Structural basis of antifolate recognition and transport by PCFT.

Folates (also known as vitamin B9) have a critical role in cellular metabolism as the starting point in the synthesis of nucleic acids, amino acids and the universal methylating agent S-adenylsmethionine1,2. Folate deficiency is associated with a number of developmental, immune and neurological disorders3-5. Mammals cannot synthesize folates de novo; several systems have therefore evolved to take up folates from the diet and distribute them within the body3,6. The proton-coupled folate transporter (PCFT) (also known as SLC46A1) mediates folate uptake across the intestinal brush border membrane and the choroid plexus4,7, and is an important route for the delivery of antifolate drugs in cancer chemotherapy8-10. How PCFT recognizes folates or antifolate agents is currently unclear. Here we present cryo-electron microscopy structures of PCFT in a substrate-free state and in complex with a new-generation antifolate drug (pemetrexed). Our results provide a structural basis for understanding antifolate recognition and provide insights into the pH-regulated mechanism of folate transport mediated by PCFT.

Capturing heterogeneous conformers of cobalamin riboswitch by cryo-EM.

RNA conformational heterogeneity often hampers its high-resolution structure determination, especially for large and flexible RNAs devoid of stabilizing proteins or ligands. The adenosylcobalamin riboswitch exhibits heterogeneous conformations under 1 mM Mg2+ concentration and ligand binding reduces conformational flexibility. Among all conformers, we determined one apo (5.3 Å) and four holo cryo-electron microscopy structures (overall 3.0-3.5 Å, binding pocket 2.9-3.2 Å). The holo dimers exhibit global motions of helical twisting and bending around the dimer interface. A backbone comparison of the apo and holo states reveals a large structural difference in the P6 extension position. The central strand of the binding pocket, junction 6/3, changes from an 'S'- to a 'U'-shaped conformation to accommodate ligand. Furthermore, the binding pocket can partially form under 1 mM Mg2+ and fully form under 10 mM Mg2+ within the bound-like structure in the absence of ligand. Our results not only demonstrate the stabilizing ligand-induced conformational changes in and around the binding pocket but may also provide further insight into the role of the P6 extension in ligand binding and selectivity.

Type 9 secretion system structures reveal a new protein transport mechanism.

The type 9 secretion system (T9SS) is the protein export pathway of bacteria of the Gram-negative Fibrobacteres-Chlorobi-Bacteroidetes superphylum and is an essential determinant of pathogenicity in severe periodontal disease. The central element of the T9SS is a so-far uncharacterized protein-conducting translocon located in the bacterial outer membrane. Here, using cryo-electron microscopy, we provide structural evidence that the translocon is the T9SS protein SprA. SprA forms an extremely large (36-strand) single polypeptide transmembrane ß-barrel. The barrel pore is capped on the extracellular end, but has a lateral opening to the external membrane surface. Structures of SprA bound to different components of the T9SS show that partner proteins control access to the lateral opening and to the periplasmic end of the pore. Our results identify a protein transporter with a distinctive architecture that uses an alternating access mechanism in which the two ends of the protein-conducting channel are open at different times.

An inhibitor of complement C5 provides structural insights into activation.

The complement system is a crucial part of innate immune defenses against invading pathogens. The blood-meal of the tick Rhipicephalus pulchellus lasts for days, and the tick must therefore rely on inhibitors to counter complement activation. We have identified a class of inhibitors from tick saliva, the CirpT family, and generated detailed structural data revealing their mechanism of action. We show direct binding of a CirpT to complement C5 and have determined the structure of the C5-CirpT complex by cryoelectron microscopy. This reveals an interaction with the peripheral macro globulin domain 4 (C5_MG4) of C5. To achieve higher resolution detail, the structure of the C5_MG4-CirpT complex was solved by X-ray crystallography (at 2.7 Å). We thus present the fold of the CirpT protein family, and provide detailed mechanistic insights into its inhibitory function. Analysis of the binding interface reveals a mechanism of C5 inhibition, and provides information to expand our biological understanding of the activation of C5, and thus the terminal complement pathway.

A signal sequence suppressor mutant that stabilizes an assembled state of the twin arginine translocase.

The twin-arginine protein translocation (Tat) system mediates transport of folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of chloroplasts. The Tat system of Escherichia coli is made up of TatA, TatB, and TatC components. TatBC comprise the substrate receptor complex, and active Tat translocases are formed by the substrate-induced association of TatA oligomers with this receptor. Proteins are targeted to TatBC by signal peptides containing an essential pair of arginine residues. We isolated substitutions, locating to the transmembrane helix of TatB that restored transport activity to Tat signal peptides with inactivating twin arginine substitutions. A subset of these variants also suppressed inactivating substitutions in the signal peptide binding site on TatC. The suppressors did not function by restoring detectable signal peptide binding to the TatBC complex. Instead, site-specific cross-linking experiments indicate that the suppressor substitutions induce conformational change in the complex and movement of the TatB subunit. The TatB F13Y substitution was associated with the strongest suppressing activity, even allowing transport of a Tat substrate lacking a signal peptide. In vivo analysis using a TatA-YFP fusion showed that the TatB F13Y substitution resulted in signal peptide-independent assembly of the Tat translocase. We conclude that Tat signal peptides play roles in substrate targeting and in triggering assembly of the active translocase.

