scholarly journals Route, mechanism, and implications of proton import during Na+/K+ exchange by native Na+/K+-ATPase pumps

2014 ◽  
Vol 143 (4) ◽  
pp. 449-464 ◽  
Author(s):  
Natascia Vedovato ◽  
David C. Gadsby
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Conformational Changes ◽  
Outward Current ◽  
Side Chain ◽  
Ion Binding ◽  
Inward Current ◽  
Na Release ◽  
K Transport ◽  
External K

A single Na+/K+-ATPase pumps three Na+ outwards and two K+ inwards by alternately exposing ion-binding sites to opposite sides of the membrane in a conformational sequence coupled to pump autophosphorylation from ATP and auto-dephosphorylation. The larger flow of Na+ than K+ generates outward current across the cell membrane. Less well understood is the ability of Na+/K+ pumps to generate an inward current of protons. Originally noted in pumps deprived of external K+ and Na+ ions, as inward current at negative membrane potentials that becomes amplified when external pH is lowered, this proton current is generally viewed as an artifact of those unnatural conditions. We demonstrate here that this inward current also flows at physiological K+ and Na+ concentrations. We show that protons exploit ready reversibility of conformational changes associated with extracellular Na+ release from phosphorylated Na+/K+ pumps. Reversal of a subset of these transitions allows an extracellular proton to bind an acidic side chain and to be subsequently released to the cytoplasm. This back-step of phosphorylated Na+/K+ pumps that enables proton import is not required for completion of the 3 Na+/2 K+ transport cycle. However, the back-step occurs readily during Na+/K+ transport when external K+ ion binding and occlusion are delayed, and it occurs more frequently when lowered extracellular pH raises the probability of protonation of the externally accessible carboxylate side chain. The proton route passes through the Na+-selective binding site III and is distinct from the principal pathway traversed by the majority of transported Na+ and K+ ions that passes through binding site II. The inferred occurrence of Na+/K+ exchange and H+ import during the same conformational cycle of a single molecule identifies the Na+/K+ pump as a hybrid transporter. Whether Na+/K+ pump–mediated proton inflow may have any physiological or pathophysiological significance remains to be clarified.

scholarly journals Theoretical insights into the molecular mechanism of I117V mutation in neuraminidase mediated reduction of oseltamivir drug susceptibility in A/H5N1 influenza virus

2021 ◽  
Vol 3 ◽  
pp. e19
Author(s):  
Mohini Yadav ◽  
Manabu Igarashi ◽  
Norifumi Yamamoto
Keyword(s):  
Binding Site ◽  
Binding Affinity ◽  
Conformational Changes ◽  
Md Simulations ◽  
Side Chain ◽  
Electrostatic Repulsion ◽  
H5n1 Influenza Virus ◽  
Mutation Site ◽  
H5n1 Influenza ◽  
Β Sheet

The substitution of Ile to Val at residue 117 (I117V) of neuraminidase (NA) reduces the susceptibility of the A/H5N1 influenza virus to oseltamivir (OTV). However, the molecular mechanism by which the I117V mutation affects the intermolecular interactions between NA and OTV has not been fully elucidated. In this study, we performed molecular dynamics (MD) simulations to analyze the characteristic conformational changes that contribute to the reduced binding affinity of NA to OTV after the I117V mutation. The results of MD simulations revealed that after the I117V mutation in NA, the changes in the secondary structure around the mutation site had a noticeable effect on the residue interactions in the OTV-binding site. In the case of the WT NA-OTV complex, the positively charged side chain of R118, located in the β-sheet region, frequently interacted with the negatively charged side chain of E119, which is an amino acid residue in the OTV-binding site. This can reduce the electrostatic repulsion of E119 toward D151, which is also a negatively charged residue in the OTV-binding site, so that both E119 and D151 simultaneously form hydrogen bonds with OTV more frequently, which greatly contributes to the binding affinity of NA to OTV. After the I117V mutation in NA, the side chain of R118 interacted with the side chain of E119 less frequently, likely because of the decreased tendency of R118 to form a β-sheet structure. As a result, the electrostatic repulsion of E119 toward D151 is greater than that of the WT case, making it difficult for both E119 and D151 to simultaneously form hydrogen bonds with OTV, which in turn reduces the binding affinity of NA to OTV. Hence, after the I117V mutation in NA, influenza viruses are less susceptible to OTV because of conformational changes in residues of R118, E119, and D151 around the mutation site and in the binding site.

