RESUMEN
Myoregulin (MLN) is a member of the regulin family, a group of homologous membrane proteins that bind to and regulate the activity of the sarcoplasmic reticulum Ca2+-ATPase (SERCA). MLN, which is expressed in skeletal muscle, contains an acidic residue in its transmembrane domain. The location of this residue, Asp35, is unusual because the relative occurrence of aspartate is very rare (<0.2%) within the transmembrane helix regions. Therefore, we used atomistic simulations and ATPase activity assays of protein co-reconstitutions to probe the functional role of MLN residue Asp35. These structural and functional studies showed Asp35 has no effects on SERCA's affinity for Ca2+ or the structural integrity of MLN in the lipid bilayer. Instead, Asp35 controls SERCA inhibition by populating a bound-like orientation of MLN. We propose Asp35 provides a functional advantage over other members of the regulin family by populating preexisting MLN conformations required for MLN-specific regulation of SERCA. Overall, this study provides new clues about the evolution and functional divergence of the regulin family and offers novel insights into the functional role of acidic residues in transmembrane protein domains.
Asunto(s)
Calcio , Músculo Esquelético , Calcio/metabolismo , Proteínas de Unión al Calcio/química , Transporte Iónico , Conformación Molecular , Músculo Esquelético/metabolismo , Retículo Sarcoplasmático/química , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/química , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , HumanosRESUMEN
Intracellular calcium signaling is essential for all kingdoms of life. An important part of this process is the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), which maintains the low cytosolic calcium levels required for intracellular calcium homeostasis. In higher organisms, SERCA is regulated by a series of tissue-specific transmembrane subunits such as phospholamban in cardiac muscles and sarcolipin in skeletal muscles. These regulatory axes are so important for muscle contractility that SERCA, phospholamban, and sarcolipin are practically invariant across mammalian species. With the recent discovery of the arthropod sarcolambans, the family of calcium pump regulatory subunits appears to span more than 550 million years of evolutionary divergence from arthropods to humans. This evolutionary divergence is reflected in the peptide sequences, which vary enormously from one another and only vaguely resemble phospholamban and sarcolipin. The discovery of the sarcolambans allowed us to address two questions. How much sequence variation is tolerated in the regulation of mammalian SERCA activity by the transmembrane peptides? Do divergent peptide sequences mimic phospholamban or sarcolipin in their regulatory activities despite limited sequence similarity? We expressed and purified recombinant sarcolamban peptides from three different arthropods. The peptides were coreconstituted into proteoliposomes with mammalian SERCA1a and the effect of each peptide on the apparent calcium affinity and maximal activity of SERCA was measured. All three peptides were superinhibitors of SERCA, exhibiting either phospholamban-like or sarcolipin-like characteristics. Molecular modeling, protein-protein docking, and molecular dynamics simulations revealed novel features of the divergent peptides and their SERCA regulatory properties.
Asunto(s)
Calcio , Retículo Sarcoplasmático , Animales , Calcio/metabolismo , Señalización del Calcio , Proteínas de Unión al Calcio/química , Humanos , Mamíferos/metabolismo , Simulación de Dinámica Molecular , Proteínas Musculares , Péptidos/metabolismo , Péptidos/farmacología , Proteolípidos/química , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/químicaRESUMEN
The sarco-endoplasmic reticulum calcium ATPase (SERCA) is responsible for maintaining calcium homeostasis in all eukaryotic cells by actively transporting calcium from the cytosol into the sarco-endoplasmic reticulum (SR/ER) lumen. Calcium is an important signaling ion, and the activity of SERCA is critical for a variety of cellular processes such as muscle contraction, neuronal activity, and energy metabolism. SERCA is regulated by several small transmembrane peptide subunits that are collectively known as the "regulins". Phospholamban (PLN) and sarcolipin (SLN) are the original and most extensively studied members of the regulin family. PLN and SLN inhibit the calcium transport properties of SERCA and they are required for the proper functioning of cardiac and skeletal muscles, respectively. Myoregulin (MLN), dwarf open reading frame (DWORF), endoregulin (ELN), and another-regulin (ALN) are newly discovered tissue-specific regulators of SERCA. Herein, we compare the functional properties of the regulin family of SERCA transmembrane peptide subunits and consider their regulatory mechanisms in the context of the physiological and pathophysiological roles of these peptides. We present new functional data for human MLN, ELN, and ALN, demonstrating that they are inhibitors of SERCA with distinct functional consequences. Molecular modeling and molecular dynamics simulations of SERCA in complex with the transmembrane domains of MLN and ALN provide insights into how differential binding to the so-called inhibitory groove of SERCA-formed by transmembrane helices M2, M6, and M9-can result in distinct functional outcomes.