The Na ⁺ –Ca ²⁺ exchanger plays a central role in cardiac contractility by maintaining Ca ²⁺ homeostasis. Two Ca ²⁺ -binding domains, CBD1 and CBD2, located in a large intracellular loop, regulate activity of the exchanger. Ca ²⁺ binding to these regulatory domains activates the transport of Ca ²⁺ across the plasma membrane. Previously, we solved the structure of CBD1, revealing four Ca ²⁺ ions arranged in a tight planar cluster. Here, we present structures of CBD2 in the Ca ²⁺ -bound (1.7-Å resolution) and -free (1.4-Å resolution) conformations. Like CBD1, CBD2 has a classical Ig fold but coordinates only two Ca ²⁺ ions in primary and secondary Ca ²⁺ sites. In the absence of Ca ²⁺ , Lys ⁵⁸⁵ stabilizes the structure by coordinating two acidic residues (Asp ⁵⁵² and Glu ⁶⁴⁸ ), one from each of the Ca ²⁺ -binding sites, and prevents a substantial protein unfolding. We have mutated all of the acidic residues that coordinate the Ca ²⁺ ions and have examined the effects of these mutations on regulation of exchange activity. Three mutations (E516L, D578V, and E648L) at the primary Ca ²⁺ site completely remove Ca ²⁺ regulation, placing the exchanger into a constitutively active state. These are the first data defining the role of CBD2 as a regulatory domain in the Na ⁺ –Ca ²⁺ exchanger.

Most membrane proteins studies require the use of detergents, but because of the lack of a general, accurate and rapid method to quantify them, many uncertainties remain that hamper proper functional and structural data analyses. To solve this problem, we propose a method based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) that allows quantification of pure or mixed detergents in complex with membrane proteins. We validated the method with a wide variety of detergents and membrane proteins. We automated the process, thereby allowing routine quantification for a broad spectrum of usage. As a first illustration, we show how to obtain information of the amount of detergent in complex with a membrane protein, essential for liposome or nanodiscs reconstitutions. Thanks to the method, we also show how to reliably and easily estimate the detergent corona diameter and select the smallest size, critical for favoring protein-protein contacts and triggering/promoting membrane protein crystallization, and to visualize the detergent belt for Cryo-EM studies.

Lactose permease of Escherichia coli (LacY) with a single-Cys residue in place of A122 (helix IV) transports galactopyranosides and is specifically inactivated by methanethiosulfonyl-galactopyranosides (MTS-gal), which behave as unique suicide substrates. In order to study the mechanism of inactivation more precisely, we solved the structure of single-Cys122 LacY in complex with covalently bound MTS-gal. This structure exhibits an inward-facing conformation similar to that observed previously with a slight narrowing of the cytoplasmic cavity. MTS-gal is bound covalently, forming a disulfide bond with C122 and positioned between R144 and W151. E269, a residue essential for binding, coordinates the C-4 hydroxyl of the galactopyranoside moiety. The location of the sugar is in accord with many biochemical studies.

Multidrug ABC transporters translocate drugs across membranes by a mechanism for which the molecular features of drug release are so far unknown. Here, we resolved three ATP-Mg ²⁺ –bound outward-facing conformations of the Bacillus subtilis (homodimeric) BmrA by x-ray crystallography and single-particle cryo–electron microscopy (EM) in detergent solution, one of them with rhodamine 6G (R6G), a substrate exported by BmrA when overexpressed in B. subtilis . Two R6G molecules bind to the drug-binding cavity at the level of the outer leaflet, between transmembrane (TM) helices 1–2 of one monomer and TM5′–6′ of the other. They induce a rearrangement of TM1–2, highlighting a local flexibility that we confirmed by hydrogen/deuterium exchange and molecular dynamics simulations. In the absence of R6G, simulations show a fast postrelease occlusion of the cavity driven by hydrophobicity, while when present, R6G can move within the cavity, maintaining it open.