Structural basis of metabolite transport by the chloroplast outer envelope channel OEP21.

Triose phosphates (TPs) are the primary products of photosynthetic CO2 fixation in chloroplasts, which need to be exported into the cytosol across the chloroplast inner envelope (IE) and outer envelope (OE) membranes to sustain plant growth. While transport across the IE is well understood, the mode of action of the transporters in the OE remains unclear. Here we present the high-resolution nuclear magnetic resonance (NMR) structure of the outer envelope protein 21 (OEP21) from garden pea, the main exit pore for TPs in C3 plants. OEP21 is a cone-shaped ß-barrel pore with a highly positively charged interior that enables binding and translocation of negatively charged metabolites in a competitive manner, up to a size of ~1 kDa. ATP stabilizes the channel and keeps it in an open state. Despite the broad substrate selectivity of OEP21, these results suggest that control of metabolite transport across the OE might be possible.

Quantifying the insertion of membrane proteins into lipid bilayer nanodiscs using a fusion protein strategy.

A membrane protein's oligomeric state modulates its functionality in various cellular processes. Since membrane proteins have to be solubilized in an appropriate membrane mimetic, the use of classical biophysical methods to analyze protein oligomers is challenging. We here present a method to determine the number of membrane proteins inserted into lipid nanodiscs. It is based on the ability to selectively quantify the amount of a small and robust fusion protein that can be proteolytically cleaved off from a membrane protein after incorporation into lipid nanodiscs. A detailed knowledge of the number of membrane proteins per nanodisc at defined assembly conditions is essential to estimate the tendency for oligomerization, but also for guiding sample optimization for structural investigations that require the presence of a homogenous oligomeric state. We show that this method can efficiently be used to determine the number of VDAC1 channels in nanodiscs at various assembly conditions, as confirmed by negative stain EM. The presented method is suitable in particular for membrane proteins that cannot be probed easily by other methods such as single span transmembrane helices. This assay can be applied to any membrane protein that can be incorporated into a nanodisc without the requirement for special instrumentation and will thus be widely applicable and complementary to other methods that quantify membrane protein insertion in lipid nanodiscs.

Conformational processing of oncogenic v-Src kinase by the molecular chaperone Hsp90.

Hsp90 is a molecular chaperone involved in the activation of numerous client proteins, including many kinases. The most stringent kinase client is the oncogenic kinase v-Src. To elucidate how Hsp90 chaperones kinases, we reconstituted v-Src kinase chaperoning in vitro and show that its activation is ATP-dependent, with the cochaperone Cdc37 increasing the efficiency. Consistent with in vivo results, we find that Hsp90 does not influence the almost identical c-Src kinase. To explain these findings, we designed Src kinase chimeras that gradually transform c-Src into v-Src and show that their Hsp90 dependence correlates with compactness and folding cooperativity. Molecular dynamics simulations and hydrogen/deuterium exchange of Hsp90-dependent Src kinase variants further reveal increased transitions between inactive and active states and exposure of specific kinase regions. Thus, Hsp90 shifts an ensemble of conformations of v-Src toward high activity states that would otherwise be metastable and poorly populated.