Membrane co-transport proteins that use a five-helix inverted repeat motif have recently emerged as one of the largest structural classes of secondary active transporters. However, despite many ...structural advances there is no clear evidence of how ion and substrate transport are coupled. Here we report a comprehensive study of the sodium/galactose transporter from Vibrio parahaemolyticus (vSGLT), consisting of molecular dynamics simulations, biochemical characterization and a new crystal structure of the inward-open conformation at a resolution of 2.7 Å. Our data show that sodium exit causes a reorientation of transmembrane helix 1 that opens an inner gate required for substrate exit, and also triggers minor rigid-body movements in two sets of transmembrane helical bundles. This cascade of events, initiated by sodium release, ensures proper timing of ion and substrate release. Once set in motion, these molecular changes weaken substrate binding to the transporter and allow galactose readily to enter the intracellular space. Additionally, we identify an allosteric pathway between the sodium-binding sites, the unwound portion of transmembrane helix 1 and the substrate-binding site that is essential in the coupling of co-transport.
NADPH oxidases (NOX) are transmembrane proteins, widely spread in eukaryotes and prokaryotes, that produce reactive oxygen species (ROS). Eukaryotes use the ROS products for innate immune defense and ...signaling in critical (patho)physiological processes. Despite the recent structures of human NOX isoforms, the activation of electron transfer remains incompletely understood. SpNOX, a homolog from
, can serves as a robust model for exploring electron transfers in the NOX family thanks to its constitutive activity. Crystal structures of SpNOX full-length and dehydrogenase (DH) domain constructs are revealed here. The isolated DH domain acts as a flavin reductase, and both constructs use either NADPH or NADH as substrate. Our findings suggest that hydride transfer from NAD(P)H to FAD is the rate-limiting step in electron transfer. We identify significance of F397 in nicotinamide access to flavin isoalloxazine and confirm flavin binding contributions from both DH and Transmembrane (TM) domains. Comparison with related enzymes suggests that distal access to heme may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity, contrary to previous suggestions. It rather suggests requirement of an internal rearrangement, within the DH domain, to switch from a resting to an active state. Thus, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2's requirement for activation.
The transporter Multidrug Resistance Protein 1 (MRP1, ABCC1) is implicated in multidrug resistant (MDR) phenotype of cancer cells. Glutathione (GSH) plays a key role in MRP1 transport activities. In ...addition, a ligand-stimulated GSH transport which triggers the death of cells overexpressing MRP1, by collateral sensitivity (CS), has been described. This CS could be a way to overcome the poor prognosis for patients suffering from a chemoresistant cancer. The molecular mechanism of such massive GSH transport and its connection to the other transport activities of MRP1 are unknown. In this context, we generated MRP1/MRP2 chimeras covering different regions, MRP2 being a close homolog that does not trigger CS. The one encompassing helices 16 and 17 led to the loss of CS and MDR phenotype without altering basal GSH transport. Within this region, the sole restoration of the original G1228 (D1236 in MRP2) close to the extracellular loop between the two helices fully rescued the CS (massive GSH efflux and cell death) but not the MDR phenotype. The flexibility of that loop and the binding of a CS agent like verapamil could favor a particular conformation for the massive transport of GSH, not related to other transport activities of MRP1.
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.
Human P‐glycoprotein (P‐gp) controls drugs bioavailability by pumping structurally unrelated drugs out of cells. The X‐ray structure of the mouse P‐gp ortholog has been solved, with two SSS ...enantiomers or one RRR enantiomer of the selenohexapeptide inhibitor QZ59, found within the putative drug‐binding pocket (Aller SG, Yu J, Ward A, Weng Y, Chittaboina S, Zhuo R, Harrell PM, Trinh YT, Zhang Q, Urbatsch IL et al. (2009). Science 323, 1718–1722). This offered the first opportunity to localize the well‐known H and R drug‐binding sites with respect to the QZ59 inhibition mechanisms of Hoechst 33342 and daunorubicin transports, characterized here in cellulo. We found that QZ59‐SSS competes efficiently with both substrates, with KI,app values of 0.15 and 0.3 μm, which are 13 and 2 times lower, respectively, than the corresponding Km,app values. In contrast, QZ59‐RRR non‐competitively inhibited daunorubicin transport with moderate efficacy (KI,app = 1.9 μm); it also displayed a mixed‐type inhibition of the Hoechst 33342 transport, resulting from a main non‐competitive tendency (Ki2,app = 1.6 μm) and a limited competitive tendency (Ki1,app = 5 μm). These results suggest a positional overlap of QZ59 and drugs binding sites: full for the SSS enantiomer and partial for the RRR enantiomer. Crystal structure analysis suggests that the H site overlaps both QZ59‐SSS locations while the R site overlaps the most embedded location.
The P‐glycoprotein expels out of the cells hundreds of structurally unrelated compounds. We bring here a molecular basis of this polyspecificity by localizing the H and R drug‐binding sites. We achieved this by establishing in cellulo the mechanism of Hoechst 33342 and daunorubicin efflux inhibitions by the selenohexapeptide inhibitor QZ59 enantiomers, previously co‐crystallized with the mouse P‐gp.