scholarly journals The crystal structure of the Ca2+-ATPase 1 from Listeria monocytogenes reveals a pump primed for dephosphorylation

2020 ◽  
Author(s):  
Sara Basse Hansen ◽  
Mateusz Dyla ◽  
Caroline Neumann ◽  
Jacob Lauwring Andersen ◽  
Magnus Kjaergaard ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Binding Site ◽  
Single Molecule ◽  
Calcium Binding ◽  
Side Chain ◽  
Evolutionary Divergence ◽  
Calcium Binding Site ◽  
Single Molecule Studies ◽  
Ion Binding Sites ◽  
P Type

AbstractBacteria regulate intracellular calcium concentrations by exporting calcium from the cell using active transporters. These transporters include homologues of the mammalian sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), which has served as a paradigm for the structure and mechanism of P-type ATPase ion transport. Here we present three crystal structures of the Ca2+-ATPase 1 from Listeria monocytogenes (LMCA1). Structures with BeF3− mimicking a phosphoenzyme state reveal an intermediate between the outward-open E2P and the proton-occluded E2-P* conformations known for SERCA. This suggests that LMCA1 pre-organizes for dephosphorylation already at the E2P state, consistent with the rapid dephosphorylation of this pump and observations from single-molecule studies. Comparison of ion binding sites show that an arginine side-chain occupies the position equivalent to the calcium binding site I in SERCA leaving a single Ca2+-binding site in LMCA1, corresponding to SERCA site II. Absence of putative proton pathways suggest a direct mechanism of proton counter transport through the Ca2+ exchange pathways. In total, the new structures provide insight into the evolutionary divergence and conserved features of an important class of ion transporters.

scholarly journals An intracellular pathway controlled by the N-terminus of the pump subunit inhibits the bacterial KdpFABC ion pump in high K+ conditions

2021 ◽  
Author(s):  
Himanshu Khandelia ◽  
David Stokes ◽  
Bjørn Panyella Pedersen ◽  
Vikas Dubey
Keyword(s):  
Binding Site ◽  
Chemical Potential ◽  
Transmembrane Protein ◽  
Ion Binding ◽  
Ion Pump ◽  
High K ◽  
N Terminus ◽  
P Type ◽  
Intracellular Pathway ◽  
External K

AbstractThe heterotetrameric bacterial KdpFABC transmembrane protein complex is an ion channel-pump hybrid that consumes ATP to import K+ against its transmembrane chemical potential gradient in low external K+ environments. The KdpB ion-pump subunit of KdpFABC is a P-type ATPase, and catalyses ATP hydrolysis. Under high external K+ conditions, K+ can diffuse into the cells through passive ion channels. KdpFABC must therefore be inhibited in high K+ conditions to conserve cellular ATP. Inhibition is thought to occur via unusual phosphorylation of residue Ser162 of the TGES motif of the cytoplasmic A domain. It is proposed that phosphorylation most likely traps KdpB in an inactive E1-P like conformation, but the molecular mechanism of phosphorylation-mediated inhibition and the allosteric links between phosphorylation on the A domain and the inactivation of the pump remain unknown. Here, we employ molecular dynamics (MD) simulations of the dephosphorylated and phosphorylated versions of KdpFABC to demonstrate that phosphorylated KdpB is trapped in a conformation where the ion-binding site is hydrated by an intracellular pathway between transmembrane helices M1 and M2 which opens in response to the rearrangement of cytoplasmic domains resulting from phosphorylation. Cytoplasmic access of water to the ion-binding site is accompanied by a remarkable loss of secondary structure of the KdpB N-terminus and disruption of a key salt bridge between Glu87 in the A domain and Arg212 in the P domain. Our results provide the molecular basis of a unique mechanism of regulation amongst P-type ATPases, and suggest that the N-terminus has a significant role to play in the conformational cycle and regulation of KdpFABC

scholarly journals Mutations in an intracellular vestibule that bifurcates from the pore of ClC-1 chloride ion channels affect anion permeation

