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Creatine is a crucial metabolite that plays a fundamental role in ATP homeostasis in tissues with high-energy demands. The creatine transporter (CreaT, SLC6A8) belongs to the solute carrier 6 (SLC6) transporters family, and more particularly to the GABA transporters (GATs) subfamily. Understanding the molecular determinants of specificity within the SLC6 transporters in general, and the GATs in particular is very challenging due to the high similarity of these proteins. In the study presented here, our efforts focused on finding key structural features involved in binding selectivity for CreaT using structure-based computational methods. Due to the lack of three-dimensional structures of SLC6A8, our approach was based on the realization of two reliable homology models of CreaT using the structures of two templates, i.e. the human serotonin transporter (hSERT) and the prokaryotic leucine transporter (LeuT). Our models reveal that an optimal complementarity between the shape of the binding site and the size of the ligands is necessary for transport. These findings provide a framework for a deeper understanding of substrate selectivity of the SLC6 family and other LeuT fold transporters.

The translocase of the outer mitochondrial membrane (TOM) is the main entry gate for proteins 1 – 4 . Here we use cryo-electron microscopy to report the structure of the yeast TOM core complex 5 – 9 at 3.8-Å resolution. The structure reveals the high-resolution architecture of the translocator consisting of two Tom40 β-barrel channels and α-helical transmembrane subunits, providing insight into critical features that are conserved in all eukaryotes 1 – 3 . Each Tom40 β-barrel is surrounded by small TOM subunits, and tethered by two Tom22 subunits and one phospholipid. The N-terminal extension of Tom40 forms a helix inside the channel; mutational analysis reveals its dual role in early and late steps in the biogenesis of intermembrane-space proteins in cooperation with Tom5. Each Tom40 channel possesses two precursor exit sites. Tom22, Tom40 and Tom7 guide presequence-containing preproteins to the exit in the middle of the dimer, whereas Tom5 and the Tom40 N extension guide preproteins lacking a presequence to the exit at the periphery of the dimer. The high-resolution cryo-electron microscopy structure of the yeast translocase of the outer mitochondrial membrane reveals key features of mitochondrial protein import that are conserved in all eukaryotes.

Many proteins must translocate through the protein-conducting Sec61 channel in the eukaryotic endoplasmic reticulum membrane or the SecY channel in the prokaryotic plasma membrane 1 , 2 . Proteins with highly hydrophobic signal sequences are first recognized by the signal recognition particle (SRP) 3 , 4 and then moved co-translationally through the Sec61 or SecY channel by the associated translating ribosome. Substrates with less hydrophobic signal sequences bypass the SRP and are moved through the channel post-translationally 5 , 6 . In eukaryotic cells, post-translational translocation is mediated by the association of the Sec61 channel with another membrane protein complex, the Sec62–Sec63 complex 7 – 9 , and substrates are moved through the channel by the luminal BiP ATPase 9 . How the Sec62–Sec63 complex activates the Sec61 channel for post-translational translocation is not known. Here we report the electron cryo-microscopy structure of the Sec complex from Saccharomyces cerevisiae , consisting of the Sec61 channel and the Sec62, Sec63, Sec71 and Sec72 proteins. Sec63 causes wide opening of the lateral gate of the Sec61 channel, priming it for the passage of low-hydrophobicity signal sequences into the lipid phase, without displacing the channel’s plug domain. Lateral channel opening is triggered by Sec63 interacting both with cytosolic loops in the C-terminal half of Sec61 and transmembrane segments in the N-terminal half of the Sec61 channel. The cytosolic Brl domain of Sec63 blocks ribosome binding to the channel and recruits Sec71 and Sec72, positioning them for the capture of polypeptides associated with cytosolic Hsp70 10 . Our structure shows how the Sec61 channel is activated for post-translational protein translocation. The cryo-EM structure of the post-translational protein translocation machinery of the endoplasmic reticulum membrane shows that Sec63 opens the channel, enabling insertion of low-hydrophobicity signal sequences into the lipid phase.

