α-synuclein (αS) is a protein involved in neurotransmitter release in presynaptic terminals, and whose aberrant aggregation is associated with Parkinson’s disease. In dopaminergic neurons, αS exists in a tightly regulated equilibrium between water-soluble and membrane-associated forms. Here we used a combination of solid-state and solution-state NMR spectroscopy to characterize the conformations of αS bound to lipid membranes mimicking the composition and physical properties of synaptic vesicles. The study evidences three αS regions possessing distinct structural and dynamical properties, including an N-terminal helical segment having a role of membrane-anchor, an unstructured C-terminal region that is weakly associated with the membrane, and a central region acting as a sensor of the lipid properties and determining the affinity of αS membrane binding. Taken together, our data define the nature of the interactions of αS with biological membranes and provide insights into their roles in the function and in the molecular processes leading the aggregation of this protein.

The membrane protein complex between the sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) and phospholamban (PLN) controls Ca 2+ transport in cardiomyocytes, thereby modulating cardiac contractility. β-Adrenergic-stimulated phosphorylation of PLN at Ser-16 enhances SERCA activity via an unknown mechanism. Using solid-state nuclear magnetic resonance spectroscopy, we mapped the physical interactions between SERCA and both unphosphorylated and phosphorylated PLN in membrane bilayers. We found that the allosteric regulation of SERCA depends on the conformational equilibrium of PLN, whose cytoplasmic regulatory domain interconverts between three different states: a ground T state (helical and membrane associated), an excited R state (unfolded and membrane detached), and a B state (extended and enzymebound), which is noninhibitory. Phosphorylation at Ser-16 of PLN shifts the populations toward the B state, increasing SERCA activity. We conclude that PLN's conformational equilibrium is central to maintain SERCA's apparent Ca 2+ affinity within a physiological window. This model represents a paradigm shift in our understanding of SERCA regulation by posttranslational phosphorylation and suggests strategies for designing innovative therapeutic approaches to enhance cardiac muscle contractility. -ATPase (SERCA)/phospholamban (PLN) complex regulates Ca 2+ translocation into the sarcoplasmic reticulum (SR) of cardiomyocytes and constitutes the main mechanism of cardiac relaxation (diastole) (1-3). SERCA is a P-type ATPase that translocates two Ca 2+ ions per ATP molecule hydrolyzed in exchange for three H + ( Fig. 1) (4, 5). PLN binds and allosterically inhibits SERCA function, decreasing its apparent affinity for Ca 2+ ions (3, 6). On β-adrenergic stimulation, cAMP-dependent protein kinase A phosphorylates PLN at Ser-16, reversing the inhibition and augmenting cardiac output (3). Disruptions in this regulatory mechanism degenerate into Ca 2+ mishandling and heart failure (3). Several X-ray structures of SERCA have been determined along its enzymatic coordinates, providing atomic details on the structural transitions in the absence of PLN (4, 5). The first image of the SERCA/PLN complex resulted from cryo-EM studies (7), but the low-resolution data prevent an atomic view of PLN structure and architecture within the complex. In addition, mutagenesis and cross-linking data were used to model the complex, suggesting that the inhibitory transmembrane (TM) region of PLN is positioned into a binding groove far from the putative Ca 2+ entry, as well as the ATP binding site, and located between TM helices M2, M4, M6, and M9 of SERCA. The location of PLN's TM domain agrees with a recent crystal structure of the SERCA/PLN complex (8) and is remarkably similar to the one recently identified for a PLN homolog, sarcolipin, in complex with SERCA (9, 10) (Fig. 1).In the SERCA/PLN model, which was further refined using NMR constraints (11), the loop bridging the TM and cytoplasmic domain of PLN adopts an unfolded configuration, stretching tow...

The effects of specific drug-polymer interactions (ionic or hydrogen-bonding) on the molecular mobility of model amorphous solid dispersions (ASDs) were investigated. ASDs of ketoconazole (KTZ), a weakly basic drug, with each of poly(acrylic acid) (PAA), poly(2-hydroxyethyl methacrylate) (PHEMA), and polyvinylpyrrolidone (PVP) were prepared. Drug-polymer interactions in the ASDs were evaluated by infrared and solid-state NMR, the molecular mobility quantified by dielectric spectroscopy, and crystallization onset monitored by differential scanning calorimetry (DSC) and variable temperature X-ray diffractometry (VTXRD). KTZ likely exhibited ionic interactions with PAA, hydrogen-bonding with PHEMA, and weaker dipole-dipole interactions with PVP. On the basis of dielectric spectroscopy, the α-relaxation times of the ASDs followed the order: PAA > PHEMA > PVP. In addition, the presence of ionic interactions also translated to a dramatic and disproportionate decrease in mobility as a function of polymer concentration. On the basis of both DSC and VTXRD, an increase in strength of interaction translated to higher crystallization onset temperature and a decrease in extent of crystallization. Stronger drug-polymer interactions, by reducing the molecular mobility, can potentially delay the crystallization onset temperature as well as crystallization extent.

SUMMARY TLQP-21, a VGF-encoded peptide is emerging as a novel target for obesity-associated disorders. TLQP-21 is found in the sympathetic nerve terminals in the adipose tissue and targets the G-protein-coupled-receptor (GPCR) Complement-3a-Receptor1 (C3aR1). So far, the mechanisms of TLQP-21-induced receptor activation remained unexplored. Here, we report that TLQP-21 is intrinsically disordered and undergoes a disorder-to-order transition, adopting an α-helical conformation, upon targeting cells expressing the C3aR1. We determined that the hot spots for TLQP-21 are located at the C-terminus, with mutations in the last four amino acids progressively reducing the bioactivity and, a single site mutation (R21A) or C-terminal amidation abolishing its function completely. Interestingly, the human TLQP-21 sequence carrying a S20A substitution activates the human C3aR1 receptor with lower potency compared to the rodent sequence. These studies reveal the mechanism of action of TLQP-21 and provide molecular templates for designing agonists and antagonists to modulate C3aR1 functions.

Fast data collection: A general method for dual data acquisition of multidimensional magic‐angle spinning solid‐state NMR experiments is presented (see picture). The method uses a simultaneous Hartmann–Hahn cross‐polarization from 1H to 13C and 15N nuclei and exploits the long‐living 15N polarization for parallel acquisition of two multidimensional experiments.