Distinct Structural Identities of Catalytic and Ca&lt;sup&gt;2+&lt;/sup&gt; Binding Domains in the Sarcoplasmic Reticulum ATPase

Inesi, Giuseppe; Lu, Li-Fan; Kirtley, Mary E.

doi:10.1159/000154717

Cited by 8 publications

(3 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In particular, the ten predicted transmembrane helices have been recently fitted into a low-resolution structure ). In addition, the detailed liganding of two calcium ions by side chains from six specific residues (identified by site-directed mutagenesis) has been modeled based on the calcium-binding site of thermolysin, which uses six analogous residues for cooperative binding of two calcium ions (Inesi et al, 1994).…”

Section: Introductionmentioning

confidence: 99%

Lamellar stacking in three-dimensional crystals of Ca(2+)-ATPase from sarcoplasmic reticulum

Cheong

Young

Ogawa

et al. 1996

Biophysical Journal

View full text Add to dashboard Cite

Electron microscopy of multilamellar crystals of CA(2+)-ATPase currently offers the best opportunity for obtaining a high-resolution structure of this ATP-driven ion pump. Under certain conditions small, wormlike crystals are formed and provide views parallel to the lamellar plane, from which parameters of lamellar stacking can be directly measured. Assuming that molecular packing is the same, data from these views could supplement those obtained by tilting large, thin platelike crystals. However, we were surprised to discover that the lamellar spacing was variable and depended on the amount of glycerol present during crystallization (20% versus 5%). Projection maps (h,0,l) from these womklike crystals suggest different molecular contacts that give rise to the different lamellar spacings. Based on an orthogonal projection map (h,k,0) from collapsed, wormlike crystals and on x-ray powder patterns, we conclude that molecular packing within the lamellar plane is the same as that in thin, platelike crystals and is unaffected by glycerol. Finally, the orientation of molecules in the lamellar plane was characterized from freeze-dried, shadowed crystals. Comparing the profile of molecules in these multilamellar crystals with that previously observed in helical tubes induced by vanadate gives structural evidence of the conformational change that accompanies binding of calcium of Ca(2+)-ATPase.

show abstract

Section: Introductionmentioning

confidence: 99%

Lamellar stacking in three-dimensional crystals of Ca(2+)-ATPase from sarcoplasmic reticulum

Cheong

Young

Ogawa

et al. 1996

Biophysical Journal

View full text Add to dashboard Cite

show abstract

“…A more recent reconstruction from frozen-hydrated, unstained specimens revealed the structure of the transmembrane domain (Toyoshima et al, 1993) and allowed a first attempt at fitting helices into the slightly higher resolution (14 A) envelope (Stokes et al, 1994). A more detailed model for the calciumbinding site (Inesi, 1994;Inesi et al, 1994) was constructed by juxtaposing transmembrane helices that contained the six residues previously implicated in calcium binding by sitedirected mutagenesis (Clark et al, 1989). As would be expected for Ca2+-ATPase, this model provides sites for cooperative binding of two calcium ions in addition to a narrow access for these ions to the cytoplasmic side of the membrane.…”

Section: Introductionmentioning

confidence: 99%

Preparation and analysis of large, flat crystals of Ca(2+)-ATPase for electron crystallography

Shi

Hsiung

Pace

et al. 1995

Biophysical Journal

View full text Add to dashboard Cite

Obtaining large, flat, well ordered crystals represents the key to structure determination by electron crystallography. Multilamellar crystals of Ca(2+)-ATPase are a good candidate for this methodology, and we have optimized methods of crystallization and of preparation for cryoelectron microscopy. In particular, high concentrations of glycerol were found to prevent nucleation and to reduce stacking; thus, by seeding solutions containing 40% glycerol, we obtained thin crystals that were 5-30 microns in diameter and 2-10 unit cells thick. We found that removing vesicles and minimizing concentrations of divalent cations were critical to preparing flat crystals in the frozen-hydrated state. Finally, we developed two methods for determining the number of lamellae composing individual crystals, information that is required for structure determination of this crystal form. The first method, using low magnification images of freeze-dried crystals, is more practical in our case. Nevertheless, the alternative method, involving analysis of Laue zones from electron diffraction patterns of slightly tilted crystals, may be of general use in structure determination from thin, three-dimensional crystals.

show abstract

“…Ca2+ binding to the sarcoplasmic reticulum (SR) Ca2+-ATPase protein is a prerequisite for the phosphoryl transfer from ATP to the enzyme (for reviews, see [1,2]). This fundamental step in the enzyme activation requires a long-range transmission of the Ca2+ signal from the transmembrane sector (transport sites) to the cytoplasmic head region (catalytic centre).…”

Section: Introductionmentioning

confidence: 99%

Functional properties of a sarcoplasmic reticulum Ca2+-ATPase with an altered Ca2+-binding mechanism

Martı́nez-Azorı́n

Soler

Gómez‐Fernández

et al. 1995

Biochemical Journal

View full text Add to dashboard Cite

Treatment of sarcoplasmic reticulum vesicles with diethylpyrocarbonate in the presence of a large excess of reagent, at pH 6.2 and at room temperature, reveals both a fast- and a slow-reacting population of protein residues. The loss of the Ca(2+)-ATPase activity is mainly associated with the fast-reacting population being partially sensitive to hydroxylamine. There is also an effect on the Ca(2+)-binding mechanism. Shorter derivatization times (5 min) produce a loss of the positive cooperativity of Ca2+ binding. When the treatment was prolonged for 30 min there was an additional decrease in the overall Ca2+ affinity. Curve-fitting procedures applied to the non-cooperative binding isotherms provide the equilibrium constants for the two Ca2+ sites, although they cannot discriminate between interacting and independent site mechanisms. Prestationary kinetics assays show 2 Ca2+:1 ATP ratios, at any extent of Ca2+ saturation, indicating that the Ca2+ sites are not independent. The Ca2+ dissociation profile after derivatization shows a decrease in the dissociation constant for the release of the second Ca2+, which is consistent with interacting sites. Isotopic exchange experiments show fast and slow components of equal amplitude even at subsaturating Ca2+ concentrations, which is incompatible with independent binding sites. The experimental data suggest a modification of the equilibrium binding constants making them more similar, but keeping the interacting character. The structural position of the external (cytoplasmic) and the internal (lumenal) Ca2+ sites remains unaltered in the absence of positive cooperativity.

show abstract

Distinct Structural Identities of Catalytic and Ca<sup>2+</sup> Binding Domains in the Sarcoplasmic Reticulum ATPase

Cited by 8 publications

References 20 publications

Lamellar stacking in three-dimensional crystals of Ca(2+)-ATPase from sarcoplasmic reticulum

Lamellar stacking in three-dimensional crystals of Ca(2+)-ATPase from sarcoplasmic reticulum

Preparation and analysis of large, flat crystals of Ca(2+)-ATPase for electron crystallography

Functional properties of a sarcoplasmic reticulum Ca2+-ATPase with an altered Ca2+-binding mechanism

Contact Info

Product

Resources

About