Displacement of the Na+/K+ pump's transmembrane domains demonstrates conserved conformational changes in P-type 2 ATPases.
Ontology highlight
ABSTRACT:
SUBMITTER: Young VC
PROVIDER: S-EPMC7923365 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Type-I transmembrane proteins represent a large group of 1,412 proteins in humans with a multitude of functions in cells and tissues. They are characterized by an extracellular, or luminal, N-terminus followed by a single transmembrane helix and a cytosolic C-terminus. The domain composition and structures of the extracellular and intercellular segments differ substantially amongst its members. Most of the type-I transmembrane proteins have roles in cell signaling processes, as ligands or receptors, and in cellular adhesion. The extracellular segment often determines specificity and can control signaling and adhesion. Here we focus on recent structural understanding on how the extracellular segments of several diverse type-I transmembrane proteins engage in interactions and can undergo conformational changes for their function. Interactions at the extracellular side by proteins on the same cell or between cells are enhanced by the transmembrane setting. Extracellular conformational domain rearrangement and structural changes within domains alter the properties of the proteins and are used to regulate signaling events. The combination of structural properties and interactions can support the formation of larger-order assemblies on the membrane surface that are important for cellular adhesion and intercellular signaling.
| S-EPMC7427315 | biostudies-literature
Project description:Zn(2+) is an essential transition metal required in trace amounts by all living organisms. However, metal excess is cytotoxic and leads to cell damage. Cells rely on transmembrane transporters, with the assistance of other proteins, to establish and maintain Zn(2+) homeostasis. Metal coordination during transport is key to specific transport and unidirectional translocation without the backward release of free metal. The coordination details of Zn(2+) at the transmembrane metal binding site responsible for transport have now been established. Escherichia coli ZntA is a well-characterized Zn(2+)-ATPase responsible for intracellular Zn(2+) efflux. A truncated form of the protein lacking regulatory metal sites and retaining the transport site was constructed. Metrical parameters of the metal-ligand coordination geometry for the zinc bound isolated form were characterized using x-ray absorption spectroscopy (XAS). Our data support a nearest neighbor ligand environment of (O/N)(2)S(2) that is compatible with the proposed invariant metal coordinating residues present in the transmembrane region. This ligand identification and the calculated bond lengths support a tetrahedral coordination geometry for Zn(2+) bound to the TM-MBS of P-type ATPase transporters.
| S-EPMC3319165 | biostudies-literature
Project description:Metabotropic glutamate receptors (mGluRs) are class C, synaptic G-protein-coupled receptors (GPCRs) that contain large extracellular ligand binding domains (LBDs) and form constitutive dimers. Despite the existence of a detailed picture of inter-LBD conformational dynamics and structural snapshots of both isolated domains and full-length receptors, it remains unclear how mGluR activation proceeds at the level of the transmembrane domains (TMDs) and how TMD-targeting allosteric drugs exert their effects. Here, we use time-resolved functional and conformational assays to dissect the mechanisms by which allosteric drugs activate and modulate mGluR2. Single-molecule subunit counting and inter-TMD fluorescence resonance energy transfer measurements in living cells reveal LBD-independent conformational rearrangements between TMD dimers during receptor modulation. Using these assays along with functional readouts, we uncover heterogeneity in the magnitude, direction, and the timing of the action of both positive and negative allosteric drugs. Together our experiments lead to a three-state model of TMD activation, which provides a framework for understanding how inter-subunit rearrangements drive class C GPCR activation.
| S-EPMC6588349 | biostudies-literature
Project description:Basic architecture of ABC transporters includes two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). Although the transport process takes place in the TMDs, which provide the substrate translocation pathway across the cell membrane and control its accessibility between the two sides of the membrane, the energy required for the process is provided by conformational changes induced in the NBDs by binding and hydrolysis of ATP. Nucleotide-dependent conformational changes in the NBDs, therefore, need to be coupled to structural changes in the TMDs. Using molecular dynamics simulations, we have investigated the structural elements involved in the conformational coupling between the NBDs and the TMDs in the Escherichia coli maltose transporter, an ABC importer for which an intact structure is available both in inward-facing and outward-facing conformations. The prevailing model of coupling is primarily based on a single structural motif, known as the coupling helices, as the main structural element for the NBD-TMD coupling. Surprisingly, we find that in the absence of the NBDs the coupling helices can be conformationally decoupled from the rest of the TMDs, despite their covalent connection. That is, the structural integrity of the coupling helices and their tight coupling to the core of the TMDs rely on the contacts provided by the NBDs. Based on the conformational and dynamical analysis of the simulation trajectories, we propose that the core coupling elements in the maltose transporter involve contributions from several structural motifs located at the NBD-TMD interface, namely, the EAA loops from the TMDs, and the Q-loop and the ENI motifs from the NBDs. These three structural motifs in small ABC importers show a high degree of correlation in motion and mediate the necessary conformational coupling between the core of TMDs and the helical subdomains of NBDs. A comprehensive analysis of the structurally known ABC transporters shows a high degree of conservation of the identified 3-motif coupling elements only in the subfamily of small ABC importers, suggesting a distinct mode of NBD-TMD coupling from the other two major ABC transporter folds, namely large ABC importers and ABC exporters.
