión , Transporte Biológico , Proteínas de Transporte de Catión/metabolismo , Modelos Moleculares , ATPasas Tipo P/clasificación , Conformación Proteica , Rhodobacteraceae/clasificación , Zinc/metabolismoRESUMEN
P4-ATPases, a subfamily of P-type ATPases, translocate cell membrane phospholipids from the exoplasmic/luminal leaflet to the cytoplasmic leaflet to generate and maintain membrane lipid asymmetry. Exposure of phosphatidylserine (PS) in the exoplasmic leaflet is well known to transduce critical signals for apoptotic cell clearance and platelet coagulation. PS exposure is also involved in many other biological processes, including myoblast and osteoclast fusion, and the immune response. Moreover, mounting evidence suggest that PS exposure is critical for neuronal regeneration and degeneration. In apoptotic cells, PS exposure is induced by irreversible activation of scramblases and inactivation of P4-ATPases. However, how PS is reversibly exposed and restored in viable cells during other biological processes remains poorly understood. In the present review, we discuss the physiological significance of reversible PS exposure in living cells, and the putative roles of flippases, floppases, and scramblases.
Asunto(s)
Membrana Celular/metabolismo , Citoplasma/metabolismo , ATPasas Tipo P/metabolismo , Fosfatidilserinas/metabolismo , Animales , Apoptosis/fisiología , Supervivencia Celular/fisiología , Humanos , Membrana Dobles de Lípidos/metabolismo , ATPasas Tipo P/clasificación , Proteínas de Transferencia de Fosfolípidos/metabolismo , Activación Plaquetaria/fisiología , Especificidad por SustratoRESUMEN
P-type ATPases are integral membrane transporters that play important roles in transmembrane transport in plants. However, a comprehensive analysis of the P-type ATPase gene family has not been conducted in Chinese white pear (Pyrus bretschneideri) or other Rosaceae species. Here, we identified 419 P-type ATPase genes from seven Rosaceae species (Pyrus bretschneideri, Malus domestica, Prunus persica, Fragaria vesca, Prunus mume, Pyrus communis and Pyrus betulifolia). Structural and phylogenetic analyses revealed that P-type ATPase genes can be divided into five subfamilies. Different subfamilies have different conserved motifs and cis-acting elements, which may lead to functional divergence within one gene family. Dispersed duplication and whole-genome duplication may play critical roles in the expansion of the P-type ATPase family. Purifying selection was the primary force driving the evolution of P-type ATPase family genes. Based on the dynamic transcriptome analysis and transient transformation of Chinese white pear fruit, Pbr029767.1 in the P3A subfamily were found to be associated with malate accumulation during pear fruit development. Using a co-expression network, we identified several transcription factors that may have regulatory relationships with the P-type ATPase gene family. Overall, this study lays a solid foundation for understanding the evolution and functions of P-type ATPase genes in Chinese white pear and six other Rosaceae species.
Asunto(s)
ATPasas Tipo P/genética , Proteínas de Plantas/genética , Pyrus/genética , Mapeo Cromosómico , Evolución Molecular , Duplicación de Gen , Redes Reguladoras de Genes , Malatos/metabolismo , Familia de Multigenes , Motivos de Nucleótidos , ATPasas Tipo P/clasificación , ATPasas Tipo P/metabolismo , Proteínas de Plantas/clasificación , Proteínas de Plantas/metabolismo , Regiones Promotoras Genéticas , Pyrus/crecimiento & desarrollo , Pyrus/metabolismo , Rosaceae/genéticaRESUMEN
P-type ATPases are critical to the maintenance and regulation of cellular ion homeostasis and membrane lipid asymmetry due to their ability to move ions and phospholipids against a concentration gradient by utilizing the energy of ATP hydrolysis. P-type ATPases are particularly relevant in human pathogenic trypanosomatids which are exposed to abrupt and dramatic changes in their external environment during their life cycles. This review describes the complete inventory of ion-motive, P-type ATPase genes in the human pathogenic Trypanosomatidae; eight Leishmania species (L. aethiopica, L. braziliensis, L. donovani, L. infantum, L. major, L. mexicana, L. panamensis, L. tropica), Trypanosoma cruzi and three Trypanosoma brucei subspecies (Trypanosoma brucei brucei TREU927, Trypanosoma brucei Lister strain 427, Trypanosoma brucei gambiense DAL972). The P-type ATPase complement in these trypanosomatids includes the P1B (metal pumps), P2A (SERCA, sarcoplasmic-endoplasmic reticulum calcium ATPases), P2B (PMCA, plasma membrane calcium ATPases), P2D (Na+ pumps), P3A (H+ pumps), P4 (aminophospholipid translocators), and P5B (no assigned specificity) subfamilies. These subfamilies represent the P-type ATPase transport functions necessary for survival in the Trypanosomatidae as P-type ATPases for each of these seven subfamilies are found in all Leishmania and Trypanosoma species included in this analysis. These P-type ATPase subfamilies are correlated with current molecular and biochemical knowledge of their function in trypanosomatid growth, adaptation, infectivity, and survival.
