The Third Sodium Binding Site of Na,K-ATPase Is Functionally Linked to Acidic pH-Activated Inward Current

Active transport of sugars into bacteria occurs through symporters driven by ion gradients. LacY is the most well-studied proton sugar symporter, whereas vSGLT is the most characterized sodium sugar symporter. These are members of the major facilitator (MFS) and the amino acid-Polyamine organocation (APS) transporter superfamilies. While there is no structural homology between these transporters, they operate by a similar mechanism. They are nano-machines driven by their respective ion electrochemical potential gradients across the membrane. LacY has 12 transmembrane helices (TMs) organized in two 6-TM bundles, each containing two 3-helix TM repeats. vSGLT has a core structure of 10 TM helices organized in two inverted repeats (TM 1–5 and TM 6–10). In each case, a single sugar is bound in a central cavity and sugar selectivity is determined by hydrogen- and hydrophobic- bonding with side chains in the binding site. In vSGLT, the sodium-binding site is formed through coordination with carbonyl- and hydroxyl-oxygens from neighboring side chains, whereas in LacY the proton (H3O+) site is thought to be a single glutamate residue (Glu325). The remaining challenge for both transporters is to determine how ion electrochemical potential gradients drive uphill sugar transport.

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

The Journal of General Physiology ◽

10.1085/jgp.201311148 ◽

2014 ◽

Vol 143 (4) ◽

pp. 449-464 ◽

Cited By ~ 45

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.

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Dominant Thermodynamic Role of the Third Independent Receptor Binding Site in the Receptor-Associated Protein RAP†

Biochemistry ◽

10.1021/bi0110692 ◽

2001 ◽

Vol 40 (50) ◽

pp. 15408-15417 ◽

Cited By ~ 31

Author(s):

Olav M. Andersen ◽

Frederick P. Schwarz ◽

Edward Eisenstein ◽

Christian Jacobsen ◽

Søren K. Moestrup ◽

...

Keyword(s):

Binding Site ◽

Receptor Binding ◽

Receptor Binding Site ◽

Receptor Associated Protein ◽

The Third

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Second-Site Revertants of a Low-Sodium-Affinity Mutant of the Na+/H+Exchanger Reveal the Participation of TM4 into a Highly Constrained Sodium-Binding Site†

Biochemistry ◽

10.1021/bi0025464 ◽

2001 ◽

Vol 40 (16) ◽

pp. 5095-5101 ◽

Cited By ~ 35

Author(s):

Nicolas Touret ◽

Philippe Poujeol ◽

Laurent Counillon

Keyword(s):

Binding Site ◽

Low Sodium ◽

Sodium Binding

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Dynamic plasticity of the lipid antigen-binding site of CD1d is crucially favoured by acidic pH and helper proteins

Scientific Reports ◽

10.1038/s41598-020-62833-y ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Bruno Cuevas-Zuviría ◽

Marina Mínguez-Toral ◽

Araceli Díaz-Perales ◽

María Garrido-Arandia ◽

Luis F. Pacios

Keyword(s):

Binding Site ◽

Antigen Binding ◽

Acidic Ph ◽

Antigen Binding Site ◽

Lipid Antigen ◽

Dynamic Plasticity

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Conformational changes in the third PDZ domain of the neuronal postsynaptic density protein 95

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798319001980 ◽

2019 ◽

Vol 75 (4) ◽

pp. 381-391 ◽

Cited By ~ 6

Author(s):

Ana Camara-Artigas ◽

Javier Murciano-Calles ◽

Jose C. Martínez

Keyword(s):