Membrane Protein Complex ExbB4-ExbD1-TonB1 from Escherichia coli Demonstrates Conformational Plasticity.

UNLABELLED: Iron acquisition at the outer membrane (OM) of Gram-negative bacteria is powered by the proton motive force (PMF) of the cytoplasmic membrane (CM), harnessed by the CM-embedded complex of ExbB, ExbD, and TonB. Its stoichiometry, ensemble structural features, and mechanism of action are unknown. By panning combinatorial phage libraries, periplasmic regions of dimerization between ExbD and TonB were predicted. Using overexpression of full-length His6-tagged exbB-exbD and S-tagged tonB, we purified detergent-solubilized complexes of ExbB-ExbD-TonB from Escherichia coli. Protein-detergent complexes of ∼230 kDa with a hydrodynamic radius of ∼6.0 nm were similar to previously purified ExbB4-ExbD2 complexes. Significantly, they differed in electronegativity by native agarose gel electrophoresis. The stoichiometry was determined to be ExbB4-ExbD1-TonB1. Single-particle electron microscopy agrees with this stoichiometry. Two-dimensional averaging supported the phage display predictions, showing two forms of ExbD-TonB periplasmic heterodimerization: extensive and distal. Three-dimensional (3D) particle classification showed three representative conformations of ExbB4-ExbD1-TonB1. Based on our structural data, we propose a model in which ExbD shuttles a proton across the CM via an ExbB interprotein rearrangement. Proton translocation would be coupled to ExbD-mediated collapse of extended TonB in complex with ligand-loaded receptors in the OM, followed by repositioning of TonB through extensive dimerization with ExbD. Here we present the first report for purification of the ExbB-ExbD-TonB complex, molar ratios within the complex (4:1:1), and structural biology that provides insights into 3D organization. IMPORTANCE: Receptors in the OM of Gram-negative bacteria allow entry of iron-bound siderophores that are necessary for pathogenicity. Numerous iron-acquisition strategies rely upon a ubiquitous and unique protein for energization: TonB. Complexed with ExbB and ExbD, the Ton system links the PMF to OM transport. Blocking iron uptake by targeting a vital nanomachine holds promise in therapeutics. Despite much research, the stoichiometry, structural arrangement, and molecular mechanism of the CM-embedded ExbB-ExbD-TonB complex remain unreported. Here we demonstrate in vitro evidence of ExbB4-ExbD1-TonB1 complexes. Using 3D EM, we reconstructed the complex in three conformational states that show variable ExbD-TonB heterodimerization. Our structural observations form the basis of a model for TonB-mediated iron acquisition.

Purification and interaction analyses of two human lysosomal vitamin B12 transporters: LMBD1 and ABCD4.

Mutations in human LMBRD1 and ABCD4 prevent lysosomal export of vitamin B(12) to the cytoplasm, impairing the vitamin B(12)-dependent enzymes methionine synthase and methylmalonyl-CoA mutase. The gene products of LMBRD1 and ABCD4 are implicated in vitamin B(12) transport at the lysosomal membrane and are proposed to act in complex. To address the mechanism for lysosomal vitamin B(12) transport, we report the novel recombinant production of LMBD1 and ABCD4 for detailed biophysical analyses. Using blue native PAGE, chemical crosslinking, and size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), we show that both detergent-solubilized LMBD1 and detergent-solubilized ABCD4 form homodimers. To examine the functional binding properties of these proteins, label-free surface plasmon resonance (SPR) provides direct in vitro evidence that: (i) LMBD1 and ABCD4 interact with low nanomolar affinity; and (ii) the cytoplasmic vitamin B(12)-processing protein MMACHC also interacts with LMBD1 and ABCD4 with low nanomolar affinity. Accordingly, we propose a model whereby membrane-bound LMBD1 and ABCD4 facilitate the vectorial delivery of lysosomal vitamin B(12) to cytoplasmic MMACHC, thus preventing cofactor dilution to the cytoplasmic milieu and protecting against inactivating side reactions.

Structural basis of directional switching by the bacterial flagellum.