TITLE: Les ATPases de transport de type P chez Leishmania et Trypanosoma. ABSTRACT: Les ATPases de type P sont essentielles au maintien et à la régulation de l'homéostasie des ions cellulaires et de l'asymétrie des lipides membranaires en raison de leur capacité à déplacer les ions et les phospholipides contre un gradient de concentration en utilisant l'énergie de l'hydrolyse de l'ATP. Les ATPases de type P sont particulièrement utiles chez les trypanosomatidés pathogènes pour l'homme, qui sont exposés à des changements abrupts et dramatiques de leur environnement externe au cours de leur cycle de vie. Cette revue décrit l'inventaire complet des gènes d'ATPase de type P à motif ionique chez les Trypanosomatidae pathogènes pour l'homme ; huit espèces de Leishmania (L. aethiopica, L. braziliensis, L. donovani, L. infantum, L. major, L. mexicana, L. panamensis, L. tropica), Trypanosoma cruzi et trois sous-espèces de Trypanosoma brucei (Trypanosoma brucei brucei TREU927, Trypanosoma brucei Lister souche 427, Trypanosoma brucei gambiense DAL972). Le complément ATPase de type P dans ces trypanosomatidés comprend les sous-familles P1B (pompes métalliques), P2A (SERCA, ATPases calciques du réticulum sarcoplasmo-endoplasmique), P2B (PMCA, ATPases calciques de la membrane plasmique), P2D (pompes Na+), P3A (pompes H+), P4 (translocateurs des aminophospholipides) et P5B (sans spécificité attribuée). Ces sous-familles représentent les fonctions de transport des ATPases de type P nécessaires à la survie des trypanosomatidés, car les ATPases de type P de chacune de ces sept sous-familles sont présentes chez toutes les espèces de Leishmania et de Trypanosoma incluses dans cette analyse. Ces sous-familles d'ATPases de type P sont corrélées aux connaissances moléculaires et biochimiques actuelles sur leur fonction dans la croissance, l'adaptation, l'infectivité et la survie des trypanosomatidés.
Asunto(s)
Leishmania/enzimología , Leishmania/genética , ATPasas Tipo P/genética , Trypanosoma/enzimología , Trypanosoma/genética , Genoma de Protozoos , ATPasas Tipo P/clasificaciónRESUMEN
The P-type ATPases (P-ATPases) are present in all living cells where they mediate ion transport across membranes on the expense of ATP hydrolysis. Different ions which are transported by these pumps are protons like calcium, sodium, potassium, and heavy metals such as manganese, iron, copper, and zinc. Maintenance of the proper gradients for essential ions across cellular membranes makes P-ATPases crucial for cell survival. In this study, characterization of two families of P-ATPases including P-ATPase 13A1 and P-ATPase 13A3 protein was compared in two different insect species from different orders. According to the conserved motifs found with MEME, nine motifs were shared by insects of 13A1 family but eight in 13A3 family. Seven different insect species from 13A1 and five samples from 13A3 family were selected as the representative samples for functional and structural analyses. The structural and functional analyses were performed with ProtParam, SOPMA, SignalP 4.1, TMHMM 2.0, ProtScale and ProDom tools in the ExPASy database. The tertiary structure of Bombus terrestris as a sample of each family of insects were predicted by the Phyre2 and TM-score servers and their similarities were verified by SuperPose server. The tertiary structures were predicted via the "c3b9bA" model (PDB Accession Code: 3B9B) in P-ATPase 13A1 family and "c2zxeA" model (PDB Accession Code: 2ZXE) in P-ATPase 13A3 family. A phylogenetic tree was constructed with MEGA 6.06 software using the Neighbor-joining method. According to the results, there was a high identity of P-ATPase families so that they should be derived from a common ancestor however they belonged to separate groups. In protein-protein interaction analysis by STRING 10.0, six common enriched pathways of KEGG were identified in B. terrestris in both families. The obtained data provide a background for bioinformatic studies of the function and evolution of other insects and organisms.