Nearly all mitochondrial proteins are encoded by the nuclear genome and imported into mitochondria after synthesis on cytosolic ribosomes. These precursor proteins are translocated into mitochondria by the TOM complex, a protein-conducting channel in the mitochondrial outer membrane. We have determined high-resolution cryo-EM structures of the core TOM complex from Saccharomyces cerevisiae in dimeric and tetrameric forms. Dimeric TOM consists of two copies each of five proteins arranged in two-fold symmetry: pore-forming β-barrel protein Tom40 and four auxiliary α-helical transmembrane proteins. The pore of each Tom40 has an overall negatively charged inner surface attributed to multiple functionally important acidic patches. The tetrameric complex is essentially a dimer of dimeric TOM, which may be capable of forming higher-order oligomers. Our study reveals the detailed molecular organization of the TOM complex and provides new insights about the mechanism of protein translocation into mitochondria.

Electron cryo-microscopy structures of the human peptide-loading complex shed light on its operation and on the onset of adaptive immune responses. Structure of a peptide loader The peptide-loading complex (PLC) is a dynamic membrane complex in the endoplasmic reticulum that regulates the transport and loading of antigenic peptides onto major histocompatibility complex class I (MHC-I) molecules. As such, this complex has a key role in important adaptive immune responses to infections and tumour progression. Here, Robert Tampé and colleagues report the structure of the human PLC by electron cryo-microscopy. The editing modules of the complex are centred around the TAP transporter, which delivers the peptides from the cytosol, and peptide loading appears to induce changes in the structure of MHC-I, releasing the stable peptide/MHC-I complexes from the PLC. This provides glimpses into the mechanism of the PLC, antigen processing and the onset of MHC-I-mediated immunity. The peptide-loading complex (PLC) is a transient, multisubunit membrane complex in the endoplasmic reticulum that is essential for establishing a hierarchical immune response. The PLC coordinates peptide translocation into the endoplasmic reticulum with loading and editing of major histocompatibility complex class I (MHC-I) molecules. After final proofreading in the PLC, stable peptide–MHC-I complexes are released to the cell surface to evoke a T-cell response against infected or malignant cells 1 , 2 . Sampling of different MHC-I allomorphs requires the precise coordination of seven different subunits in a single macromolecular assembly, including the transporter associated with antigen processing (TAP1 and TAP2, jointly referred to as TAP), the oxidoreductase ERp57, the MHC-I heterodimer, and the chaperones tapasin and calreticulin 3 , 4 . The molecular organization of and mechanistic events that take place in the PLC are unknown owing to the heterogeneous composition and intrinsically dynamic nature of the complex. Here, we isolate human PLC from Burkitt’s lymphoma cells using an engineered viral inhibitor as bait and determine the structure of native PLC by electron cryo-microscopy. Two endoplasmic reticulum-resident editing modules composed of tapasin, calreticulin, ERp57, and MHC-I are centred around TAP in a pseudo-symmetric orientation. A multivalent chaperone network within and across the editing modules establishes the proofreading function at two lateral binding platforms for MHC-I molecules. The lectin-like domain of calreticulin senses the MHC-I glycan, whereas the P domain reaches over the MHC-I peptide-binding pocket towards ERp57. This arrangement allows tapasin to facilitate peptide editing by clamping MHC-I. The translocation pathway of TAP opens out into a large endoplasmic reticulum lumenal cavity, confined by the membrane entry points of tapasin and MHC-I. Two lateral windows channel the antigenic peptides to MHC-I. Structures of PLC captured at distinct assembly states provide mechanistic insight into the recruitment and release of MHC-I. Our work defines the molecular symbiosis of an ABC transporter and an endoplasmic reticulum chaperone network in MHC-I assembly and provides insight into the onset of the adaptive immune response.