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Proteínas Musculares/metabolismo , Proteolípidos/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Animales , Proteínas de Unión al Calcio/genética , Humanos , Modelos Moleculares , Proteínas Musculares/genética , Proteolípidos/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genéticaRESUMEN
Myoregulin (MLN) is a protein that regulates the activity of the sarcoplasmic reticulum Ca2+-ATPase (SERCA) without affecting its affinity for Ca2+. MLN's residue Lys27 is located at a site where other SERCA regulators control Ca2+ affinity. Therefore, we conducted atomistic simulations and ATPase activity experiments to determine whether replacing Lys27 with asparagine, a conserved residue found in various muscle SERCA regulators, would enable MLN to modulate both the Ca2+ affinity and catalytic activity of SERCA. Our findings indicate that replacing Lys27 with Asn significantly enhances the inhibitory potency of MLN, but it does not affect SERCA's affinity for Ca2+. We suggest that the SERCA site modulating Ca2+ affinity also acts as a catalytic activity switch. Therefore, this site is a key element contributing to the functional divergence among homologous SERCA regulators. This study paves the way for future investigations to explore how biological function diverges during the evolution of the SERCA regulator family.
Asunto(s)
Asparagina , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Asparagina/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Retículo Sarcoplasmático/metabolismoRESUMEN
The reversible association of transmembrane helices is a fundamental mechanism in how living cells convey information and respond to physiological events. The cardiac calcium transport regulator phospholamban (PLN) is an example of a single-span transmembrane protein that populates a variety of reversible and competing oligomeric states. PLN primarily forms monomers and pentamers in the membrane, where the PLN pentamer is a storage form and the PLN monomer forms a hetero-dimeric inhibitory complex with SERCA. The binding affinity and free-energy of formation of the SERCA-PLN complex in a membrane have not been determined. As is the case for most transmembrane protein interactions, measuring these quantities experimentally is extremely challenging. In this study, we estimated binding affinities by employing in silico alchemical free-energy calculations for all PLN transmembrane alanine substitutions in a membrane bilayer. The binding affinities were calculated separately for the SERCA-PLN complex, a PLN monomer, and a PLN pentamer and compared to in vitro functional measurements of SERCA regulation by the PLN alanine substitutions. Initially, the changes in SERCA inhibition by PLN alanine substitutions were compared to the changes in free energy for the SERCA-PLN complex formed from the PLN monomer. However, the functional data for the PLN alanine substitutions were better explained by the formation of the SERCA-PLN complex directly from the PLN pentamer. This finding points to an inhibitory mechanism favoring conformational selection of SERCA and the interaction of a PLN pentamer with SERCA for 'delivery' of a PLN monomer to the inhibitory site. The implications of these findings suggest that the energetics of helix exchange between homo- and hetero-oligomeric signaling complexes is favored over an intermediate involving a free monomeric helix in the membrane bilayer.
RESUMEN
The sarco-plasmic reticulum calcium pump (SERCA) plays a critical role in the contraction-relaxation cycle of muscle. In cardiac muscle, SERCA is regulated by the inhibitor phospholamban. A new regulator, dwarf open reading frame (DWORF), has been reported to displace phospholamban from SERCA. Here, we show that DWORF is a direct activator of SERCA, increasing its turnover rate in the absence of phospholamban. Measurement of in-cell calcium dynamics supports this observation and demonstrates that DWORF increases SERCA-dependent calcium reuptake. These functional observations reveal opposing effects of DWORF activation and phospholamban inhibition of SERCA. To gain mechanistic insight into SERCA activation, fluorescence resonance energy transfer experiments revealed that DWORF has a higher affinity for SERCA in the presence of calcium. Molecular modeling and molecular dynamics simulations provide a model for DWORF activation of SERCA, where DWORF modulates the membrane bilayer and stabilizes the conformations of SERCA that predominate during elevated cytosolic calcium.