Determining the structure of the bacterial voltage-gated sodium channel NaChBac embedded in liposomes by cryo electron tomography and subtomogram averaging.

Voltage-gated sodium channels shape action potentials that propagate signals along cells. When the membrane potential reaches a certain threshold, the channels open and allow sodium ions to flow through the membrane depolarizing it, followed by the deactivation of the channels. Opening and closing of the channels is important for cellular signalling and regulates various physiological processes in muscles, heart and brain. Mechanistic insights into the voltage-gated channels are difficult to achieve as the proteins are typically extracted from membranes for structural analysis which results in the loss of the transmembrane potential that regulates their activity. Here, we report the structural analysis of a bacterial voltage-gated sodium channel, NaChBac, reconstituted in liposomes under an electrochemical gradient by cryo electron tomography and subtomogram averaging. We show that the small channel, most of the residues of which are embedded in the membrane, can be localized using a genetically fused GFP. GFP can aid the initial alignment to an average resulting in a correct structure, but does not help for the final refinement. At a moderate resolution of ˜16 Å the structure of NaChBac in an unrestricted membrane bilayer is 10% wider than the structure of the purified protein previously solved in nanodiscs, suggesting the potential movement of the peripheral voltage-sensing domains. Our study explores the limits of structural analysis of membrane proteins in membranes.

KAHRP dynamically relocalizes to remodeled actin junctions and associates with knob spirals in Plasmodium falciparum-infected erythrocytes.

The knob-associated histidine-rich protein (KAHRP) plays a pivotal role in the pathophysiology of Plasmodium falciparum malaria by forming membrane protrusions in infected erythrocytes, which anchor parasite-encoded adhesins to the membrane skeleton. The resulting sequestration of parasitized erythrocytes in the microvasculature leads to severe disease. Despite KAHRP being an important virulence factor, its physical location within the membrane skeleton is still debated, as is its function in knob formation. Here, we show by super-resolution microscopy that KAHRP initially associates with various skeletal components, including ankyrin bridges, but eventually colocalizes with remnant actin junctions. We further present a 35 Å map of the spiral scaffold underlying knobs and show that a KAHRP-targeting nanoprobe binds close to the spiral scaffold. Single-molecule localization microscopy detected ~60 KAHRP molecules/knob. We propose a dynamic model of KAHRP organization and a function of KAHRP in attaching other factors to the spiral scaffold.

Structure of the merozoite surface protein 1 from Plasmodium falciparum.

The merozoite surface protein 1 (MSP-1) is the most abundant protein on the surface of the erythrocyte-invading Plasmodium merozoite, the causative agent of malaria. MSP-1 is essential for merozoite formation, entry into and escape from erythrocytes, and is a promising vaccine candidate. Here, we present monomeric and dimeric structures of full-length MSP-1. MSP-1 adopts an unusual fold with a large central cavity. Its fold includes several coiled-coils and shows structural homology to proteins associated with membrane and cytoskeleton interactions. MSP-1 formed dimers through these domains in a concentration-dependent manner. Dimerization is affected by the presence of the erythrocyte cytoskeleton protein spectrin, which may compete for the dimerization interface. Our work provides structural insights into the possible mode of interaction of MSP-1 with erythrocytes and establishes a framework for future investigations into the role of MSP-1 in Plasmodium infection and immunity.