The identification of SERCA (Sarco/Endoplasmic Reticulum Calcium ATPase) as a target for modulating gain-of-function NOTCH1 mutations in Notch-dependent cancers has spurred the development of this compound class for cancer therapeutics. Despite the innate toxicity challenge associated with SERCA inhibition, we identified CAD204520 a small molecule with better drug-like properties and reduced off-target Ca 2+ toxicity compared to the SERCA inhibitor thapsigargin.In this work, we describe the properties and complex structure of CAD204520 and show that CAD204520 preferentially targets mutated over wild type NOTCH1 proteins in T-cell acute lymphoblastic leukemia (T-ALL) and mantle cell lymphoma (MCL). Uniquely among SERCA inhibitors, CAD204520 suppresses NOTCH1 mutated leukemic cells in a T-ALL xenografted model without causing cardiac toxicity.This study supports the development of SERCA inhibitors for Notch-dependent cancers and extends their application to cases with isolated mutations in the PEST degradation domain of NOTCH1, such as MCL or chronic lymphocytic leukemia (CLL).

The structure of the hexameric fungal proton pump Pma1 reveals its mechanism of autoinhibition and interactions with lipids.

CYBASC proteins are ascorbate (AscH − ) reducible, diheme b-containing integral membrane cytochrome b 561 proteins (cytb 561 ), which are proposed to be involved in AscH − recycling and facilitation of iron absorption. Two distinct CYBASC paralogs from the plant Arabidopsis thaliana, Atcytb 561 -A (A-paralog) and Atcytb 561 -B (B-paralog), have been found to differ in their visiblespectral characteristics and their interaction with AscH − and ferric iron chelates. A previously determined crystal structure of the Bparalog provides the first insights into the structural organization of a CYBASC member and implies hydrogen bonding between the substrate AscH − and the conserved lysine residues at positions 77 (B-K77) and 81 (B-K81). The function of the highly conserved tyrosine at position 70 (B-Y70) is not obvious in the crystal structure, but its localization indicates the possible involvement in proton-coupled electron transfer. Here we show that B-Y70 plays a major role in the modulation of the oxidation−reduction midpoint potential of the high-potential heme, E M (b H ), as well as in AscH − oxidation. Our results support the involvement of the functionally conserved B-K77 in the stabilization of the dianion Asc 2− . These findings are supported by the crystal structure of the Bparalog, but a comparative biochemical and biophysical characterization of the A-and B-paralogs implied distinct and more complex functions of the corresponding residues A-Y69 and A-K76 in the A-paralog. Our results emphasize the need for a high-resolution crystal structure of the A-paralog to illuminate the differences in functional organization between the two paralogs.

An intricate case of charge migration in proteins is represented by the diheme‐containing class of succinate:quinone oxidoreductases, where, depending on the species and the direction of the reaction catalyzed in vivo, transmembrane electron transfer is either coupled to transmembrane proton transfer or not. The absence of compensatory transmembrane proton transfer results in an electrogenic overall reaction, as has been demonstrated for diheme‐containing succinate:menaquinone reductases. The presence of such a compensatory transmembrane proton transfer renders the overall reaction electroneutral, as has been shown to be the case for diheme‐containing menaquinol:fumarate reductases (QFR). The availability of high‐resolution X‐ray crystal structures of the diheme‐containing QFR from Wolinella succinogenes has inspired and enabled several theoretical and experimental studies providing proof and further characterization of these electron and proton transfer events.

The fungal plasma membrane H+-ATPase Pma1 is a vital enzyme, generating a proton-motive force that drives the import of essential nutrients. Auto-inhibited Pma1 hexamers in starving fungi are activated by glucose signalling resulting in phosphorylation of the auto-inhibitory domain. As related P-type ATPases are not known to oligomerise, the physiological relevance of Pma1 hexamers remains unknown. We have determined the structure of hexameric Pma1 from Neurospora crassa by cryo-EM at 3.3 Å resolution, elucidating the molecular basis for hexamer formation and auto-inhibition, and providing a basis for structure-based drug development. Coarse-grained molecular dynamics simulations in a lipid bilayer suggest lipid-mediated contacts between monomers and a substantial protein-induced membrane deformation that could act as a proton-attracting funnel.