Nedd4-2 E3 ligase regulates Na+ homeostasis by ubiquitinating various channels and membrane transporters, including the epithelial sodium channel ENaC. In turn, Nedd4-2 dysregulation leads to various conditions, including electrolytic imbalance, respiratory distress, hypertension, and kidney diseases. However, Nedd4-2 regulation remains mostly unclear. The present study aims at elucidating Nedd4-2 regulation by structurally characterizing Nedd4-2 and its complexes using several biophysical techniques. Our cryo-EM reconstruction shows that the C2 domain blocks the E2-binding surface of the HECT domain. This blockage, ubiquitin-binding exosite masking by the WW1 domain, catalytic C922 blockage and HECT domain stabilization provide the structural basis for Nedd4-2 autoinhibition. Furthermore, Ca2+-dependent C2 membrane binding disrupts C2/HECT interactions, but not Ca2+ alone, whereas 14-3-3 protein binds to a flexible region of Nedd4-2 containing the WW2 and WW3 domains, thereby inhibiting its catalytic activity and membrane binding. Overall, our data provide key mechanistic insights into Nedd4-2 regulation toward fostering the development of strategies targeting Nedd4-2 function.

• MeSH
• Cryoelectron Microscopy MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Models, Molecular MeSH
• Protein Domains MeSH
• 14-3-3 Proteins * metabolism   chemistry   MeSH
• Ubiquitination MeSH
• Nedd4 Ubiquitin Protein Ligases * metabolism   chemistry   genetics   ultrastructure   MeSH
• Calcium * metabolism   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Nedd4 protein, human MeSH Browser
• Nedd4L protein, human MeSH Browser
• 14-3-3 Proteins * MeSH
• Nedd4 Ubiquitin Protein Ligases * MeSH
• Calcium * MeSH

The ubiquitous CLC membrane transporters are unique in their ability to exchange anions for cations. Despite extensive study, there is no mechanistic model that fully explains their 2:1 Cl‒/H+ stoichiometric exchange mechanism. Here, we provide such a model. Using differential hydrogen-deuterium exchange mass spectrometry, cryo-EM structure determination, and molecular dynamics simulations, we uncovered new conformational dynamics in CLC-ec1, a bacterial CLC homolog that has served as a paradigm for this family of transporters. Simulations based on a cryo-EM structure at pH 3 revealed critical steps in the transport mechanism, including release of Cl‒ ions to the extracellular side, opening of the inner gate, and novel water wires that facilitate H+ transport. Surprisingly, these water wires occurred independently of Cl‒ binding, prompting us to reassess the relationship between Cl‒ binding and Cl‒/H+ coupling. Using isothermal titration calorimetry and quantitative flux assays on mutants with reduced Cl‒ binding affinity, we conclude that, while Cl‒ binding is necessary for coupling, even weak binding can support Cl‒/H+ coupling. By integrating our findings with existing literature, we establish a complete and efficient CLC 2:1 Cl‒/H+ exchange mechanism.

• Publication type
• Journal Article MeSH
• Preprint MeSH

In proteomics, postproline cleaving enzymes (PPCEs), such as Aspergillus niger prolyl endopeptidase (AnPEP) and neprosin, complement proteolytic tools because proline is a stop site for many proteases. But while aiming at using AnPEP in online proteolysis, we found that this enzyme also displayed specificity to reduced cysteine. By LC-MS/MS, we systematically analyzed AnPEP sources and conditions that could affect this cleavage preference. Postcysteine cleavage was blocked by cysteine modifications, including disulfide bond formation, oxidation, and alkylation. The last modification explains why this activity has remained undetected so far. In the same experimental paradigm, neprosin mimicked this cleavage specificity. Based on these findings, PPCEs cleavage preferences should be redefined from post-Pro/Ala to post-Pro/Ala/Cys. Moreover, this evidence demands reconsidering PPCEs applications, whether cleaving Cys-rich proteins or assessing Cys status in proteins, and calls for revisiting the proposed enzymatic mechanism of these proteases.

• Publication type
• Journal Article MeSH

MICAL proteins play a crucial role in cellular dynamics by binding and disassembling actin filaments, impacting processes like axon guidance, cytokinesis, and cell morphology. Their cellular activity is tightly controlled, as dysregulation can lead to detrimental effects on cellular morphology. Although previous studies have suggested that MICALs are autoinhibited, and require Rab proteins to become active, the detailed molecular mechanisms remained unclear. Here, we report the cryo-EM structure of human MICAL1 at a nominal resolution of 3.1 Å. Structural analyses, alongside biochemical and functional studies, show that MICAL1 autoinhibition is mediated by an intramolecular interaction between its N-terminal catalytic and C-terminal coiled-coil domains, blocking F-actin interaction. Moreover, we demonstrate that allosteric changes in the coiled-coil domain and the binding of the tripartite assembly of CH-L2α1-LIM domains to the coiled-coil domain are crucial for MICAL activation and autoinhibition. These mechanisms appear to be evolutionarily conserved, suggesting a potential universality across the MICAL family.