2020 ◽  
Author(s):  
Brett Bennetts ◽  
Craig J. Morton ◽  
Michael W. Parker
Keyword(s):  
Ion Transport ◽  
Binding Site ◽  
Binding Sites ◽  
Structural Data ◽  
Passive Diffusion ◽  
Side Chain ◽  
Ion Binding ◽  
Transport Pathway ◽  
Anion Permeation ◽  
Structural Differences

AbstractThe ubiquitous CLC protein superfamily consists of channels, that permit passive diffusion of Cl ions across biological membranes, and pumps, that can actively transport Cl ions against their electrochemical gradient; yet, puzzlingly, both types share a strongly conserved Cl ion transport pathway comprised of three consecutive binding sites. This raises the question; how does the same pathway support passive diffusion in CLC channels and active transport in CLC pumps? Based on high-resolution structural data current theories suggest that subtle structural differences in the conserved pathway allow CLC channels to ‘leak’ Cl ions. A recent cryo-electron microscopy structure of the human ClC-1 channel does not show occupancy of the central Cl ion binding site but reveals a wide intracellular vestibule that bifurcates from the conserved pathway in this region. Here we show that replacing residues that line the ClC-1 intracellular vestibule with the corresponding residues of CLC pumps resulted in interactions between permeating anions at neighbouring binding sites and altered anion selectivity. Removing the side chain of a strictly conserved tyrosine residue, that coordinates Cl ion at the central binding site of CLC pumps, removed multi-ion behaviour in ClC-1 mutants. In contrast, removing the side chain of a highly conserved glutamate residue that transiently occupies Cl ion binding sites, as part of the transport mechanism of CLC pumps and the mechanism that opens and closes CLC channels, only partially removed multi-ion behaviour in ClC-1 mutants. Our findings show that structural differences between CLC channels and pumps, outside of the conserved Cl ion transport pathway, fundamentally affect anion permeation in ClC-1 channels.SummarySome CLC proteins are passive Cl- channels while others are active Cl- pumps but, paradoxically, both share a conserved, canonical, Cl- permeation pathway. Here Bennetts, Morton and Parker show that ‘pump-like’ mutations in a poorly conserved region, located remotely from the canonical pathway, affect anion permeation in human ClC-1 channels.

scholarly journals Permeation and interaction of monovalent cations with the cGMP-gated channel of cone photoreceptors.

1992 ◽  
Vol 100 (4) ◽  
pp. 647-673 ◽  
Author(s):  
A Picones ◽  
J I Korenbrot
Keyword(s):  
Binding Site ◽  
Ion Selectivity ◽  
Membrane Surface ◽  
Outward Current ◽  
Rate Theory ◽  
Ion Binding ◽  
Membrane Thickness ◽  
Monovalent Cations ◽  
Cone Photoreceptors ◽  
Ionic Solutions

