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 use a combination of solid-state and solution NMR spectroscopy to characterize the conformations of αS bound to lipid membranes mimicking the composition and physical properties of synaptic vesicles. The study shows 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 of this protein and in the molecular processes leading to its aggregation.

Protein kinase A (PKA) is a ubiquitous phosphoryl transferase that mediates hundreds of cell signaling events. During turnover, its catalytic subunit (PKA-C) interconverts between three major conformational states (open, intermediate, and closed) that are dynamically and allosterically activated by nucleotide binding. We show that the structural transitions between these conformational states are minimal and allosteric dynamics encode the motions from one state to the next. NMR and molecular dynamics simulations define the energy landscape of PKA-C, with the substrate allowing the enzyme to adopt a broad distribution of conformations (dynamically committed state) and the inhibitors (high magnesium and pseudosubstrate) locking it into discrete minima (dynamically quenched state), thereby reducing the motions that allow turnover. These results unveil the role of internal dynamics in both kinase function and regulation.

Phospholamban (PLN) is an essential regulator of cardiac muscle contractility. The homopentameric assembly of PLN is the reservoir for active monomers that, upon deoligomerization form 1:1 complexes with the sarco(endo)plasmic reticulum Ca ²⁺ -ATPase (SERCA), thus modulating the rate of calcium uptake. In lipid bilayers and micelles, monomeric PLN exists in equilibrium between a bent (or resting) T state and a more dynamic (or active) R state. Here, we report the high-resolution structure and topology of the T state of a monomeric PLN mutant in lipid bilayers, using a hybrid of solution and solid-state NMR restraints together with molecular dynamics simulations in explicit lipid environments. Unlike the previous structural ensemble determined in micelles, this approach gives a complete picture of the PLN monomer structure in a lipid bilayer. This hybrid ensemble exemplifies the tilt, rotation, and depth of membrane insertion, revealing the interaction with the lipids for all protein domains. The N-terminal amphipathic helical domain Ia (residues 1–16) rests on the surface of the lipid membrane with the hydrophobic face of domain Ia embedded in the membrane bilayer interior. The helix comprised of domain Ib (residues 23–30) and transmembrane domain II (residues 31–52) traverses the bilayer with a tilt angle of ≈24°. The specific interactions between PLN and lipid membranes may represent an additional regulatory element of its inhibitory function. We propose this hybrid method for the simultaneous determination of structure and topology for membrane proteins with compact folds or proteins whose spatial arrangement is dictated by their specific interactions with lipid bilayers.

Phospholamban (PLN) is a type II membrane protein that inhibits the sarcoplasmic reticulum Ca ²⁺ -ATPase (SERCA), thereby regulating calcium homeostasis in cardiac muscle. In membranes, PLN forms pentamers that have been proposed to function either as a storage for active monomers or as ion channels. Here, we report the T-state structure of pentameric PLN solved by a hybrid solution and solid-state NMR method. In lipid bilayers, PLN adopts a pinwheel topology with a narrow hydrophobic pore, which excludes ion transport. In the T state, the cytoplasmic amphipathic helices (domains Ia) are absorbed into the lipid bilayer with the transmembrane domains arranged in a left-handed coiled-coil configuration, crossing the bilayer with a tilt angle of approximately 11° with respect to the membrane normal. The tilt angle difference between the monomer and pentamer is approximately 13°, showing that intramembrane helix–helix association forces dominate over the hydrophobic mismatch, driving the overall topology of the transmembrane assembly. Our data reveal that both topology and function of PLN are shaped by the interactions with lipids, which fine-tune the regulation of SERCA.

Significance: The sarcoplasmic reticulum Ca ²⁺ -ATPase (SERCA)/phospholamban complex regulates cardiac muscle contractility by controlling Ca ²⁺ transport from the cytosol to the lumen of the sarcoplasmic reticulum. By mapping the interactions between these two membrane proteins, we found that SERCA function depends on the equilibria between transient conformational states of phospholamban. Phosphorylation of phospholamban shifts the equilibria, enhancing SERCA function. This mechanism explains why tuning phospholamban’s structural dynamics can modulate SERCA function and may aid in designing innovative therapeutic approaches to heart failure.