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

Abstract

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...

Influence of decavanadate on rat synaptic plasma membrane ATPases activity

“…It indicates that V 10 as a reversible mixed inhibitor of Na + /K + -ATPase activity binds to enzyme sites participating in both substrate binding and catalysis. Obtained mixed inhibition is in agreement with previously reported results that there are two binding sites for V 10 on Ca 2+ -ATPase which belongs, as well as Na + /K + -ATPase, to P-type of ATPases Stokes et al 2005). Decrease in apparent affinity for MgATP 2-is in agreement with competition of V 10 with fluorescein isothocyanate for ATP binding site of Ca 2+ -ATPase .…”

“…It could be summarized that the obtained dose-dependent inhibition of PMCA and sodium pump by V 10 , as well as kinetic analysis, is in agreement with previously reported findings that decameric vanadate species block the active side of P-type ATPases and consequently affect phosphorylation step in the enzyme cycle of P-type ATPases (sodium and calcium pump) (Stokes et al 2005). However, this mechanism could not be responsible for obtained ecto-ATPase inhibition (a member of ecto-NTPDases) and probably occurs via different mechanism resulting in less sensitivity compared to P-type ATPases (sodium and calcium pump).…”

“…It was reported that decavanadate (V 10 ) is the major protein-bound species of vanadium and have a stronger effect on various enzymes, when compared to other vanadate oligomers (Aureliano and Gândara 2005). Recently reported V 10 studies include the possibility of its use as a tool in the understanding of the transducing chemical energy into conformational energy for Ca 2+ transport by Ca 2+ -ATPase (Stokes et al 2005) as well as molecular mechanism of muscle contraction (Tiago et al 2004). Inhibitions of several adenosine triphosphatases (ATPases) such as P-type ATPases, ABC-ATPases and ribonucleases by V 10 suggest that V 10 interactions with these proteins probably favored by the existence of an ATP binding site (Messmore and Raines 2000;Pezza et al 2002).…”

Application of the iterative helical real-space reconstruction method to large membranous tubular crystals of P-type ATPases

“…In previous work, we followed the conventional strategies of Fourier-Bessel reconstruction, increasing the numbers of tubular crystals in several symmetry groups and using real-space averaging in order to maximize the resolution. In contrast to Ca 2+ -ATPase where we were able to achieve 7 Å resolution with as few as 13 tubes (Stokes et al, 2005), a 12 Å structure of Na + /K + -ATPase required 27 tubes (Rice et al, 2001) and further increases in this number have not improved this resolution significantly (unpublished results). This suggests the presence of disorder such as short range bending or variable twist that cannot be compensated by Fourier-Bessel methods, but may be tractable by IHRSR.…”

“…This data set consisted of 635 subimages with dimensions of 360×120 pixels, a relative shift of 4 pixels, and comprising a total of ~1500 unit cells. Initially, we used a reference from a Fourier-Bessel reconstruction from 13 tubular crystals with the same helical symmetry and with an estimated resolution of ~7 Å (Stokes et al, 2005). We easily obtained a well-defined minimum at the expected helical parameters Δφ and Δz during the first round of refinement, even though we did not employ layer line filtering for this data set.…”