The Ton and Tol motor proteins use the proton gradient at the inner membrane of Gram-negative bacteria as an energy source. The generated force is transmitted through the periplasmic space to protein components associated with the outer membrane, either to maintain the outer membrane integrity for the Tol system, or to allow essential nutrients to enter the cell for Ton. We have solved the high-resolution structures of the E. coli TonB-ExbB-ExbD and TolA-TolQ-TolR complexes, revealing the inner membrane embedded engine parts of the Ton and Tol systems, and showing how TonB and TolA interact with the ExbBD and TolQR subcomplexes. Structural similarities between the two motor complexes suggest a common mechanism for the opening of the proton channel and the propagation of the proton motive force into movement of the TonB and TolA subunits. Because TonB and TolA bind at preferential ExbB or TolQ subunits, we propose a new mechanism of assembly of TonB and TolA with their respective ExbBD and TolQR subcomplexes and discuss its impact on the mechanism of action for the Ton and Tol systems. The Ton and Tol systems are bacterial energy-transducing complexes that use the proton motive force at the inner membrane to exert force on outer membrane proteins. Here the authors present the high-resolution cryoEM structures of the inner membrane engine part of these two complexes.

The emergence and spread of drug-resistant Plasmodium falciparum impedes global efforts to control and eliminate malaria. For decades, treatment of malaria has relied on chloroquine (CQ), a safe and affordable 4-aminoquinoline that was highly effective against intra-erythrocytic asexual blood-stage parasites, until resistance arose in Southeast Asia and South America and spread worldwide 1 . Clinical resistance to the chemically related current first-line combination drug piperaquine (PPQ) has now emerged regionally, reducing its efficacy 2 . Resistance to CQ and PPQ has been associated with distinct sets of point mutations in the P. falciparum CQ-resistance transporter PfCRT, a 49-kDa member of the drug/metabolite transporter superfamily that traverses the membrane of the acidic digestive vacuole of the parasite 3 – 9 . Here we present the structure, at 3.2 Å resolution, of the PfCRT isoform of CQ-resistant, PPQ-sensitive South American 7G8 parasites, using single-particle cryo-electron microscopy and antigen-binding fragment technology. Mutations that contribute to CQ and PPQ resistance localize primarily to moderately conserved sites on distinct helices that line a central negatively charged cavity, indicating that this cavity is the principal site of interaction with the positively charged CQ and PPQ. Binding and transport studies reveal that the 7G8 isoform binds both drugs with comparable affinities, and that these drugs are mutually competitive. The 7G8 isoform transports CQ in a membrane potential- and pH-dependent manner, consistent with an active efflux mechanism that drives CQ resistance 5 , but does not transport PPQ. Functional studies on the newly emerging PfCRT F145I and C350R mutations, associated with decreased PPQ susceptibility in Asia and South America, respectively 6 , 9 , reveal their ability to mediate PPQ transport in 7G8 variant proteins and to confer resistance in gene-edited parasites. Structural, functional and in silico analyses suggest that distinct mechanistic features mediate the resistance to CQ and PPQ in PfCRT variants. These data provide atomic-level insights into the molecular mechanism of this key mediator of antimalarial treatment failures. Structural, functional and in silico analyses of the chloroquine-resistance transporter PfCRT of Plasmodium falciparum suggest that distinct mechanistic features mediate the resistance to chloroquine and piperaquine in drug-resistant parasites.

