A structural model for the catalytic cycle of Ca2+-ATPase

We showed earlier that the kinetic behavior of the ␣2 isoform of the Na,K-ATPase differs from the ubiquitous ␣1 isoform primarily by a shift in the steady-state E 1 /E 2 equilibrium of ␣2 in favor of E 1 form(s). The aim of the present study was to identify regions of the ␣ chain that confer the ␣1/␣2 distinct behavior using a mutagenesis and chimera approach. Criteria to assess shifts in conformational equilibrium included (i) K ؉ sensitivity of Na-ATPase measured at micromolar ATP, under which condition E 2 (K ؉ ) 3 E 1 ؉ K ؉ becomes rate-limiting, (ii) changes in K ATP for low affinity ATP binding, (iii) vanadate sensitivity of Na,K-ATPase activity, and (iv) the rate of the partial reaction E 1 P 3 E 2 P. We first confirmed that interactions between the cytoplasmic domains of ␣2 that modulate conformational shifts are fundamentally similar to those of ␣1, suggesting that the predilection of ␣2 for E 1 state(s) is due to differences in primary structure of the two isoforms. Kinetic behavior of the ␣1/␣2 chimeras indicates that the difference in E 1 /E 2 poise of the two isoforms cannot be accounted for by their notably distinct N termini, but rather by the front segment extending from the cytoplasmic N terminus to the C-terminal end of the extracellular loop between transmembranes 3 and 4, with a lesser contribution of the ␣1/␣2 divergent portion within the M4-M5 loop near the ATP binding domain. In addition, we show that the E 1 shift of ␣2 results primarily from differences in the conformational transition of the dephosphoenzyme, (E 2 (K ؉ ) 3 E 1 ؉ K ؉ ), rather than phosphoenzyme (E 1 P 3 E 2 P).The Na,K-ATPase or sodium pump is an integral membrane protein complex found in the plasma membrane of virtually all animal cells. It catalyzes the exchange of three intracellular Na ϩ ions for two extracellular K ϩ ions using the energy of hydrolysis of one molecule of ATP. Consequently, the sodium pump plays an essential role in the maintenance of the electrochemical alkali cation gradients, providing the driving force for the transport of various nutrients into the cell. The Na,KATPase is a member of the family of P-type ATPases, which during the course of their catalytic cycle undergo phosphorylation and dephosphorylation of a conserved aspartate residue located in the large catalytic loop between transmembrane segments 4 and 5 of the catalytic ␣ subunit. During the catalytic cycle both dephospho-and phosphoenzymes undergo conformational transitions commonly referred to as E 1 7 E 2 and E 1 P 7 E 2 P, respectively. In addition to the large catalytic ␣ subunit, the Na,K-ATPase comprises a smaller, highly glycosylated ␤ subunit that is important for the proper folding of ␣ and its insertion into the plasma membrane. At present, four isoforms of ␣ and three isoforms of ␤ have been described, and these are distributed in a tissue-and developmentally dependent manner.The ␣2 isoform is located primarily in skeletal muscle and in brain, predominantly in glial cells. Our earlier studies indicated that it differs from t...

Section: Discussionsupporting

confidence: 60%

Structural Basis for α1 Versus α2 Isoform-distinct Behavior of the Na,K-ATPase

Segall¹,

Javaid²,

Carl³

et al. 2003

“…The Hinge Movement between N-and P-domain-The hinge movement upon nucleotide binding seems, however, to be less pronounced than anticipated in the structural models (61,62). Fluorescence energy transfer experiments show that distances between fluorescence labels in the N-and the P-domain do not change between E1Ca 2 or an E2 conformation, as reviewed in Ref.…”

Section: Discussionmentioning

confidence: 90%

Mapping Interactions between the Ca2+-ATPase and Its Substrate ATP with Infrared Spectroscopy