• MeSH
• Actins metabolism   chemistry   MeSH
• Allosteric Regulation MeSH
• Calponins MeSH
• Cryoelectron Microscopy * MeSH
• Humans MeSH
• Actin Cytoskeleton metabolism   ultrastructure   MeSH
• Microfilament Proteins metabolism   chemistry   ultrastructure   MeSH
• Models, Molecular MeSH
• Mixed Function Oxygenases MeSH
• Protein Domains MeSH
• LIM Domain Proteins metabolism   chemistry   genetics   MeSH
• Protein Binding * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Actins MeSH
• Calponins MeSH
• MICAL1 protein, human MeSH Browser
• Microfilament Proteins MeSH
• Mixed Function Oxygenases MeSH
• LIM Domain Proteins MeSH

Covalent labeling in combination with mass spectrometry is a powerful approach used in structural biology to study protein structures, interactions, and dynamics. Recently, the toolbox of covalent labeling techniques has been expanded with fast fluoroalkylation of proteins (FFAP). FFAP is a novel radical labeling method that utilizes fluoroalkyl radicals generated from hypervalent Togni reagents for targeting aromatic residues. This report further demonstrates the benefits of FFAP as a new method for structural characterization of therapeutic antibodies and interaction interfaces of antigen-antibody complexes. The results obtained from human trastuzumab and its complex with human epidermal growth factor receptor 2 (HER2) correlate well with previously published structural data and demonstrate the potential of FFAP in structural biology.

• MeSH
• Alkylation MeSH
• Protein Footprinting methods   MeSH
• Halogenation MeSH
• Antigen-Antibody Complex chemistry   MeSH
• Humans MeSH
• Epitope Mapping * methods   MeSH
• Antibodies, Monoclonal chemistry   immunology   MeSH
• Receptor, ErbB-2 * chemistry   immunology   MeSH
• Trastuzumab * chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Cytosolic Ca2+ and Na+ allosterically regulate Na+/Ca2+ exchanger (NCX) proteins to vary the NCX-mediated Ca2+ entry/exit rates in diverse cell types. To resolve the structure-based dynamic mechanisms underlying the ion-dependent allosteric regulation in mammalian NCXs, we analyze the apo, Ca2+, and Na+-bound species of the brain NCX1.4 variant using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations. Ca2+ binding to the cytosolic regulatory domains (CBD1 and CBD2) rigidifies the intracellular regulatory loop (5L6) and promotes its interaction with the membrane domains. Either Na+ or Ca2+ stabilizes the intracellular portions of transmembrane helices TM3, TM4, TM9, TM10, and their connecting loops (3L4 and 9L10), thereby exposing previously unappreciated regulatory sites. Ca2+ or Na+ also rigidifies the palmitoylation domain (TMH2), and neighboring TM1/TM6 bundle, thereby uncovering a structural entity for modulating the ion transport rates. The present analysis provides new structure-dynamic clues underlying the regulatory diversity among tissue-specific NCX variants.

• MeSH
• Sodium-Calcium Exchanger * chemistry   MeSH
• Mammals * MeSH
• Protein Structure, Secondary MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Sodium-Calcium Exchanger * MeSH

Chloride Intracellular Channel (CLIC) family members uniquely transition between soluble and membrane-associated conformations. Despite decades of extensive functional and structural studies, CLICs' function as ion channels remains debated, rendering our understanding of their physiological role incomplete. Here, we expose the function of CLIC5 as a fusogen. We demonstrate that purified CLIC5 directly interacts with the membrane and induces fusion, as reflected by increased liposomal diameter and lipid and content mixing between liposomes. Moreover, we show that this activity is facilitated by acidic pH, a known trigger for CLICs' transition to a membrane-associated conformation, and that increased exposure of the hydrophobic inter-domain interface is crucial for this process. Finally, mutation of a conserved hydrophobic interfacial residue diminishes the fusogenic activity of CLIC5 in vitro and impairs excretory canal extension in C. elegans in vivo. Together, our results unravel the long-sought physiological role of these enigmatic proteins.