The Na⁺-Ca²⁺ exchanger plays a central role in cardiac contractility by maintaining Ca²⁺ homeostasis. Two Ca²⁺-binding domains, CBD1 and CBD2, located in a large intracellular loop, regulate activity ...of the exchanger. Ca²⁺ binding to these regulatory domains activates the transport of Ca²⁺ across the plasma membrane. Previously, we solved the structure of CBD1, revealing four Ca²⁺ ions arranged in a tight planar cluster. Here, we present structures of CBD2 in the Ca²⁺-bound (1.7-Å resolution) and -free (1.4-Å resolution) conformations. Like CBD1, CBD2 has a classical Ig fold but coordinates only two Ca²⁺ ions in primary and secondary Ca²⁺ sites. In the absence of Ca²⁺, Lys⁵⁸⁵ stabilizes the structure by coordinating two acidic residues (Asp⁵⁵² and Glu⁶⁴⁸), one from each of the Ca²⁺-binding sites, and prevents a substantial protein unfolding. We have mutated all of the acidic residues that coordinate the Ca²⁺ ions and have examined the effects of these mutations on regulation of exchange activity. Three mutations (E516L, D578V, and E648L) at the primary Ca²⁺ site completely remove Ca²⁺ 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⁺-Ca²⁺ exchanger.
Despite the growing interest in membrane proteins, their crystallization remains a major challenge. In the course of a crystallographic study on the multidrug ATP-binding cassette transporter BmrA, ...mass spectral analyses on samples purified with six selected detergents revealed unexpected protein contamination visible for the most part on overloaded SDS-PAGE. A major contamination from the outer membrane protein OmpF was detected in purifications with Foscholine 12 (FC12) but not with Lauryldimethylamine-N-oxide (LDAO) or any of the maltose-based detergents. Consequently, in the FC12 purified BmrA, OmpF easily crystallized over BmrA in a new space group, and whose structure is reported here. We therefore devised an optimized protocol to eliminate OmpF during the FC12 purification of BmrA. On the other hand, an additional band visible at ∼110 kDa was detected in all samples purified with the maltose-based detergents. It contained AcrB that crystallized over BmrA despite its trace amounts. Highly pure BmrA preparations could be obtained using either a ΔacrAB E. coli strain and n-dodecyl-β-D-maltopyranoside, or a classical E. coli strain and lauryl maltose neopentyl glycol for the overexpression and purification, respectively. Overall our results urge to incorporate a proteomics-based purity analysis into quality control checks prior to commencing crystallization assays of membrane proteins that are notoriously arduous to crystallize. Moreover, the strategies developed here to selectively eliminate obstinate contaminants should be applicable to the purification of other membrane proteins overexpressed in E. coli.
Detergents wrap around membrane proteins to form a belt covering the hydrophobic part of the protein serving for membrane insertion and interaction with lipids. The number of detergent monomers ...forming this belt is usually unknown to investigators, unless dedicated detergent quantification is undertaken, which for many projects is difficult to setup. Yet, having an approximate knowledge of the amount of detergent forming the belt is extremely useful, to better grasp the protein of interest in interaction with its direct environment rather than picturing the membrane protein "naked". We created the Det.Belt server to dress up membrane proteins and represent in 3D the bulk made by detergent molecules wrapping in a belt. Many detergents are included in a database, allowing investigators to screen in silico the effect of different detergents around their membrane protein. The input number of detergents is changeable with fast recomputation of the belt for interactive usage. Metrics representing the belt are readily available together with scripts to render quality 3D images for publication. The Det.Belt server is a tool for biochemists to better grasp their sample.
The mammalian Na+/Ca2+ exchanger, NCX1.1, serves as the main mechanism for Ca2+ efflux across the sarcolemma following cardiac contraction. In addition to transporting Ca2+, NCX1.1 activity is also ...strongly regulated by Ca2+ binding to two intracellular regulatory domains, CBD1 and CBD2. The structures of both of these domains have been solved by NMR spectroscopy and x-ray crystallography, greatly enhancing our understanding of Ca2+ regulation. Nevertheless, the mechanisms by which Ca2+ regulates the exchanger remain incompletely understood. The initial NMR study showed that the first regulatory domain, CBD1, unfolds in the absence of regulatory Ca2+. It was further demonstrated that a mutation of an acidic residue involved in Ca2+ binding, E454K, prevents this structural unfolding. A contradictory result was recently obtained in a second NMR study in which Ca2+ removal merely triggered local rearrangements of CBD1. To address this issue, we solved the crystal structure of the E454K-CBD1 mutant and performed electrophysiological analyses of the full-length exchanger with mutations at position 454. We show that the lysine substitution replaces the Ca2+ ion at position 1 of the CBD1 Ca2+ binding site and participates in a charge compensation mechanism. Electrophysiological analyses show that mutations of residue Glu-454 have no impact on Ca2+ regulation of NCX1.1. Together, structural and mutational analyses indicate that only two of the four Ca2+ ions that bind to CBD1 are important for regulating exchanger activity.
Diffraction anisotropy is a phenomenon that impacts more specifically membrane proteins, compared to soluble ones, but the reasons for this discrepancy remained unclear. Often, it is referred to a ...difference in resolution limits between highest and lowest diffraction limits as a signature for anisotropy. We show in this article that there is no single correlation between anisotropy and difference in resolution limits, with notably a substantial number of structures displaying various anisotropy with no difference in resolution limits. We further investigated diffraction intensity profiles, and observed a peak centred on 4.9 Å resolution more predominant in membrane proteins. Since this peak is in the region corresponding to secondary structures, we investigated the influence of secondary structure ratio. We showed that secondary structure content has little influence on this profile, while secondary structure collinearity in membrane proteins correlate with a stronger peak. Finally, we could further show that the presence of this peak is linked to higher diffraction anisotropy. These results bring to light a specific diffraction of membrane protein crystals, which calls for a specific handling by crystallographic software. It also brings an explanation for investigators struggling with their anisotropic data.
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•Membrane proteins diffract X-rays more anisotropically than soluble proteins.•Membrane proteins display a peak around 4.9 Å resolution in intensity profiles.•Secondary structures in membrane proteins are more collinear than soluble proteins.
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