Biology subjects

Aguayo-Ortiz, R.

Publications and source records attributed to Aguayo-Ortiz, R..

3 recordsLinked to original sources

bioRxiv2020

Sarcolambans are phospholamban- and sarcolipin-like regulators of the sarcoplasmic reticulum calcium pump SERCA

From insects to humans, calcium signaling is essential for life. An important part of this process is the sarco-endoplasmic reticulum calcium pump SERCA, which maintains low cytosolic calcium levels required for intracellular calcium homeostasis. In higher organisms, this is a tightly controlled system where SERCA interacts with tissuespecific regulatory subunits such as phospholamban in cardiac muscle and sarcolipin in skeletal muscle. With the recent discovery of the sarcolambans, the family of calcium pump regulatory subunits also appears to be ancient, spanning more than 550 million years of evolutionary divergence from insects to humans. This evolutionary divergence is reflected in the peptide sequences, which vary enormously from one another and range from vaguely phospholamban-like to vaguely sarcolipin-like. Here, our goal was to investigate select sarcolamban peptides for their ability to regulate calcium pump activity. For a side-by-side comparison of diverse sarcolamban peptides, we tested them against mammalian skeletal muscle SERCA1a. This allowed us to determine if the sarcolamban peptides mimic phospholamban and sarcolipin in their regulatory activities. Four sarcolamban peptides were chosen from different invertebrate species. Of these, we were able to express and purify sarcolamban peptides from bumble bee, water flea, and tadpole shrimp. Sarcolamban peptides were co-reconstituted into proteoliposomes with mammalian SERCA1a and the effect of each peptide on the apparent calcium affinity and maximal activity of SERCA was measured. While all peptides were super-inhibitors of SERCA, they exhibited either phospholamban-like or sarcolipin-like characteristics. Molecular modeling, protein-protein docking, and molecular dynamics simulations were used to reveal novel features of insect versus mammalian calcium pumps and the sarcolamban regulatory subunits.

biochemistry↗

bioRxiv2020

Dynamics-driven allostery underlies pre-activation of the regulatory Ca2+-ATPase/phospholamban complex

Sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) and phospholamban (PLB) are essential for intracellular Ca2+ transport in myocytes. Ca2+-dependent activation of SERCA-PLB provides a rheostat function that regulates cytosolic and SR Ca2+ levels. While experimental and computational studies alone have led to a greater insight into the mechanisms for SERCA-PLB regulation, the structural changes induced by Ca2+ binding and how those are communicated to couple enzymatic activity with active transport remain poorly understood. Therefore, we have performed atomistic simulations totaling 32.7 s and cell-based intramolecular fluorescence resonance energy transfer (FRET) experiments to determine structural changes of PLB-bound SERCA in response to Ca2+ binding. Complementary simulations and experiments showed structural disorder underlies PLB inhibition of SERCA, and Ca2+ binding is sufficient to shift the protein population toward a structurally ordered state of the complex. This structural transition results in a redistribution of structural states toward a partially closed conformation of SERCAs cytosolic headpiece. Closure is accompanied by functional interactions between the N-domain {beta}5-{beta}6 loop and the A-domain. Regulation of these key structural elements indicate that Ca2+ is a critical mediator of allosteric signaling that dictates structural changes and motions that pre-activate SERCA-PLB. These findings provide direct support that dynamically driven protein allostery underlies PLB regulation of SERCA. These functional insights at unprecedented spatiotemporal resolution suggest a general modular architecture mechanism for dynamic regulation of the SERCA-PLB complex. Understanding these mechanisms is of paramount importance to guide therapeutic modulation of SERCA and other evolutionarily related ion-motive ATPases.

biophysics↗

bioRxiv2020

Conserved luminal C-terminal domain dynamically controls interdomain communication in sarcolipin

ABSTRACTSarcolipin (SLN) mediates Ca2+ transport and metabolism in muscle by regulating the activity of the Ca2+ pump SERCA. SLN has a conserved luminal C-terminal domain that contributes to the its functional divergence among homologous SERCA regulators, but the precise mechanistic role of this domain remains poorly understood. We used all-atom molecular dynamics (MD) simulations of SLN totaling 77.5 μs to show that the N- (NT) and C-terminal (CT) domains function in concert. Analysis of the MD simulations showed that serial deletions of SLN C-terminus does not affect the stability of the peptide nor induce dissociation of SLN from the membrane but promotes a gradual decrease in both tilt angle of the transmembrane helix and the local thickness of the lipid bilayer. Mutual information analysis showed that the NT and CT domains communicate with each other in SLN, and that interdomain communication is partially or completely abolished upon deletion of the conserved segment Tyr29-Tyr31 as well as by serial deletions beyond this domain. Phosphorylation of SLN at residue Thr5 also induces changes in the communication between the CT and NT domains, thus providing additional evidence for interdomain communication within SLN. We found that interdomain communication is independent of the force field used and lipid composition, thus demonstrating that communication between the NT and CT domains is an intrinsic functional feature of SLN. We propose the novel hypothesis that the conserved C-terminus is an essential element required for dynamic control of SLN regulatory function.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