SERCA Structural Dynamics Induced by ATP and Calcium

Mueller, Benjamin K.; Zhao, Min; Negrashov, Igor V.; Bennett, Roberta; Thomas, David D.

doi:10.1021/bi0489457

Cited by 25 publications

(30 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Consider that stabilization by multiple weak interactions depends on anisotropy, and thermally induced motion reduces electrostatic forces because of averaging effects by dipole rotations. In fact, spectroscopic studies have shown conformational heterogeneity of the ATPase protein (37), as well as prominent effects of ligands on the internal dynamics of the enzyme protein (17). Strong restraint of fluctuations is then observed upon acquisition of intermediate catalytic states.…”

Section: Discussionmentioning

confidence: 99%

“…3) does not involve a primary effect on the digestion pattern but rather a delay of the time course of digestion occurring with the same pattern (E1 or E2, respectively). This suggests that protection may be due to constraint of conformational fluctuations related to internal protein dynamics (17) and required for suitable interaction of the ATPase with the proteolytic enzyme. This is best demonstrated by comparing the ATPase digestion with trypsin at various temperatures.…”

Section: Temperature Dependence Of Proteolytic Digestion and Effects mentioning

confidence: 99%

See 1 more Smart Citation

Conformational Fluctuations of the Ca2+-ATPase in the Native Membrane Environment

Inesi

Lewis

Toyoshima

et al. 2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

2؉ to the E2-P pattern, whereby alkaline pH will limit this conformational transition. Complementary experiments on digestion with trypsin exhibit high temperature dependence, indicating that, in the E1 and E2 ground states, the ATPase conformation undergoes strong fluctuations related to internal protein dynamics. The fluctuations are tightly constrained by ATP binding and phosphoenzyme formation, and this constraint must be overcome by thermal activation and substrate-free energy to allow enzyme turnover. In fact, a substantial portion of ATP free energy is utilized for conformational work related to the E1ϳP⅐2Ca 2؉ to E2-P transition, thereby disrupting high affinity binding and allowing luminal diffusion of Ca 2؉ . The E2 state and luminal path closure follow removal of conformational constraint by phosphate.The Ca 2ϩ -ATPase of sarcoendoplasmic reticulum membranes (SERCA) 2 includes multiple isoforms and splice variants with variable tissue distribution. In this study, we used the SERCA1a isoform of skeletal muscle, a well characterized enzyme (1, 2) that utilizes the free energy of ATP for Ca 2ϩ transport against a concentration gradient. The functional unit is a protein monomer consisting of 994 amino acid residues. The sequence is folded into a cluster of 10 segments forming a transmembrane region, and three relatively large domains ("N", "P," and "A") protruding from the cytosolic surface of the membrane (3, 4). The ATPase cycle begins with high affinity binding of two Ca 2ϩ derived from the cytosolic medium ("outside"), followed by ATP utilization to form a phosphorylated enzyme intermediate. Isomerization of the phosphoenzyme intermediate is then coupled to active transport of the bound Ca 2ϩ across the membrane ("inside"). Hydrolytic cleavage of the phosphoenzyme is the final step that allows enzyme turnover.The cooperative character of Ca 2ϩ binding as well as the relatively large distance between the catalytic site in the headpiece and the Ca 2ϩ -binding sites of the ATPase within the transmembrane region imply that conformational rearrangements of the ATPase protein are involved in the mechanism of catalytic activation and energy transduction. Within the general context of cation transport, these rearrangements were envisioned as interconversions of the E1 and E2 conformations in the ground state of the enzyme and the E1-P to E2-P conformations of the phosphorylated intermediate. In fact, conformational changes were initially detected by spectroscopic experimentation (5). High resolution structures were then obtained by crystallographic studies and attributed to different catalytic intermediates (6). On the other hand, the occurrence of conformational transitions in the native membrane environment is revealed by changes in the patterns of proteolysis (7). We report here a series of experiments on limited proteolysis with proteinase K or trypsin, yielding complementary information on the conformational effects of pH, temperature, catalytic ligands, and the specific inhibitor thapsigargin (TG). The ...

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Temperature Dependence Of Proteolytic Digestion and Effects mentioning

confidence: 99%

Conformational Fluctuations of the Ca2+-ATPase in the Native Membrane Environment

Inesi

Lewis

Toyoshima

et al. 2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Rabbit light skeletal SR vesicles were prepared using a method previously reported. 13 Rabbit muscles were harvested from the hind leg of New Zealand White rabbits and purified using a sucrose gradient. The result light skeletal SR contains 80% SERCA.…”

Section: Methodsmentioning

confidence: 99%

Discovery of Enzyme Modulators via High-Throughput Time-Resolved FRET in Living Cells

et al. 2014

Self Cite

View full text Add to dashboard Cite

We have used a “2-color” SERCA (sarco/endoplasmic reticulum calcium ATPase) biosensor and a unique high-throughput fluorescence lifetime plate-reader (FLT-PR) to develop a high-precision live-cell assay designed to screen for small molecules that perturb SERCA structure. A SERCA construct, in which red fluorescent protein (RFP) was fused to the N terminus and green fluorescent protein (GFP) to an interior loop, was stably expressed in an HEK cell line that grows in monolayer or suspension. Fluorescence resonance energy transfer (FRET) from GFP to RFP was measured in the FLT-PR, which increases precision 30-fold over intensity-based plate-readers without sacrificing throughput. FRET was highly sensitive to known SERCA modulators. We screened a small chemical library and identified ten compounds that significantly affected 2-color SERCA FLT. Three of these compounds reproducibly lowered FRET and inhibited SERCA in a dose-dependent manner. This assay is ready for large-scale HTS campaigns, and is adaptable to many other targets.

show abstract

“…2,[8][9][10][11][12][13] However, fluorescence resonance energy transfer (FRET) in functionally reconstituted membranes has shown that PLB binds tightly to SERCA in both the presence and absence of micromolar [Ca 2C ], so Ca-dependent relief of inhibition must be due to structural rearrangement 0022-2836/$ -see front matter q 2006 Elsevier Ltd. All rights reserved.…”

Section: Relief Of Inhibition Does Not Require Plb Dissociation From mentioning

confidence: 99%

Phosphorylation-dependent Conformational Switch in Spin-labeled Phospholamban Bound to SERCA

Karim

Zhang

Howard

et al. 2006

Journal of Molecular Biology

Self Cite

105

214

View full text Add to dashboard Cite

SERCA Structural Dynamics Induced by ATP and Calcium

Cited by 25 publications

References 38 publications

Conformational Fluctuations of the Ca2+-ATPase in the Native Membrane Environment

Conformational Fluctuations of the Ca2+-ATPase in the Native Membrane Environment

Discovery of Enzyme Modulators via High-Throughput Time-Resolved FRET in Living Cells

Phosphorylation-dependent Conformational Switch in Spin-labeled Phospholamban Bound to SERCA

Contact Info

Product

Resources

About