The Molecular Mechanism of Human Voltage-Dependent Anion Channel 1 Blockade by the Metallofullerenol Gd@C82(OH)22: An In Silico Study

The endohedral metallofullerenol Gd@C82(OH)22 has been identified as a possible antineoplastic agent that can inhibit both the growth and metastasis of cancer cells. Despite these potentially important effects, our understanding of the interactions between Gd@C82(OH)22 and biomacromolecules remains incomplete. Here, we study the interaction between Gd@C82(OH)22 and the human voltage-dependent anion channel 1 (hVDAC1), the most abundant porin embedded in the mitochondrial outer membrane (MOM), and a potential druggable target for novel anticancer therapeutics. Using in silico approaches, we observe that Gd@C82(OH)22 molecules can permeate and form stable interactions with the pore of hVDAC1. Further, this penetration can occur from either side of the MOM to elicit blockage of the pore. The binding between Gd@C82(OH)22 and hVDAC1 is largely driven by long-range electrostatic interactions. Analysis of the binding free energies indicates that it is thermodynamically more favorable for Gd@C82(OH)22 to bind to the hVDAC1 pore when it enters the channel from inside the membrane rather than from the cytoplasmic side of the protein. Multiple factors contribute to the preferential penetration, including the surface electrostatic landscape of hVDAC1 and the unique physicochemical properties of Gd@C82(OH)22. Our findings provide insights into the potential molecular interactions of macromolecular biological systems with the Gd@C82(OH)22 nanodrug.

Boar spermadhesin AWN: Novel insights in its binding behavior and localization on sperm

As a major spermadhesin first found in the seminal plasma of boars, AWN is described to fulfil a variety of reproduction related tasks. Although being the best investigated boar spermadhesin, information about its interaction with membranes is inconsistent. In this regard, previous reports locate AWN either inside or on the surface of sperm cells and at different regions, depending on the method and antibody used. Here, we localize native AWN in/on epididymal, ejaculated, capacitated and acrosome-reacted boar sperm using epifluorescence and electron microscopy, as well as an analysis of potential lipid binding partners of native and recombinant AWN. By applying a custom-made anti-AWN antibody, localization of AWN in the equatorial segment of ejaculated, capacitated and acrosome-reacted boar sperm was discovered. Electron microscopy showed that AWN is localized both on the sperm surface and on the cytoplasmic side of the plasma membrane, and in close vicinity to the nuclear and both acrosomal membranes of sperm. Analysis of epididymal sperm indicated migration of AWN from the retral postacrosomal part to the equatorial segment during the epididymal passage. In contrast to hypotheses claiming a specific association of AWN to phosphatidylethanolamine and in line with our previous study describing an interaction with phosphatidic acid, the current results show a rather electrostatically-driven binding mechanism of AWN to negative lipids. In conclusion, this work provides new insights into the arrangement of AWN in the equatorial segment that suggest a possible role in sperm-oocyte fusion.

Unidirectional ion transport mechanism of a light-driven chloride pump revealed using X-ray free electron lasers

Light-driven chloride-pumping rhodopsins actively transport anions, including various halide ions, across cell membranes. Recent studies using time-resolved serial femtosecond crystallography (TR-SFX) have uncovered the structural changes and ion transfer mechanisms in light-driven cation-pumping rhodopsins. However, the mechanism by which the conformational changes pump an anion to achieve unidirectional ion transport, from the extracellular side to the cytoplasmic side, in anion-pumping rhodopsins remains enigmatic. We have collected TR-SFX data of Nonlabens marinus rhodopsin-3 (NM-R3), derived from a marine flavobacterium, at 10 µs and 1 ms time-points after photoexcitation. Our structural analysis reveals the conformational alterations during ion transfer and after ion release. Movements of the retinal chromophore initially displace a conserved tryptophan to the cytoplasmic side of NM-R3, accompanied with a slight shift of the halide ion bound to the retinal. After ion release, the inward movements of helix C and helix G and the lateral displacements of the retinal block access to the extracellular side of NM-R3. Anomalous signal data have also been obtained from NM-R3 crystals containing iodide ions. The anomalous density maps provide insight into the halide binding site for ion transfer in NM-R3.