| S-EPMC3145290 | biostudies-literature
Project description:Powered by ATP hydrolysis, P(IB) -ATPases drive the energetically uphill transport of transition metals. These high affinity pumps are essential for heavy metal detoxification and delivery of metal cofactors to specific cellular compartments. Amino acid sequence alignment of the trans-membrane (TM) helices of P(IB)-ATPases reveals a high degree of conservation, with ? 60-70 fully conserved positions. Of these conserved positions, 6-7 were previously identified to be important for transport. However, the functional importance of the majority of the conserved TM residues remains unclear. To investigate the role of conserved TM residues of P(IB)-ATPases we conducted an extensive mutagenesis study of a Zn(2+)/Cd(2+) P(IB)-ATPase from Rhizobium radiobacter (rrZntA) and seven other P(IB)-ATPases. Of the 38 conserved positions tested, 24 had small effects on metal tolerance. Fourteen mutations compromised in vivo metal tolerance and in vitro metal-stimulated ATPase activity. Based on structural modelling, the functionally important residues line a constricted 'channel', tightly surrounded by the residues that were found to be inconsequential for function. We tentatively propose that the distribution of the mutable and immutable residues marks a possible trans-membrane metal translocation pathway. In addition, by substituting six trans-membrane amino acids of rrZntA we changed the in vivo metal specificity of this pump from Zn(2+)/Cd(2+) to Ag(+).
| S-EPMC4285229 | biostudies-literature
Project description:Soil salinity is one of the major factors obstructing the growth and development of agricultural crops. Eukaryotes have two main transport systems involved in active Na+ removal: cation/H+ antiporters and Na+-P-type ATPases. Key transport proteins, Na+/K+-P-ATPases, are widely distributed among the different taxa families of pumps which are responsible for keeping cytosolic Na+ concentrations below toxic levels. Na+/K+-P-ATPases are considered to be absent in flowering plants. The data presented here are a complete inventory of P-type Na+/K+-P-ATPases in the major branches of the plant kingdom. We also attempt to elucidate the evolution of these important membrane pumps in plants in comparison with other organisms. We were able to observe the gradual replacement of the Na+-binding site to the Ca2+-binding site, starting with cyanobacteria and moving to modern land plants. Our results show that the ?-subunit likely evolved from one common ancestor to bacteria, fungi, plants, and mammals, whereas the ?-subunit did not evolve in green algae. In conclusion, our results strongly suggest the significant differences in the domain architecture and subunit composition of plant Na+/K+-P-ATPases depending on plant taxa and the salinity of the environment. The obtained data clarified and broadened the current views on the evolution of Na+/K+-P-ATPases. The results of this work would be helpful for further research on P-type ATPase functionality and physiological roles.
| S-EPMC7911474 | biostudies-literature
Project description:Na(+)-pumping NADH:ubiquinone oxidoreductase (Na(+)-NQR) is responsible for maintaining a sodium gradient across the inner bacterial membrane. This respiratory enzyme, which couples sodium pumping to the electron transfer between NADH and ubiquinone, is not present in eukaryotes and as such could be a target for antibiotics. In this paper it is shown that the site of ubiquinone reduction is conformationally coupled to the NqrB subunit, which also hosts the final cofactor in the electron transport chain, riboflavin. Previous work showed that mutations in conserved NqrB glycine residues 140 and 141 affect ubiquinone reduction and the proper functioning of the sodium pump. Surprisingly, these mutants did not affect the dissociation constant of ubiquinone or its analog HQNO (2-n-heptyl-4-hydroxyquinoline N-oxide) from Na(+)-NQR, which indicates that these residues do not participate directly in the ubiquinone binding site but probably control its accessibility. Indeed, redox-induced difference spectroscopy showed that these mutations prevented the conformational change involved in ubiquinone binding but did not modify the signals corresponding to bound ubiquinone. Moreover, data are presented that demonstrate the NqrA subunit is able to bind ubiquinone but with a low non-catalytically relevant affinity. It is also suggested that Na(+)-NQR contains a single catalytic ubiquinone binding site and a second site that can bind ubiquinone but is not active.
