scholarly journals Impact of an electric field on P-type ATPases

2008 ◽  
Vol 22 (4) ◽  
pp. 319-325 ◽  
Author(s):  
Christian Weidemüller ◽  
Karin Hauser
Keyword(s):  
Electric Field ◽  
Ion Transport ◽  
Transmembrane Helix ◽  
Energy Transduction ◽  
Transmembrane Helices ◽  
Electrical Measurements ◽  
Transduction Mechanism ◽  
Charge Translocation ◽  
P Type ◽  
Hydrolysis Of

P-type ATPases are membrane proteins acting as ion pumps that drive an active transport of cations across the membrane against a concentration gradient. The required energy for the ion transport is provided by binding and hydrolysis of ATP. A reaction mechanism of ion transport and energy transduction is assumed to be common for all P-type ATPases and generally described by the Post-Albers cycle. Transient currents and charge translocation of P-type ATPases were extensively investigated by electrical measurements that apply voltage jumps to initiate the reaction cycle. In this study, we simulate an applied voltage across the membrane by an electric field and perform electrostatic calculations in order to verify the experimentally-driven hypothesis that the energy transduction mechanism is regulated by specific structural elements. Side chain conformational and ionization changes induced by the electric field are evaluated for each transmembrane helix and the selectivity in response is qualitatively analyzed for the Ca2+-ATPase as well as for structural models of the Na+/K+-ATPase. Helix M5 responds with more conformer changes as compared to the other transmembrane helices what is even more emphasized when the stalk region is included. Thus our simulations support experimental results and indicate a crucial role for the highly conserved transmembrane helix M5 in the energy transduction mechanism of P-type ATPases.

scholarly journals Systematic Comparison of Molecular Conformations of H+,K+-ATPase Reveals an Important Contribution of the A-M2 Linker for the Luminal Gating

2014 ◽  
Vol 289 (44) ◽  
pp. 30590-30601 ◽  
Author(s):  
Kazuhiro Abe ◽  
Kazutoshi Tani ◽  
Yoshinori Fujiyoshi
Keyword(s):  
Molecular Conformation ◽  
Transmembrane Helix ◽  
Transmembrane Helices ◽  
Competitive Antagonist ◽  
Molecular Conformations ◽  
Systematic Comparison ◽  
Azimuthal Position ◽  
Cryo Electron Microscopy ◽  
Open Conformation ◽  
P Type

Gastric H+,K+-ATPase, an ATP-driven proton pump responsible for gastric acidification, is a molecular target for anti-ulcer drugs. Here we show its cryo-electron microscopy (EM) structure in an E2P analog state, bound to magnesium fluoride (MgF), and its K+-competitive antagonist SCH28080, determined at 7 Å resolution by electron crystallography of two-dimensional crystals. Systematic comparison with other E2P-related cryo-EM structures revealed that the molecular conformation in the (SCH)E2·MgF state is remarkably distinguishable. Although the azimuthal position of the A domain of the (SCH)E2·MgF state is similar to that in the E2·AlF (aluminum fluoride) state, in which the transmembrane luminal gate is closed, the arrangement of transmembrane helices in the (SCH)E2·MgF state shows a luminal-open conformation imposed on by bound SCH28080 at its luminal cavity, based on observations of the structure in the SCH28080-bound E2·BeF (beryllium fluoride) state. The molecular conformation of the (SCH)E2·MgF state thus represents a mixed overall structure in which its cytoplasmic and luminal half appear to be independently modulated by a phosphate analog and an antagonist bound to the respective parts of the enzyme. Comparison of the molecular conformations revealed that the linker region connecting the A domain and the transmembrane helix 2 (A-M2 linker) mediates the regulation of luminal gating. The mechanistic rationale underlying luminal gating observed in H+,K+-ATPase is consistent with that observed in sarcoplasmic reticulum Ca2+-ATPase and other P-type ATPases and is most likely conserved for the P-type ATPase family in general.

scholarly journals Oligomerization of the antimalarial drug target PfATP4 is essential for parasite survival

2019 ◽  
Author(s):  
Aarti A. Ramanathan ◽  
Joanne M. Morrisey ◽  
Thomas M. Daly ◽  
Lawrence W. Bergman ◽  
Michael W. Mather ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasmodium Falciparum ◽  
Antimalarial Drug ◽  
Efflux Pump ◽  
Transmembrane Helix ◽  
Aromatic Amino Acids ◽  
Transmembrane Helices ◽  
Parasite Survival ◽  
Oligomeric Complex ◽  
P Type