The bacterial flagellum is a macromolecular protein complex that harvests energy from uni-directional ion flow across the inner membrane to power bacterial swimming via rotation of the flagellar filament. Rotation is bi-directional, with binding of a cytoplasmic chemotactic response regulator controlling reversal, though the structural and mechanistic bases for rotational switching are not well understood. Here we present cryoelectron microscopy structures of intact Salmonella flagellar basal bodies (3.2-5.5 Å), including the cytoplasmic C-ring complexes required for power transmission, in both counter-clockwise and clockwise rotational conformations. These reveal 180° movements of both the N- and C-terminal domains of the FliG protein, which, when combined with a high-resolution cryoelectron microscopy structure of the MotA5B2 stator, show that the stator shifts from the outside to the inside of the C-ring. This enables rotational switching and reveals how uni-directional ion flow across the inner membrane is used to accomplish bi-directional rotation of the flagellum.

Structural insights into the mechanism of protein transport by the Type 9 Secretion System translocon.

Secretion systems are protein export machines that enable bacteria to exploit their environment through the release of protein effectors. The Type 9 Secretion System (T9SS) is responsible for protein export across the outer membrane (OM) of bacteria of the phylum Bacteroidota. Here we trap the T9SS of Flavobacterium johnsoniae in the process of substrate transport by disrupting the T9SS motor complex. Cryo-EM analysis of purified substrate-bound T9SS translocons reveals an extended translocon structure in which the previously described translocon core is augmented by a periplasmic structure incorporating the proteins SprE, PorD and a homologue of the canonical periplasmic chaperone Skp. Substrate proteins bind to the extracellular loops of a carrier protein within the translocon pore. As transport intermediates accumulate on the translocon when energetic input is removed, we deduce that release of the substrate-carrier protein complex from the translocon is the energy-requiring step in T9SS transport.

Subcellular location of MMACHC and MMADHC, two human proteins central to intracellular vitamin B(12) metabolism.

MMACHC and MMADHC are the genes responsible for cblC and cblD defects of vitamin B(12) metabolism, respectively. Patients with cblC and cblD defects present with various combinations of methylmalonic aciduria (MMA) and homocystinuria (HC). Those with cblC mutations have both MMA and HC whereas cblD patients can present with one of three distinct biochemical phenotypes: isolated MMA, isolated HC, or combined MMA and HC. Based on the subcellular localization of these enzymatic pathways it is thought that MMACHC functions in the cytoplasm while MMADHC functions downstream of MMACHC in both the cytoplasm and the mitochondrion. In this study we determined the subcellular location of MMACHC and MMADHC by immunofluorescence and subcellular fractionation. We show that MMACHC is cytoplasmic while MMADHC is both mitochondrial and cytoplasmic, consistent with the proposal that MMADHC acts as a branch point for vitamin B(12) delivery to the cytoplasm and mitochondria. The factors that determine the distribution of MMADHC between the cytoplasm and mitochondria remain unknown. Functional complementation experiments showed that retroviral expression of the GFP tagged constructs rescued all biochemical defects in cblC and cblD fibroblasts except propionate incorporation in cblD-MMA cells, suggesting that the endogenous mutant protein interferes with the function of the transduced wild type construct.

Structure and assembly of the NOT10:11 module of the CCR4-NOT complex.

NOT1, NOT10, and NOT11 form a conserved module in the CCR4-NOT complex, critical for post-transcriptional regulation in eukaryotes, but how this module contributes to the functions of the CCR4-NOT remains poorly understood. Here, we present cryo-EM structures of human and chicken NOT1:NOT10:NOT11 ternary complexes to sub-3 Å resolution, revealing an evolutionarily conserved, flexible structure. Through biochemical dissection studies, which include the Drosophila orthologs, we show that the module assembly is hierarchical, with NOT11 binding to NOT10, which then organizes it for binding to NOT1. A short proline-rich motif in NOT11 stabilizes the entire module assembly.

Structural Basis of Directional Switching by the Bacterial Flagellum.

The bacterial flagellum is a macromolecular protein complex that harvests energy from ion-flow across the inner membrane to power bacterial swimming in viscous fluids via rotation of the flagellar filament. Bacteria such as Salmonella enterica are capable of bi-directional flagellar rotation even though ion flow is uni-directional. How uni-directional ion-movement through the inner membrane is utilized by this macromolecular machine to drive bi-directional flagellar rotation is not understood, but a chemotactic response regulator in the cytoplasm is known to reverse the direction of rotation. We here present cryo-EM structures of intact Salmonella flagellar basal bodies, including the cytoplasmic complexes required for power transmission, in conformations representing both directions of rotation. The structures reveal that the conformational changes required for switching the direction of rotation involve 180 degree rotations of both the N- and C-terminal domains of the FliG protein. Combining these models with a new, high-resolution, cryo-EM structure of the MotA5B2 stator, in complex with the C-terminal domain of FliG, reveals how uni-directional ion-flow across the inner membrane is used to accomplish bi-directional rotation of the flagellum.