| S-EPMC4156058 | biostudies-literature
Project description:To gain insight into the steady-state and dynamic characteristics of structural rearrangements of an electrogenic secondary-active cotransporter during its transport cycle, two measures of conformational change (pre-steady-state current relaxations and intensity of fluorescence emitted from reporter fluorophores) were investigated as a function of membrane potential and external substrate. Cysteines were substituted at three believed-new sites in the type IIb Na(+)-coupled inorganic phosphate cotransporter (SLC34A2 flounder isoform) that were predicted to be involved in conformational changes. Labeling at one site resulted in substantial suppression of transport activity, whereas for the other sites, function remained comparable to the wild-type. For these mutants, the properties of the pre-steady-state charge relaxations were similar for each, whereas fluorescence intensity changes differed significantly. Fluorescence changes could be accounted for by simulations using a five-state model with a unique set of apparent fluorescence intensities assigned to each state according to the site of labeling. Fluorescence reported from one site was associated with inward and outward conformations, whereas for the other sites, including four previously indentified sites, emissions were associated principally with one or the other orientation of the transporter. The same membrane potential change induced complementary changes in fluorescence at some sites, which suggested that the microenvironments of the respective fluorophores experience concomitant changes in polarity. In response to step changes in voltage, the pre-steady-state current relaxation and the time course of change in fluorescence intensity were described by single exponentials. For one mutant the time constants matched well with and without external Na(+), providing direct evidence that this label reports conformational changes accompanying intrinsic charge movement and cation interactions.
| S-EPMC4008805 | biostudies-literature
Project description:Tryptophan residues critical to function are frequently located at the lipid-water interface of transmembrane domains. All members of the epithelial Na+ channel (ENaC)/Degenerin (Deg) channel superfamily contain an absolutely conserved Trp at the base of their first transmembrane domain. Here, we test the importance of this conserved Trp to ENaC/Deg function. Targeted substitution of this Trp in mouse ENaC and rat ASIC subunits decrease channel activity. Differential substitution with distinct amino acids in alpha-mENaC shows that it is loss of this critical Trp rather than introduction of residues having novel properties that changes channel activity. Surprisingly, Trp substitution unmasks voltage sensitivity. Mutant ENaC has increased steady-state activity at hyperpolarizing compared with depolarizing potentials associated with transient activation and deactivation times, respectively. The times of activation and deactivation change 1 ms/mV in a linear manner with rising and decreasing slopes, respectively. Increases in macroscopic currents at hyperpolarizing potentials results from a voltage-dependent increase in open probability. Voltage sensitivity is not influenced by divalent cations; however, it is Na+-dependent with a 63-mV decrease in voltage required to reach half-maximal activity per log increase in [Na+]. Mutant channels are particularly sensitive to intracellular [Na+] for removing this sodium abolishes voltage dependence. We conclude that the conserved Trp at the base of TM1 in ENaC/Deg channels protects against voltage by masking an inhibitory allosteric or pore block mechanism, which decreases activity in response to intracellular Na+.
| S-EPMC2757952 | biostudies-literature
Project description:The C4-dicarboxylate sensor kinase DcuS is membrane integral because of the transmembrane (TM) helices TM1 and TM2. Fumarate-induced movement of the helices was probed in vivo by Cys accessibility scanning at the membrane-water interfaces after activation of DcuS by fumarate at the periplasmic binding site. TM1 was inserted with amino acid residues 21-41 in the membrane in both the fumarate-activated (ON) and inactive (OFF) states. In contrast, TM2 was inserted with residues 181-201 in the OFF state and residues 185-205 in the ON state. Replacement of Trp 185 by an Arg residue caused displacement of TM2 toward the outside of the membrane and a concomitant induction of the ON state. Results from Cys cross-linking of TM2/TM2' in the DcuS homodimer excluded rotation; thus, data from accessibility changes of TM2 upon activation, either by ligand binding or by mutation of TM2, and cross-linking of TM2 and the connected region in the periplasm suggest a piston-type shift of TM2 by four residues to the periplasm upon activation (or fumarate binding). This mode of function is supported by the suggestion from energetic calculations of two preferred positions for TM2 insertion in the membrane. The shift of TM2 by four residues (or 4-6 Å) toward the periplasm upon activation is complementary to the periplasmic displacement of 3-4 Å of the C-terminal part of the periplasmic ligand-binding domain upon ligand occupancy in the citrate-binding domain in the homologous CitA sensor kinase.
| S-EPMC4568206 | biostudies-literature