Structures of ABCG2 under turnover conditions reveal a key step in the drug transport mechanism

ABCG2 is a multidrug transporter that affects drug pharmacokinetics and contributes to multidrug resistance of cancer cells. In previously reported structures, the reaction cycle was halted by the absence of substrates or ATP, mutation of catalytic residues, or the presence of small-molecule inhibitors or inhibitory antibodies. Here we present cryo-EM structures of ABCG2 under turnover conditions containing either the endogenous substrate estrone-3-sulfate or the exogenous substrate topotecan. We find two distinct conformational states in which both the transport substrates and ATP are bound. Whereas the state turnover-1 features more widely separated NBDs and an accessible substrate cavity between the TMDs, turnover-2 features semi-closed NBDs and an almost fully occluded substrate cavity. Substrate size appears to control which turnover state is mainly populated. The conformational changes between turnover-1 and turnover-2 states reveal how ATP binding is linked to the closing of the cytoplasmic side of the TMDs. The transition from turnover-1 to turnover-2 is the likely bottleneck or rate-limiting step of the reaction cycle, where the discrimination of substrates and inhibitors occurs.

The respiratory supercomplex from C. glutamicum

Corynebacterium glutamicum is a preferentially aerobic Gram-positive bacterium belonging to the Actinobacteria phylum, which also includes the pathogen Mycobacterium tuberculosis. In the respiratory chain of these bacteria, complexes III (CIII) and IV (CIV) form a CIII2CIV2 supercomplex that catalyzes oxidation of menaquinol and reduction of dioxygen to water. Electron transfer within the CIII2CIV2 supercomplex is linked to transmembrane proton translocation, which maintains an electrochemical proton gradient that drives ATP synthesis and transport processes. We isolated the C. glutamicum supercomplex and used cryo EM to determine its structure at 2.9 Å resolution. The structure shows a central CIII2 dimer flanked by a CIV on each side. One menaquinone is bound in each of the QN and QP sites in each CIII, near the cytoplasmic and periplasmic sides, respectively. In addition, we identified a menaquinone positioned ~14 Å from heme bL on the periplasmic side. A di-heme cyt. cc subunit provides an electronic connection between each CIII monomer and the adjacent CIV. In CIII2, the Rieske iron-sulfur (FeS) proteins are positioned with the iron near heme bL. Multiple subunits interact to form a convoluted sub-structure at the cytoplasmic side of the supercomplex, which defines a novel path that conducts protons into CIV.

Cryo-EM structures of the human sodium-potassium pump revealing the gating mechanism on the cytoplasmic side

Na+/K+-ATPase (NKA) is a membrane-bound ion pump that generates electrochemical gradient of sodium ion and potassium ion across the plasma membrane via hydrolyzing ATP. During each so-called Post-Albers cycle, NKA exchanges three cytoplasmic sodium ions for two extracellular potassium ions through alternating E1 and E2 states. Hitherto, there are several steps remained unknown during the complete working cycle of NKA. Here, we report cryo-electron microscopy (cryo-EM) structures of recombinant over-expressed human NKA in three distinct states at 3.1–3.4 Å resolution, representing the E1·3Na state, in which the cytosolic gate is open, and the E1·3Na·ATP state preceding ATP hydrolysis and a basic E2·[2K] state. These structures reveal the ATP-dependent Na+-binding site remodeling for the close of the cytoplasmic gate, filling a gap in the structural elucidation of the Post-Albers cycle of NKA and providing structural basis for understanding the cytoplasmic Na+ entrance pathway.

The structure of MgtE in the absence of magnesium provides new insights into channel gating

MgtE is a Mg2+ channel conserved in organisms ranging from prokaryotes to eukaryotes, including humans, and plays an important role in Mg2+ homeostasis. The previously determined MgtE structures in the Mg2+-bound, closed-state, and structure-based functional analyses of MgtE revealed that the binding of Mg2+ ions to the MgtE cytoplasmic domain induces channel inactivation to maintain Mg2+ homeostasis. There are no structures of the transmembrane (TM) domain for MgtE in Mg2+-free conditions, and the pore-opening mechanism has thus remained unclear. Here, we determined the cryo-electron microscopy (cryo-EM) structure of the MgtE-Fab complex in the absence of Mg2+ ions. The Mg2+-free MgtE TM domain structure and its comparison with the Mg2+-bound, closed-state structure, together with functional analyses, showed the Mg2+-dependent pore opening of MgtE on the cytoplasmic side and revealed the kink motions of the TM2 and TM5 helices at the glycine residues, which are important for channel activity. Overall, our work provides structure-based mechanistic insights into the channel gating of MgtE.