We measured the ion selectivity of cGMP-dependent currents in detached membrane patches from the outer segment of cone photoreceptors isolated from the retina of striped bass. In inside-out patches excised from either single or twin cones the amplitude of these currents, under symmetric ionic solutions, changed with the concentration of cGMP with a dependence described by a Hill equation with average values, at +80 mV, of Km = 42.6 microM and n = 2.49. In the absence of divalent cations, and under symmetric ionic solutions, the I-V curves of the currents were linear over the range of -80 to +80 mV. The addition of Ca altered the form of the I-V curve to a new function well described by an empirical equation that also describes the I-V curve of the photocurrent measured in intact photoreceptors. The monovalent cation permeability sequence of the cGMP-gated channels in the absence of divalent ions was PK > PNa = PLi = PRb > PCs (1.11 > 1.0 = 0.99 = 0.96 > 0.82). The conductance selectivity sequence at +80 mV was GNa = GK > GRb > GCs > GLi (1.0 = 0.99 > 0.88 > 0.74 > 0.60). The organic cations tetramethylammonium (TMA) and arginine partially blocked the current, but the larger ion, arginine, was permeant, whereas the smaller ion, TMA, was not. The amplitude of the outward current through the channels increased with the concentration of monovalent cations on the cytoplasmic membrane surface, up to a saturating value. The increase was well described by the adsorption isotherm of a single ion binding site within the channel with average binding constants, at +80 mV, of 104 mM for Na and 37.6 mM for Li. By assuming that the ion channel contains a single ion binding site in an energy trough separated from each membrane surface by an energy barrier, and using Eyring rate theory, we simulated I-V curves that fit the experimental data measured under ionic concentration gradients. From this fit we conclude that the binding site interacts with one ion at a time and that the energy barriers are asymmetrically located within the membrane thickness. Comparison of the quantitative features of ion permeation and interaction between the cGMP-gated channels of rod and cone photoreceptors reveals that the ion binding sites are profoundly different in the two types of channels. This molecular difference may be particularly important in explaining the differences in the transduction signal of each receptor type.

scholarly journals Ligand binding remodels protein side chain conformational heterogeneity

2021 ◽  
Author(s):  
Stephanie A. Wankowicz ◽  
Saulo H.P. de Oliveira ◽  
Daniel W. Hogan ◽  
Henry van den Bedem ◽  
James S. Fraser
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Conformational Changes ◽  
Biological Function ◽  
Side Chain ◽  
Conformational Heterogeneity ◽  
X Ray Diffraction ◽  
Nmr Studies ◽  
Static Structure ◽  
X Ray

ABSTRACTWhile protein conformational heterogeneity plays an important role in many aspects of biological function, including ligand binding, its impact has been difficult to quantify. Macromolecular X-ray diffraction is commonly interpreted with a static structure, but it can provide information on both the anharmonic and harmonic contributions to conformational heterogeneity. Here, through multiconformer modeling of time- and space-averaged electron density, we measure conformational heterogeneity of 743 stringently matched pairs of crystallographic datasets that reflect unbound/apo and ligand-bound/holo states. When comparing the conformational heterogeneity of side chains, we observe that when binding site residues become more rigid upon ligand binding, distant residues tend to become more flexible, especially in non-solvent exposed regions. Among ligand properties, we observe increased protein flexibility as the number of hydrogen bonds decrease and relative hydrophobicity increases. Across a series of 13 inhibitor bound structures of CDK2, we find that conformational heterogeneity is correlated with inhibitor features and identify how conformational changes propagate differences in conformational heterogeneity away from the binding site. Collectively, our findings agree with models emerging from NMR studies suggesting that residual side chain entropy can modulate affinity and point to the need to integrate both static conformational changes and conformational heterogeneity in models of ligand binding.

Schistosomal Sulfotransferase Interaction with Oxamniquine Involves Hybrid Mechanism of Induced-fit and Conformational Selection

2020 ◽  
Vol 16 (4) ◽  
pp. 451-459 ◽  
Author(s):  
Fortunatus C. Ezebuo ◽  
Ikemefuna C. Uzochukwu
Keyword(s):  
Molecular Dynamics ◽  
Amino Acid ◽  
Binding Energy ◽  
Binding Site ◽  
Conformational Dynamics ◽  
Side Chain ◽  
Amino Acid Residues ◽  
Conformational Selection ◽  
Hybrid Mechanism ◽  
Dynamics Simulations

