Atomistic Structure and Dynamics of the Ca2+-ATPase Bound to Phosphorylated Phospholamban

Sarcolipin (SLN), a single-spanning membrane protein, is a regulator of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA1a). Chemically synthesized SLN, palmitoylated or not (pSLN or SLN), and recombinant wild-type rabbit SERCA1a expressed in S. cerevisiae design experimental conditions that provide a deeper understanding of the functional role of SLN on the regulation of SERCA1a. Our data show that chemically synthesized SLN interacts with recombinant SERCA1a, with calcium-deprived E2 state as well as with calcium-bound E1 state. This interaction hampers the binding of calcium in agreement with published data. Unexpectedly, SLN has also an allosteric effect on SERCA1a transport activity by impairing the binding of ATP. Our results reveal that SLN significantly slows down the E2 to Ca2.E1 transition of SERCA1a while it affects neither phosphorylation nor dephosphorylation. Comparison with chemically synthesized SLN deprived of acylation demonstrates that palmitoylation is not necessary for either inhibition or association with SERCA1a. However, it has a small but statistically significant effect on SERCA1a phosphorylation when various ratios of SLN-SERCA1a or pSLN-SERCA1a are tested.

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Sarcolipin alters SERCA1a interdomain communication by impairing binding of both calcium and ATP

Montigny

Huang

Beswick

et al. 2021

“…The angle between E 2P ( E 2·BeF 3 − ) and E 1Ca 2 was calculated 56.4°, 51.0°, 53.0°, and 54.8° from structures of 3B9B 2 for E 2·BeF 3 − and 1SU4 6 , 2C9M 52 , 3J7T 53 , and 5XA7 54 for E 1Ca 2 , respectively. Note that 1SU4 6 , 2C9M 52 , 3J7T 53 , and 5XA7 54 were categorized E 1Ca 2 state in Ref 55 . The angle between E 1PCa 2 ( E 1Ca 2 ·AlF 4 − ·ADP) and E 2P ( E 2·BeF 3 − ) was calculated 58.8° from structures: (PDB ID code: 1T5T 56 and 2ZBE 17 ).…”

Section: Methodsmentioning

confidence: 99%

Angle change of the A-domain in a single SERCA1a molecule detected by defocused orientation imaging

Katoh

Daiho

Yamasaki

et al. 2021

The sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) transports Ca2+ ions across the membrane coupled with ATP hydrolysis. Crystal structures of ligand-stabilized molecules indicate that the movement of actuator (A) domain plays a crucial role in Ca2+ translocation. However, the actual structural movements during the transitions between intermediates remain uncertain, in particular, the structure of E2PCa2 has not been solved. Here, the angle of the A-domain was measured by defocused orientation imaging using isotropic total internal reflection fluorescence microscopy. A single SERCA1a molecule, labeled with fluorophore ReAsH on the A-domain in fixed orientation, was embedded in a nanodisc, and stabilized on Ni–NTA glass. Activation with ATP and Ca2+ caused angle changes of the fluorophore and therefore the A-domain, motions lost by inhibitor, thapsigargin. Our high-speed set-up captured the motion during EP isomerization, and suggests that the A-domain rapidly rotates back and forth from an E1PCa2 position to a position close to the E2P state. This is the first report of the detection in the movement of the A-domain as an angle change. Our method provides a powerful tool to investigate the conformational change of a membrane protein in real-time.

“…Consequently, pharmacological stimulation of SERCA2a activity has been proposed as a therapeutic strategy to improve cardiac function 10 – 13 . There is extensive evidence indicating that key effector sites are localized in the transmembrane domain of the pump, including regulatory and inhibitory binding sites 14 . More recently, we have shown that the membrane protein dwarf open reading frame (DWORF) binds to the transmembrane domain of SERCA2a and directly stimulates the activity of the pump 15 .…”

Section: Introductionmentioning

confidence: 99%

A multiscale approach for bridging the gap between potency, efficacy, and safety of small molecules directed at membrane proteins

Aguayo‐Ortiz

Creech

Jiménez-Vázquez

et al. 2021

Self Cite

Membrane proteins constitute a substantial fraction of the human proteome, thus representing a vast source of therapeutic drug targets. Indeed, newly devised technologies now allow targeting “undruggable” regions of membrane proteins to modulate protein function in the cell. Despite the advances in technology, the rapid translation of basic science discoveries into potential drug candidates targeting transmembrane protein domains remains challenging. We address this issue by harmonizing single molecule-based and ensemble-based atomistic simulations of ligand–membrane interactions with patient-derived induced pluripotent stem cell (iPSC)-based experiments to gain insights into drug delivery, cellular efficacy, and safety of molecules directed at membrane proteins. In this study, we interrogated the pharmacological activation of the cardiac Ca2+ pump (Sarcoplasmic reticulum Ca2+-ATPase, SERCA2a) in human iPSC-derived cardiac cells as a proof-of-concept model. The combined computational-experimental approach serves as a platform to explain the differences in the cell-based activity of candidates with similar functional profiles, thus streamlining the identification of drug-like candidates that directly target SERCA2a activation in human cardiac cells. Systematic cell-based studies further showed that a direct SERCA2a activator does not induce cardiotoxic pro-arrhythmogenic events in human cardiac cells, demonstrating that pharmacological stimulation of SERCA2a activity is a safe therapeutic approach targeting the heart. Overall, this novel multiscale platform encompasses organ-specific drug potency, efficacy, and safety, and opens new avenues to accelerate the bench-to-patient research aimed at designing effective therapies directed at membrane protein domains.