AbstractPlasmodium falciparum P-type ATPase (PfATP4) is a Na+ efflux pump crucial for maintaining low [Na+]i in malaria parasites during their intraerythrocytic development cycle. In recent years, multiple studies have shown PfATP4 to be the target of a large number of chemical scaffolds, including current candidate antimalarials KAE609 and SJ733. Here we show that PfATP4 exists as a large complex. Immunopurification and proteomic studies revealed the complex to be homooligomeric in nature. The complex appears to be assembled co-translationally. Phylogenetic analysis suggests that ATP4 from apicomplexans and chromerids form a distinct class of P-type ATPases having fewer transmembrane helices compared to their orthologues. We hypothesized that reduction of transmembrane helices in PfATP4 might necessitate oligomerization to maintain its function. We further suspected potential involvement of π-π interactions between aromatic amino acids within the terminal transmembrane helix of each monomer to be critical for oligomerization. To test this hypothesis, we mutated three aromatic amino acids in the last transmembrane helix of PfATP4. Wildtype and the mutated PfATP4 genes were introduced at an ectopic locus in a P. falciparum line, in which endogenous PfATP4 was conditionally expressed. Whereas the wildtype copy of PfATP4 expressed from the ectopic locus was able to form the oligomeric complex, the mutant PfATP4 failed to do so. Strikingly, unlike the wildtype, the mutant PfATP4 failed to functionally complement the knockdown of the endogenous gene, leading to parasite demise. These results strongly suggest that co-translational oligomerization of PfATP4 is essential for its function and for parasite survival.Significance StatementPlasmodium falciparum ATP4 (PfATP4) is a Na+ efflux pump and is the target of at least two antimalarial drug candidates (KAE609, SJ733) currently in clinical trials. With a rapid parasite clearance rate (t99=12h) in initial clinical studies, PfATP4-active drugs present the prospect of taking us a step closer to malaria elimination in a world that is currently threatened by artemisinin resistance. In this study, we have established that PfATP4 exists as a homo-oligomeric complex, which is assembled co-translationally through interactions facilitated by aromatic amino acids in its last transmembrane helix. Crucially, its existence as a complex is essential for its function. This exposes a vulnerability in the target that could be potentially exploited for drug design.

scholarly journals Relative transmembrane segment rearrangements during BK channel activation resolved by structurally assigned fluorophore–quencher pairing

2012 ◽  
Vol 140 (2) ◽  
pp. 207-218 ◽  
Author(s):  
Antonios Pantazis ◽  
Riccardo Olcese
Keyword(s):  
Electric Field ◽  
Transmembrane Helix ◽  
Principal Component ◽  
Bk Channels ◽  
Bk Channel ◽  
Transmembrane Helices ◽  
Voltage Sensing ◽  
Charged Residues ◽  
Voltage Dependent ◽  
Sense And Respond

Voltage-activated proteins can sense, and respond to, changes in the electric field pervading the cell membrane by virtue of a transmembrane helix bundle, the voltage-sensing domain (VSD). Canonical VSDs consist of four transmembrane helices (S1–S4) of which S4 is considered a principal component because it possesses charged residues immersed in the electric field. Membrane depolarization compels the charges, and by extension S4, to rearrange with respect to the field. The VSD of large-conductance voltage- and Ca-activated K+ (BK) channels exhibits two salient inconsistencies from the canonical VSD model: (1) the BK channel VSD possesses an additional nonconserved transmembrane helix (S0); and (2) it exhibits a “decentralized” distribution of voltage-sensing charges, in helices S2 and S3, in addition to S4. Considering these unique features, the voltage-dependent rearrangements of the BK VSD could differ significantly from the standard model of VSD operation. To understand the mode of operation of this unique VSD, we have optically tracked the relative motions of the BK VSD transmembrane helices during activation, by manipulating the quenching environment of site-directed fluorescent labels with native and introduced Trp residues. Having previously reported that S0 and S4 diverge during activation, in this work we demonstrate that S4 also diverges from S1 and S2, whereas S2, compelled by its voltage-sensing charged residues, moves closer to S1. This information contributes spatial constraints for understanding the BK channel voltage-sensing process, revealing the structural rearrangements in a non-canonical VSD.

scholarly journals Point Mutations in Transmembrane Helices 2 and 3 of ExbB and TolQ Affect Their Activities in Escherichia coli K-12

2004 ◽  
Vol 186 (13) ◽  
pp. 4402-4406 ◽  
Author(s):  
Volkmar Braun ◽  
Christina Herrmann
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Transmembrane Helix ◽  
Point Mutations ◽  
Transmembrane Helices ◽  
The Third ◽  
Form Part ◽  
K 12

ABSTRACT Replacement of glutamate 176, the only charged amino acid in the third transmembrane helix of ExbB, with alanine (E176A) abolished ExbB activity in all determined ExbB-dependent functions of Escherichia coli. Combination of the mutations T148A in the second transmembrane helix and T181A in the third transmembrane helix, proposed to form part of a proton pathway through ExbB, also resulted in inactive ExbB. E176 and T148 are strictly conserved in ExbB and TolQ proteins, and T181 is almost strictly conserved in ExbB, TolQ, and MotA.

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