Background: Sulfotransferase family comprises key enzymes involved in drug metabolism. Oxamniquine is a pro-drug converted into its active form by schistosomal sulfotransferase. The conformational dynamics of side-chain amino acid residues at the binding site of schistosomal sulfotransferase towards activation of oxamniquine has not received attention. Objective: The study investigated the conformational dynamics of binding site residues in free and oxamniquine bound schistosomal sulfotransferase systems and their contribution to the mechanism of oxamniquine activation by schistosomal sulfotransferase using molecular dynamics simulations and binding energy calculations. Methods: Schistosomal sulfotransferase was obtained from Protein Data Bank and both the free and oxamniquine bound forms were subjected to molecular dynamics simulations using GROMACS-4.5.5 after modeling it’s missing amino acid residues with SWISS-MODEL. Amino acid residues at its binding site for oxamniquine was determined and used for Principal Component Analysis and calculations of side-chain dihedrals. In addition, binding energy of the oxamniquine bound system was calculated using g_MMPBSA. Results: The results showed that binding site amino acid residues in free and oxamniquine bound sulfotransferase sampled different conformational space involving several rotameric states. Importantly, Phe45, Ile145 and Leu241 generated newly induced conformations, whereas Phe41 exhibited shift in equilibrium of its conformational distribution. In addition, the result showed binding energy of -130.091 ± 8.800 KJ/mol and Phe45 contributed -9.8576 KJ/mol. Conclusion: The results showed that schistosomal sulfotransferase binds oxamniquine by relying on hybrid mechanism of induced fit and conformational selection models. The findings offer new insight into sulfotransferase engineering and design of new drugs that target sulfotransferase.

scholarly journals Monoclonal antibodies identify residues 199–216 of the integrin α2 vWFA domain as a functionally important region within α2β1

2000 ◽  
Vol 350 (2) ◽  
pp. 485-493 ◽  
Author(s):  
Danny S. TUCKWELL ◽  
Lyndsay SMITH ◽  
Michelle KORDA ◽  
Janet A. ASKARI ◽  
Sentot SANTOSO ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Conformational Changes ◽  
Cation Binding ◽  
Inhibitory Antibody ◽  
Collagen Binding ◽  
Collagen Peptide ◽  
Binding Residue ◽  
Important Region ◽  
Domain Mapping

Integrin α2β1 is the major receptor for collagens in the human body, and the collagen-binding site on the α2 subunit von Willebrand factor A-type domain (vWFA domain) is now well defined. However, the biologically important conformational changes that are associated with collagen binding, and the means by which the vWFA domain is integrated into the whole integrin are not completely understood. We have raised monoclonal antibodies against recombinant α2 vWFA domain for use as probes of function. Three antibodies, JA202, JA215 and JA218, inhibited binding to collagen, collagen I C-propeptide and E-cadherin, demonstrating that their function is important for structurally diverse α2β1 ligands. Cross-blocking studies grouped the epitopes into two clusters: (I) JA202, the inhibitory antibody, Gi9, and a non-inhibitory antibody, JA208; (II) JA215 and JA218. Both clusters were sensitive to events at the collagen binding site, as binding of Gi9, JA202, JA215 and JA218 were inhibited by collagen peptide, JA208 binding was enhanced by collagen peptide, and binding of JA202 was decreased after mutagenesis of the cation-binding residue Thr221 to alanine. Binding of cluster I antibodies was inhibited by the anti-functional anti-β1 antibody Mab13, and binding of Gi9 and JA218 to α2β1 was inhibited by substituting Mn2+ for Mg2+, demonstrating that these antibodies were sensitive to changes initiated outside the vWFA domain. Mapping of epitopes showed that JA202 and Gi9 bound between residues 212–216, while JA208 bound between residues 199–216. We have therefore identified two epitope clusters with novel properties; i.e. they are intimately associated with the collagen-binding site, responsive to conformational changes at the collagen-binding site and sensitive to events initiated outside the vWFA domain.

Sign in / Sign up

Export Citation Format

Share Document