SUMMARY The serotonin transporter (SERT) regulates neurotransmitter homeostasis through the sodium-and chloride-dependent recycling of serotonin into presynaptic neurons1–3. Major depression and anxiety disorders are treated using selective serotonin reuptake inhibitors (SSRIs), small molecules that competitively block substrate binding, prolonging neurotransmitter action2,4. The dopamine and norepinephrine transporters, together with SERT, are members of the neurotransmitter sodium symporter (NSS) family. Cocaine and amphetamines inhibit or modulate the transport activities of NSSs2,3 and genetic variants are associated with multiple neuropsychiatric disorders including attention deficit hyperactivity disorder, autism, and bipolar disorder2,5. Studies of bacterial NSS homologs, including LeuT, have shown how transmembrane helices (TMs) undergo conformational changes during the transport cycle, exposing a central binding site to either side of the membrane1,6–12. However, the conformational changes associated with transport in eukaryotic NSSs remain obscure. To elucidate structure-based mechanisms for transport in SERT, we turned to complexes with ibogaine, a centuries old hallucinogenic natural product with psychoactive and anti-addictive properties13,14 (Fig. 1a). Interestingly, ibogaine displays non-competitive inhibition of transport, yet it exhibits competitive binding toward SSRIs15,16. Here we report cryo-EM structures of SERT-ibogaine complexes captured in outward-open, occluded, and inward-open conformations. Ibogaine binds to the central binding site and closure of the extracellular gate largely involves movements of TMs 1b and 6a. Opening of the intracellular gate involves a hinge-like movement of TM1a and partial unwinding of TM5, which together create a permeation pathway enabling substrate and ion diffusion to the cytoplasm. These structures define the structural rearrangements that occur from outward-open to the inward-open conformations, providing insight into the mechanism of neurotransmitter transport and ibogaine inhibition.

Mitochondria are the key source of ATP that fuels cellular functions, and they are also central in cellular signaling, cell division and apoptosis. Dysfunction of mitochondria has been implicated in a wide range of diseases, including neurodegenerative and cardiac diseases, and various types of cancer. One of the key proteins that regulate mitochondrial function is the voltage-dependent anion channel 1 (VDAC1), the most abundant protein on the outer membrane of mitochondria. VDAC1 is the gatekeeper for the passages of metabolites, nucleotides, and ions; it plays a crucial role in regulating apoptosis due to its interaction with apoptotic and anti-apoptotic proteins, namely members of the Bcl-2 family of proteins and hexokinase. Therefore, regulation of VDAC1 is crucial not only for metabolic functions of mitochondria, but also for cell survival. In fact, multiple lines of evidence have confirmed the involvement of VDAC1 in several diseases. Consequently, modulation or dysregulation of VDAC1 function can potentially attenuate or exacerbate pathophysiological conditions. Understanding the role of VDAC1 in health and disease could lead to selective protection of cells in different tissues and diverse diseases. The purpose of this review is to discuss the role of VDAC1 in the pathogenesis of diseases and as a potentially effective target for therapeutic management of various pathologies.

Members of the ATP‐binding cassette (ABC) transporter superfamily translocate a broad spectrum of chemically diverse substrates. While their eponymous ATP‐binding cassette in the nucleotide‐binding domains (NBDs) is highly conserved, their transmembrane domains (TMDs) forming the translocation pathway exhibit distinct folds and topologies, suggesting that during evolution the ancient motor domains were combined with different transmembrane mechanical systems to orchestrate a variety of cellular processes. In recent years, it has become increasingly evident that the distinct TMD folds are best suited to categorize the multitude of ABC transporters. We therefore propose a new ABC transporter classification that is based on structural homology in the TMDs.

Multidrug ATP binding cassette (ABC) exporters are ubiquitous ABC transporters that extrude cytotoxic molecules across cell membranes. Despite recent progress in structure determination of these transporters, the conformational motion that transduces the energy of ATP hydrolysis to the work of substrate translocation remains undefined. Here, we have investigated the conformational cycle of BmrCD, a representative of the heterodimer family of ABC exporters that have an intrinsically impaired nucleotide binding site. We measured distances between pairs of spin labels monitoring the movement of the nucleotide binding (NBD) and transmembrane domains (TMD). The results expose previously unobserved structural intermediates of the NBDs arising from asymmetric configuration of catalytically inequivalent nucleotide binding sites. The two-state transition of the TMD, from an inward-to an outward-facing conformation, is driven exclusively by ATP hydrolysis. These findings provide direct evidence of divergence in the mechanism of ABC exporters.

Background: P-glycoprotein relies on largely unknown structural changes for its transport function.Results: EPR spectroscopy and simulations capture large-amplitude structural fluctuations for inward-facing P-glycoprotein.Conclusion: The characterized distinct dynamics of P-glycoprotein suggests mechanistic diversity of ATP-coupled transport in ABC transporters.Significance: Characterizing structural dynamics is a key step toward understanding the mechanism of this multidrug resistance transporter.

Membrane transport constitutes one of the most fundamental processes in all living cells with proteins as major players. Proteins as channels provide highly selective diffusive pathways gated by environmental factors, and as transporters furnish directed, energetically uphill transport consuming energy. X-ray crystallography of channels and transporters furnishes a rapidly growing number of atomic resolution structures, permitting molecular dynamics (MD) simulations to reveal the physical mechanisms underlying channel and transporter function. Ever increasing computational power today permits simulations stretching up to 1 μsec, i.e., to physiologically relevant time scales. Membrane protein simulations presently focus on ion channels, on aquaporins, on protein-conducting channels, as well as on various transporters. In this review we summarize recent developments in this rapidly evolving field.

Proper subcellular localization of focal adhesion kinase (FAK) is crucial for many cellular processes. It remains, however, unclear how FAK activity is regulated at subcellular compartments. To visualize the FAK activity at different membrane microdomains, we develop a fluorescence resonance energy transfer (FRET)-based FAK biosensor, and target it into or outside of detergent-resistant membrane (DRM) regions at the plasma membrane. Here we show that, on cell adhesion to extracellular matrix proteins or stimulation by platelet-derived growth factor (PDGF), the FRET responses of DRM-targeting FAK biosensor are stronger than that at non-DRM regions, suggesting that FAK activation can occur at DRM microdomains. Further experiments reveal that the PDGF-induced FAK activation is mediated and maintained by Src activity, whereas FAK activation on cell adhesion is independent of, and in fact essential for the Src activation. Therefore, FAK is activated at membrane microdomains with distinct activation mechanisms in response to different physiological stimuli.

ABC transporters constitute one of the most abundant membrane transporter families. The most common feature shared in the family is the highly conserved nucleotide binding domains (NBDs) that drive the transport process through binding and hydrolysis of ATP. Molecular dynamics simulations are used to investigate the effect of ATP hydrolysis in the NBDs. Starting with the ATP-bound, closed dimer of MalK, four simulation systems with all possible combinations of ATP or ADP-P(i) bound to the two nucleotide binding sites are constructed and simulated with equilibrium molecular dynamics for approximately 70 ns each. The results suggest that the closed form of the NBD dimer can only be maintained with two bound ATP molecules; in other words, hydrolysis of one ATP can lead to the opening of the dimer interface of the NBD dimer. Furthermore, we observed that the opening is an immediate effect of hydrolysis of ATP into ADP and P(i) rather than the dissociation of hydrolysis products. In addition, the opening is mechanistically triggered by the dissociation of the LSGGQ motif from the bound nucleotide. A metastable ADP-P(i) bound conformational state is consistently observed before the dimer opening in all the simulation systems.