Binding Site ◽

Conformational Changes ◽

Pdz Domain ◽

Postsynaptic Density ◽

Crystal Form ◽

Space Groups ◽

The Third ◽

Conformational Plasticity ◽

Α Helix ◽

Postsynaptic Density Protein

PDZ domains are protein–protein recognition modules that interact with other proteins through short sequences at the carboxyl terminus. These domains are structurally characterized by a conserved fold composed of six β-strands and two α-helices. The third PDZ domain of the neuronal postsynaptic density protein 95 has an additional α-helix (α3), the role of which is not well known. In previous structures, a succinimide was identified in the β2–β3 loop instead of Asp332. The presence of this modified residue results in conformational changes in α3. In this work, crystallographic structures of the following have been solved: a truncated form of the third PDZ domain of the neuronal postsynaptic density protein 95 from which α3 has been removed, D332P and D332G variants of the protein, and a new crystal form of this domain showing the binding of Asp332 to the carboxylate-binding site of a symmetry-related molecule. Crystals of the wild type and variants were obtained in different space groups, which reflects the conformational plasticity of the domain. Indeed, the overall analysis of these structures suggests that the conformation of the β2–β3 loop is correlated with the fold acquired by α3. The alternate conformation of the β2–β3 loop affects the electrostatics of the carboxylate-binding site and might modulate the binding of different PDZ-binding motifs.

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Defining a second epitope for heparin-induced thrombocytopenia/thrombosis antibodies using KKO, a murine HIT-like monoclonal antibody

Blood ◽

10.1182/blood.v99.4.1230 ◽

2002 ◽

Vol 99 (4) ◽

pp. 1230-1236 ◽

Cited By ~ 87

Author(s):

Zhong Q. Li ◽

Weiyi Liu ◽

Kwang S. Park ◽

Brue S. Sachais ◽

Gowthani M. Arepally ◽

...

Keyword(s):

Monoclonal Antibody ◽

Binding Site ◽

Binding Sites ◽

Heparin Therapy ◽

Common Complication ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

The Third ◽

N Terminus ◽

A Site

Heparin-induced thrombocytopenia/thrombosis (HIT/T) is a common complication of heparin therapy that is caused by antibodies to platelet factor 4 (PF4) complexed with heparin. The immune response is polyclonal and polyspecific, ie, more than one neoepitope on PF4 is recognized by HIT/T antibodies. One such epitope has been previously identified; it involves the domain between the third and fourth cysteine residues in PF4 (site 1). However, the binding sites for other HIT/T antibodies remain to be defined. To explore this issue, the binding site of KKO, an HIT/T-like murine monoclonal antibody, was defined. KKO shares a binding site with many HIT/T antibodies on PF4/heparin, but does not bind to site 1 or recognize mouse PF4/heparin. Therefore, the binding of KKO to a series of mouse/human PF4 chimeras complexed with heparin was examined. KKO recognizes a site that requires both the N terminus of PF4 and Pro34, which immediately precedes the third cysteine. Both regions lie on the surface of the PF4 tetramer in sufficient proximity (within 0.74 nm) to form a contiguous antigenic determinant. The 10 of 14 HIT/T sera that require the N terminus of PF4 for antigen recognition also require Pro34 to bind. This epitope, termed site 2, lies adjacent to site 1 in the crystal structure of the PF4 tetramer. Yet sites 1 and 2 can be recognized by distinct populations of antibodies. These studies further help to define a portion of the PF4 tetramer to which self-reactive antibodies develop in patients exposed to heparin.

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Pulmonary arterial thrombosis in a neonate with homozygous deficiency of antithrombin III: successful outcome following pulmonary thrombectomy and infusions of antithrombin III concentrate

Cardiology in the Young ◽

10.1017/s1047951100009240 ◽

2000 ◽

Vol 10 (3) ◽

pp. 275-278 ◽

Cited By ~ 5

Author(s):

K. Roman ◽

E. Rosenthal ◽

R. Razavi

Keyword(s):

Binding Site ◽

Arterial Thrombosis ◽

Antithrombin Iii ◽

Pulmonary Trunk ◽

Successful Outcome ◽

Heparin Binding ◽

The Third ◽

Heparin Binding Site ◽

Surgical Thrombectomy ◽

Pulmonary Arterial

AbstractWe report a newborn male who presented with severe central cyanosis on the third day of life. Partial thrombotic obstruction of the pulmonary trunk secondary to Antithrombin III (homozygous defect of heparin binding site) deficiency was subsequently diagnosed. Surgical thrombectomy, and infusions of Antithrombin III concentrate, led to a successful outcome. We postulate that intrauterine thrombosis occurred to give this unusual presentation.

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