Crystal structure of the high-affinity Na ⁺ ,K ⁺ -ATPase–ouabain complex with Mg ²⁺ bound in the cation binding site

Abstract

The Na + ,K + -ATPase maintains electrochemical gradients for Na + and K + that are critical for animal cells. Cardiotonic steroids (CTSs), widely used in the clinic and recently assigned a role as endogenous regulators of intracellular processes, are highly specific inhibitors of the Na

Structures and characterization of digoxin- and bufalin-bound Na + ,K + -ATPase compared with the ouabain-bound complex

“…E2P-CTS complexes is observed, as obtained from two different crystal forms and with three different compounds, validates the structural characteristics of high-affinity E2P-CTS complexes of the Na + ,K + -ATPase described here (4,5).…”

“…Other residues of both α-and β-subunits may also be in a position to form temporary water-mediated interactions with the tridigitoxose moiety exposed to the solvent (12). The impact of the aqueous environment on the protein coordination of the CTS sugar moiety has been discussed for the high-affinity E2P-ouabain complex, for which the electron density maps did not support any strong coordination of the rhamnose moiety (5).…”

“…The recently published crystal structure of the Na + ,K + -ATPase phosphoenzyme (E2P) in complex with the widely studied CTS ouabain (4,5) showed that the high-affinity CTS-binding site is constituted by the transmembrane helices αM1-6 of the catalytic α-subunit, forming a pocket exposed to the extracellular side and overlapping with the extracellular ion exchange pathway (6). The E2P-ouabain structure also revealed details on proteinligand interactions facilitating high-affinity CTS binding compared with a low-affinity ouabain complex (7).…”

Conserved molecular mechanisms underlying the effects of small molecule xenobiotic chemotherapeutics on cells

“…Thus, such small molecule xenobiotics remove CM cholesterol via cholesteroloassociation with the more exteriorly protruding cholesterol portion to decrease CM cholesterol concentration, thereby, de-stabilizing the CM sufficiently enough to concomitantly cause CM destabilization-mediated extrusion of CM cholesterol-associated proteins/receptors, and as a result, also have the potential to cause a subsequent 1ary indirect pressuromodulation (perturbomodulation)-mediated secondary increase in very high MW protein transcription (17,50,51) (Table V and Log OWPC-to-vdWD ratio, 6.75 nm -1 )], associates into the CM closely with the CM phospholipid layer-spanning portion of cholesterol that has a lipophilicity of ~7.26 nm -1 which is close to its hydrophobic core lipophilicity of 6.75 nm -1 , while transiently stabilizing itself in the layer by concomitantly interacting anisotropically with the layer phospholipid head groups viz a viz its mono-anionic hydrophilic-interacting COO -moiety, to remove cholesterol via cholesteroloassociation secondary to unstable structural association with phospholipid layer fatty acid-ester tails in context of an inability to glyceroloesterify. Therefore, chlorambucil is not an effective intra-cellularly localizing mitochondrial or nuclear DNA/RNA alkylating/bi-functional DNA crosslinking chemoxenobiotic, but instead removes CM cholesterol via cholesteroloassociation with the CM phospholipid layer-spanning interior cholesterol portion to decrease CM cholesterol concentration (52,53) and as a result, has the potential to cause a subsequent 1ary indirect pressuromodulation (perturbomodulation)-mediated secondary increase in very high MW protein transcription (17) ( Table V and associates directly into the CM among the CM phospholipid layer fatty acid-ester tails, rather than with the CM phospholipid layer-spanning interior cholesterol portion, along with more stable association within the layer by virtue of the concomitant anisotropic interaction of its cataniononeutral hydrophilic-interacting NH3 + COO -moiety with layer phospholipid head groups, that importantly results in its ability to secondarily subsequently trans-displace across CM phospholipid layers and bilayer over time into the intra-cellular compartment.…”

“…Due to the risk of endocytosis-mediated 3ary indirect pressuromodulation (17), CM pore impermeable dually-cationic small molecule xenobiotics such as AMD3100, paraquat and bleomycin are tumoropotentiators as monochemotherapies, Vincristine and doxor ubicin associate into the inter-α-helix isophilic pores of the Ca 2+ channel (92) and Na + /K + ATPase (17,50,51), respectively, to de-stabilize the CM interaction of the multi-inter-α-helix constructs of such trans-membrane proteins, that results in ligand-associated CM protein channel endocytosis (93,94), followed by intra-cellular ligand CM channel disassociation, and then, subsequent promiscuous MM VDAC channel isophilic pore association (95): thus, the primary mechanism of cellular toxicity for vincristine and doxorubicin is at the level of the mitochondria and MM, and due to the concomitant presence of sufficiently separated (SS) 1+ cationicity in the former (vincristine) and exteriorly protruding 1+ cationicity in the latter (doxorubicin), that results in non-association of microtubule γ-tubulin at MM VDAC and non-recruitment of renewed microtubule networks to the MM, mitochondrial anchorage immobility-associated MM disruption/dissolution with liberation of MM AIF and initiation of the mitochondrially-mediated nuclear apoptosis cascade, with the significant potential for inducing cellular apoptosis (96-101), which is due to no significant (in-significant) potential for CM receptor endocytosis-mediated (Pseudo) 3ary indirect pressuromodulation (17) (Table VII and Fig. 7).…”