• MeSH
• Caenorhabditis elegans * genetics   metabolism   MeSH
• Chloride Channels metabolism   MeSH
• Chlorides * metabolism   MeSH
• Liposomes MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Chloride Channels MeSH
• Chlorides * MeSH
• Liposomes MeSH

The acylated Repeats in ToXins (RTX) leukotoxins, the adenylate cyclase toxin (CyaA) or α-hemolysin (HlyA), bind β2 integrins of leukocytes but also penetrate cells lacking these receptors. We show that the indoles of conserved tryptophans in the acylated segments, W876 of CyaA and W579 of HlyA, are crucial for β2 integrin-independent membrane penetration. Substitutions of W876 by aliphatic or aromatic residues did not affect acylation, folding, or the activities of CyaA W876L/F/Y variants on cells expressing high amounts of the β2 integrin CR3. However, toxin activity of CyaA W876L/F/Y on cells lacking CR3 was strongly impaired. Similarly, a W579L substitution selectively reduced HlyA W579L cytotoxicity towards cells lacking β2 integrins. Intriguingly, the W876L/F/Y substitutions increased the thermal stability (Tm) of CyaA by 4 to 8 °C but locally enhanced the accessibility to deuteration of the hydrophobic segment and of the interface of the two acylated loops. W876Q substitution (showing no increase in Tm), or combination of W876F with a cavity-filling V822M substitution (this combination decreasing the Tm closer to that of CyaA), yielded a milder defect of toxin activity on erythrocytes lacking CR3. Furthermore, the activity of CyaA on erythrocytes was also selectively impaired when the interaction of the pyrrolidine of P848 with the indole of W876 was ablated. Hence, the bulky indoles of residues W876 of CyaA, or W579 of HlyA, rule the local positioning of the acylated loops and enable a membrane-penetrating conformation in the absence of RTX toxin docking onto the cell membrane by β2 integrins.

• Keywords
• RTX toxin, acylated segment, adenylate cyclase toxin, cytotoxicity, hydrogen/deuterium exchange, thermal stability, tryptophan residue, α-hemolysin, β(2) integrins,
• MeSH
• Adenylate Cyclase Toxin * chemistry   genetics   metabolism   MeSH
• CD18 Antigens * genetics   metabolism   MeSH
• Bordetella pertussis MeSH
• Cell Membrane metabolism   MeSH
• Erythrocytes metabolism   MeSH
• Conserved Sequence MeSH
• Tryptophan * chemistry   genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenylate Cyclase Toxin * MeSH
• CD18 Antigens * MeSH
• Tryptophan * MeSH

The conserved Tweety homolog (TTYH) family consists of three paralogs in vertebrates, displaying a ubiquitous expression pattern. Although considered as ion channels for almost two decades, recent structural and functional analyses refuted this role. Intriguingly, while all paralogs shared a dimeric stoichiometry following detergent solubilization, their structures revealed divergence in their relative subunit orientation. Here, we determined the stoichiometry of intact mouse TTYH (mTTYH) complexes in cells. Using cross-linking and single-molecule fluorescence microscopy, we demonstrate that mTTYH1 and mTTYH3 form tetramers at the plasma membrane, stabilized by interactions between their extracellular domains. Using blue-native PAGE, fluorescence-detection size-exclusion chromatography, and hydrogen/deuterium exchange mass spectrometry (HDX-MS), we reveal that detergent solubilization results in tetramers destabilization, leading to their dissolution into dimers. Moreover, HDX-MS demonstrates that the extracellular domains are stabilized in the context of the tetrameric mTTYH complex. Together, our results expose the innate tetrameric organization of TTYH complexes at the cell membrane. Future structural analyses of these assemblies in native membranes are required to illuminate their long-sought cellular function.

• MeSH
• Cell Membrane MeSH
• Detergents * MeSH
• Mice MeSH
• Hydrogen Deuterium Exchange-Mass Spectrometry * MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Detergents * MeSH

Isoprenoids are synthesized by the prenyltransferase superfamily, which is subdivided according to the product stereoisomerism and length. In short- and medium-chain isoprenoids, product length correlates with active site volume. However, enzymes synthesizing long-chain products and rubber synthases fail to conform to this paradigm, because of an unexpectedly small active site. Here, we focused on the human cis-prenyltransferase complex (hcis-PT), residing at the endoplasmic reticulum membrane and playing a crucial role in protein glycosylation. Crystallographic investigation of hcis-PT along the reaction cycle revealed an outlet for the elongating product. Hydrogen-deuterium exchange mass spectrometry analysis showed that the hydrophobic active site core is flanked by dynamic regions consistent with separate inlet and outlet orifices. Last, using a fluorescence substrate analog, we show that product elongation and membrane association are closely correlated. Together, our results support direct membrane insertion of the elongating isoprenoid during catalysis, uncoupling active site volume from product length.

• Publication type
• Journal Article MeSH