Liu

Barth

2003

Infrared spectroscopy has been used to map substrate-protein interactions: the conformational changes of the sarcoplasmic reticulum Ca 2؉ -ATPase upon nucleotide binding and ATPase phosphorylation were monitored using the substrate ATP and ATP analogues (2 -deoxy-ATP, 3 -deoxy-ATP, and inosine 5 -triphosphate), which were modified at specific functional groups of the substrate. Modifications to the 2 -OH, the 3 -OH, and the amino group of adenine reduce the extent of bindinginduced conformational change of the ATPase, with particularly strong effects observed for the latter two. This demonstrates the structural sensitivity of the nucleotide-ATPase complex to individual interactions between nucleotide and ATPase. All groups studied are important for binding and interactions of a given ligand group with the ATPase depend on interactions of other ligand groups.Phosphorylation of the ATPase was observed for ITP and 2 -deoxy-ATP, but not for 3 -deoxy-ATP. There is no direct link between the extent of conformational change upon nucleotide binding and the rate of phosphorylation showing that the full extent of the ATP-induced conformational change is not mandatory for phosphorylation. As observed for the nucleotide-ATPase complex, the conformation of the first phosphorylated ATPase intermediate E1PCa 2 also depends on the nucleotide, indicating that ATPase states have a less uniform conformation than previously anticipated.Ligand binding to proteins controls vast numbers of cellular processes and has attracted great scientific and economic interest. Protein and ligand flexibility are important determinants of the interaction and often lead to ligand binding modes that are not anticipated from structures obtained with other ligands. To these "failure(s) of the rigid receptor hypothesis" (1) is added here an impressive example: induced-fit binding of nucleotides to the Ca 2ϩ -ATPase. This finding stems from a systematic mapping of substrate-protein interactions with infrared (IR) 1 spectroscopy. New approaches like this are welcome in the field of ligand-protein recognition, since the most informative techniques, NMR and x-ray crystallography, are laborious and problematic for some systems. Methods like fluorescence and luminescence that require less expenditure also provide less molecular information. We expect that this technology gap will be bridged by IR spectroscopy.IR spectroscopy, one of the methods of vibrational spectroscopy, provides direct information on the molecular level, is cost-effective, and can be universally applied from small soluble proteins to large membrane proteins under near-physiological conditions. Work summarized in recent reviews (2-5) has shown that the vibrational spectrum changes characteristically when a ligand binds to a protein. This provides a direct observation of ligand binding: no marker compound has to be introduced to report the binding process, as with many other methods. Previous work has mostly focused on individual interactions between a ligand and a protein by monitoring the ...

“…This structure was used as a reference for a suite of programs designed to account for distortions along the tubes (27). Thereafter, a new reference was generated and the distortions were refined by a single iteration of these programs (15). Defocus values were used to correct phases and to weight amplitudes along the layer lines.…”

Section: Methodsmentioning

confidence: 99%

“…Finally, an analog of the low energy phosphoenzyme (E 2 ϳP) was crystallized with the MgF 4 2Ϫ or the AlF 4 Ϫ ligand bound to the catalytic aspartate, again including TG (13,14), which may again be expected to exert a non-physiological influence over the conformation. Similarly, lower resolution studies of the low energy phosphoenzyme employed decavanadate as a crystallization agent, which bound at the interface of the three cytoplasmic domains (15).…”

Section: Intracellular Camentioning

confidence: 99%

Structural Studies of a Stabilized Phosphoenzyme Intermediate of Ca2+-ATPase

Stokes

Delavoie

Rice

et al. 2005

Ca2؉ -ATPase belongs to the family of P-type ATPases and maintains low concentrations of intracellular Ca 2؉ . Its reaction cycle consists of four main intermediates that alternate ion binding in the transmembrane domain with phosphorylation of an aspartate residue in a cytoplasmic domain. Previous work characterized an ultrastable phosphoenzyme produced first by labeling with fluorescein isothiocyanate, then by allowing this labeled enzyme to establish a maximal Ca 2؉ gradient, and finally by removing Ca 2؉ from the solution. This phosphoenzyme is characterized by very low fluorescence and has specific enzymatic properties suggesting the existence of a high energy phosphoryl bond. To study the structural properties of this phosphoenzyme, we used cryoelectron microscopy of two-dimensional crystals formed in the presence of decavanadate and determined the structure at 8-Å resolution. To our surprise we found that at this resolution the low fluorescence phosphoenzyme had a structure similar to that of the native enzyme crystallized under equivalent conditions. We went on to use glutaraldehyde cross-linking and proteolysis for independent structural assessment and concluded that, like the unphosphorylated native enzyme, Ca 2؉ and vanadate exert a strong influence over the global structure of this low fluorescence phosphoenzyme. Based on a structural model with fluorescein isothiocyanate bound at the ATP site, we suggest that the stability as well as the low fluorescence of this phosphoenzyme is due to a fluorescein-mediated crosslink between two cytoplasmic domains that prevents hydrolysis of the aspartyl phosphate. Finally, we consider the alternative possibility that phosphate transfer to fluorescein itself could explain the properties of this low fluorescence species.Intracellular Ca 2ϩ concentrations are maintained at low levels by a group of ATP-dependent Ca 2ϩ pumps that belong to the family of P-type ATPases (1, 2). These pumps maintain low intracellular Ca 2ϩ concentrations as a baseline for signal transduction pathways initiated by opening a variety of Ca 2ϩ channels. There are distinct groups of Ca 2ϩ pumps, or Ca 2ϩ -ATPases, in the plasma membrane and in membranes of the sarcoplasmic/endoplasmic reticulum. The particular isoform from sarcoplasmic reticulum (SR) 1 of skeletal muscle is the most extensively studied and represents an archetype for the entire family. Biochemical studies have defined a series of reaction intermediates that couple the energy of ATP hydrolysis within the cytoplasmic domain to alternations in the affinity and accessibility of Ca 2ϩ binding sites within the membrane domain (3). In the key step of the reaction cycle, this energy is stored by the pump as an aspartyl phosphate and is used to drive a global conformational change that simultaneously lowers the affinity of the Ca 2ϩ sites and exposes them to the luminal side of the membrane, where the Ca 2ϩ ions are exchanged with protons. In fact, the reaction cycle appears to be driven by a series of such conformational changes, whi...