Tripartite multidrug efflux systems of Gram-negative bacteria are composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. They are assumed to form ducts inside the periplasm facilitating drug exit across the outer membrane. Here we present the reconstitution of native Pseudomonas aeruginosa MexAB–OprM and Escherichia coli AcrAB–TolC tripartite Resistance Nodulation and cell Division (RND) efflux systems in a lipid nanodisc system. Single-particle analysis by electron microscopy reveals the inner and outer membrane protein components linked together via the periplasmic adaptor protein. This intrinsic ability of the native components to self-assemble also leads to the formation of a stable interspecies AcrA–MexB–TolC complex suggesting a common mechanism of tripartite assembly. Projection structures of all three complexes emphasize the role of the periplasmic adaptor protein as part of the exit duct with no physical interaction between the inner and outer membrane components. Tripartite efflux systems consist of inner membrane, outer membrane and periplasmic components. Here, Daury et al . reconstitute native versions of RND transporters in nanodiscs and present projection structures emphasizing the role of the periplasmic adaptor in linking the inner and outer membrane proteins.

The serotonin transporter (SERT) regulates neurotransmitter homeostasis through the sodium- and chloride-dependent recycling of serotonin into presynaptic neurons 1 – 3 . Major depression and anxiety disorders are treated using selective serotonin reuptake inhibitors—small molecules that competitively block substrate binding and thereby prolong neurotransmitter action 2 , 4 . The dopamine and noradrenaline transporters, together with SERT, are members of the neurotransmitter sodium symporter (NSS) family. The transport activities of NSSs can be inhibited or modulated by cocaine and amphetamines 2 , 3 , and genetic variants of NSSs are associated with several neuropsychiatric disorders including attention deficit hyperactivity disorder, autism and bipolar disorder 2 , 5 . Studies of bacterial NSS homologues—including LeuT—have shown how their transmembrane helices (TMs) undergo conformational changes during the transport cycle, exposing a central binding site to either side of the membrane 1 , 6 – 12 . However, the conformational changes associated with transport in NSSs remain unknown. To elucidate structure-based mechanisms for transport in SERT we investigated its complexes with ibogaine, a hallucinogenic natural product with psychoactive and anti-addictive properties 13 , 14 . Notably, ibogaine is a non-competitive inhibitor of transport but displays competitive binding towards selective serotonin reuptake inhibitors 15 , 16 . Here we report cryo-electron microscopy 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 the partial unwinding of TM5, which together create a permeation pathway that enables substrate and ion diffusion to the cytoplasm. These structures define the structural rearrangements that occur from the outward-open to inward-open conformations, and provide insight into the mechanism of neurotransmitter transport and ibogaine inhibition. Cryo-electron microscopy reveals three conformations of the serotonin transporter in complex with ibogaine, detailing the structural rearrangements that occur between the different stages of its transport cycle.

To replicate inside macrophages and cause tuberculosis, Mycobacterium tuberculosis must scavenge a variety of nutrients from the host 1 , 2 . The mammalian cell entry (MCE) proteins are important virulence factors in M. tuberculosis 1 , 3 , where they are encoded by large gene clusters and have been implicated in the transport of fatty acids 4 – 7 and cholesterol 1 , 4 , 8 across the impermeable mycobacterial cell envelope. Very little is known about how cargos are transported across this barrier, and it remains unclear how the approximately ten proteins encoded by a mycobacterial mce gene cluster assemble to transport cargo across the cell envelope. Here we report the cryo-electron microscopy (cryo-EM) structure of the endogenous Mce1 lipid-import machine of Mycobacterium smegmatis —a non-pathogenic relative of M. tuberculosis . The structure reveals how the proteins of the Mce1 system assemble to form an elongated ABC transporter complex that is long enough to span the cell envelope. The Mce1 complex is dominated by a curved, needle-like domain that appears to be unrelated to previously described protein structures, and creates a protected hydrophobic pathway for lipid transport across the periplasm. Our structural data revealed the presence of a subunit of the Mce1 complex, which we identified using a combination of cryo-EM and AlphaFold2, and name LucB. Our data lead to a structural model for Mce1-mediated lipid import across the mycobacterial cell envelope. Proteins of the Mycobacterium smegmatis Mce1 system assemble to form an elongated ABC transporter complex that is long enough to span the impermeable mycobacterial cell envelope.