A nuclear export sequence promotes CRM1-dependent targeting of the nucleoporin Nup214 to the nuclear pore complex

ABSTRACT Nup214 is a major nucleoporin on the cytoplasmic side of the nuclear pore complex with roles in late steps of nuclear protein and mRNA export. It interacts with the nuclear export receptor CRM1 (also known as XPO1) via characteristic phenylalanine-glycine (FG) repeats in its C-terminal region. Here, we identify a classic nuclear export sequence (NES) in Nup214 that mediates Ran-dependent binding to CRM1. Nup214 versions with mutations in the NES, as well as wild-type Nup214 in the presence of the selective CRM1 inhibitor leptomycin B, accumulate in the nucleus of Nup214-overexpressing cells. Furthermore, physiological binding partners of Nup214, such as Nup62 and Nup88, are recruited to the nucleus together with Nup214. Nuclear export of mutant Nup214 can be rescued by artificial nuclear export sequences at the C-terminal end of Nup214, leading also to a correct localization of Nup88. Our results suggest a function of the Nup214 NES in the biogenesis of the nuclear pore complex and/or in terminal steps of CRM1-dependent protein export.

Pharmacophore Based Screening & Modification of Amiloride Analogs for Targeting the NhaP-type Cation-Proton Antiporter in Vibrio cholerae

The genome of Vibrio cholerae contains three structural genes for the NhaP-type cation-proton antiporter paralogues, Vc-NhaP1, 2 and 3 mediating exchange of K+ and or Na+ for protons across the membrane. Based on phenotype analysis of chromosomal Vc-NhaP1, 2 and 3 triple deletion mutants we suggested that Vc-NhaP paralogues might play a role in the Acid Tolerance Response (ATR) of V. cholerae as it passes through the gastric acid barrier of the stomach. Comparison of the biochemical properties of Vc-NhaP isoforms revealed that Vc-NhaP2 is the most active among all three paralogues. Therefore, Vc-NhaP2 antiporter is a plausible therapeutic target for developing novel inhibitors targeting these ion exchangers. Our structural and mutational analysis of Vc-NhaP2 identified a putative cation binding pocket formed by antiparallel extended regions of two transmembrane segments (TMSs V/XII) along with TMS VI. Molecular Dynamics (MD) simulations suggested that the flexibility of TMS-V/XII is crucial for the intra-molecular conformational events in Vc-NhaP2. In this study, we developed some putative Vc-NhaP2 inhibitors from Amiloride analogs (AAs). Amiloride is a potent inhibitor of human Na+/H+ exchanger-1 (NHE1). Based on the pharmacokinetic properties and potential binding affinity scores we chose six AAs showing high binding affinity scores to Vc-NhaP2. In silico, the six AAs interacted with the functionally important amino acid residues located in TMSs III, IV, V, VI, VIIII and IX either from the cytoplasmic side (three AAs) or the periplasmic side (three AAs) of Vc-NhaP2. Four AAs were modified to reduce their toxicity profile compared to the original AAs. Molecular docking of the modified AAs revealed promising binding. The four selected drugs interacted with functionally important amino acid residues located on the cytoplasmic side of TMS VI, the extended chain region of TMS V and TMS XII and the loop region between TMSs VIIII and IX. Molecular dynamics simulations revealed that binding of the selected drugs destabilized the Vc-NhaP2 and altered the flexibility of functionally important TMS VI.

Complexity of the eukaryotic dolichol-linked oligosaccharide scramblase suggested by activity correlation profiling mass spectrometry

The oligosaccharide required for asparagine (N)-linked glycosylation of proteins in the endoplasmic reticulum (ER) is donated by the glycolipid Glc3Man9GlcNAc2-PP-dolichol. Remarkably, whereas glycosylation occurs in the ER lumen, the initial steps of Glc3Man9GlcNAc2-PP-dolichol synthesis generate the lipid intermediate Man5GlcNAc2-PP-dolichol (M5-DLO) on the cytoplasmic side of the ER. Glycolipid assembly is completed only after M5-DLO is translocated to the luminal side. The membrane protein (M5-DLO scramblase) that mediates M5-DLO translocation across the ER membrane has not been identified, despite its importance for N-glycosylation. Building on our ability to recapitulate scramblase activity in proteoliposomes reconstituted with a crude mixture of ER membrane proteins, we developed a mass spectrometry-based 'activity correlation profiling' approach to identify scramblase candidates in the yeast Saccharomyces cerevisiae. Data curation prioritized six polytopic ER membrane proteins as scramblase candidates, but reconstitution-based assays and gene disruption in the protist Trypanosoma brucei revealed, unexpectedly, that none of these proteins is necessary for M5-DLO scramblase activity. Our results instead strongly suggest that M5-DLO scramblase activity is due to a protein, or protein complex, whose activity is regulated at the level of quaternary structure.