Delineation of functional subdomains of Huntingtin protein and their interaction with HAP40.

The huntingtin (HTT) protein plays critical roles in numerous cellular pathways by functioning as a scaffold for its many interaction partners and HTT knock out is embryonic lethal. Interrogation of HTT function is complicated by the large size of this protein so we studied a suite of structure-rationalized subdomains to investigate the structure-function relationships within the HTT-HAP40 complex. Protein samples derived from the subdomain constructs were validated using biophysical methods and cryo-electron microscopy, revealing they are natively folded and can complex with validated binding partner, HAP40. Derivatized versions of these constructs enable protein-protein interaction assays in vitro, with biotin tags, and in cells, with luciferase two-hybrid assay-based tags, which we use in proof-of-principle analyses to further interrogate the HTT-HAP40 interaction. These open-source biochemical tools enable studies of fundamental HTT biochemistry and biology, will aid the discovery of macromolecular or small-molecule binding partners and help map interaction sites across this large protein.

Structural features of recombinant MMADHC isoforms and their interactions with MMACHC, proteins of mammalian vitamin B12 metabolism.

The genes MMACHC and MMADHC encode critical proteins involved in the intracellular metabolism of cobalamin. Two clinical features, homocystinuria and methylmalonic aciduria, define inborn errors of these genes. Based on disease phenotypes, MMADHC acts at a branch point for cobalamin delivery, apparently exerting its function through interaction with MMACHC that demonstrates dealkylase and decyanase activities. Here we present biophysical analyses of MMADHC to identify structural features and to further characterize its interaction with MMACHC. Two recombinant tag-less isoforms of MMADHC (MMADHCΔ1-12 and MMADHCΔ1-61) were expressed and purified. Full length MMACHC and full length MMADHC were detected in whole cell lysates of human cells; by Western blotting, their molecular masses corresponded to purified recombinant proteins. By clear-native PAGE and by dynamic light scattering, recombinant MMADHCs were stable and monodisperse. Both species were monomeric, adopting extended conformations in solution. Circular dichroism and secondary structure predictions correlated with significant regions of disorder within the N-terminal domain of MMADHC. We found no evidence that MMADHC binds cobalamin. Phage panning against MMADHC predicted four binding regions on MMACHC, two of which overlap with predicted sites on MMACHC at which it may self-associate. Specific, concentration-dependent responses were observed for MMACHC binding to itself and to both MMADHC constructs. As estimated in the sub-micromolar range, the binding of MMACHC to itself was weaker compared to its interaction with either of the MMADHC isoforms. We propose that the function of MMADHC is exerted through its structured C-terminal domain via interactions with MMACHC.

Structures of the Type IX Secretion/Gliding Motility Motor from across the Phylum Bacteroidetes.

Gliding motility using cell surface adhesins, and export of proteins by the type IX secretion system (T9SS) are two phylum-specific features of the Bacteroidetes. Both of these processes are energized by the GldLM motor complex, which transduces the proton motive force at the inner membrane into mechanical work at the outer membrane. We previously used cryo-electron microscopy to solve the structure of the GldLM motor core from Flavobacterium johnsoniae at 3.9-Å resolution (R. Hennell James, J. C. Deme, A. Kjaer, F. Alcock, et al., Nat Microbiol 6:221-233, 2021, https://dx.doi.org/10.1038/s41564-020-00823-6). Here, we present structures of homologous complexes from a range of pathogenic and environmental Bacteroidetes species at up to 3.0-Å resolution. These structures show that the architecture of the GldLM motor core is conserved across the Bacteroidetes phylum, although there are species-specific differences at the N terminus of GldL. The resolution improvements reveal a cage-like structure that ties together the membrane-proximal cytoplasmic region of GldL and influences gliding function. These findings add detail to our structural understanding of bacterial ion-driven motors that drive the T9SS and gliding motility. IMPORTANCE Many bacteria in the Bacteroidetes phylum use the type IX secretion system to secrete proteins across their outer membrane. Most of these bacteria can also glide across surfaces using adhesin proteins that are propelled across the cell surface. Both secretion and gliding motility are driven by the GldLM protein complex, which forms a nanoscale electrochemical motor. We used cryo-electron microscopy to study the structure of the GldLM protein complex from different species, including the human pathogens Porphyromonas gingivalis and Capnocytophaga canimorsus. The organization of the motor is conserved across species, but we find species-specific structural differences and resolve motor features at higher resolution. This work improves our understanding of the type IX secretion system, which is a virulence determinant in human and animal diseases.

Nonameric structures of the cytoplasmic domain of FlhA and SctV in the context of the full-length protein.

Type three secretion is the mechanism of protein secretion found in bacterial flagella and injectisomes. At its centre is the export apparatus (EA), a complex of five membrane proteins through which secretion substrates pass the inner membrane. While the complex formed by four of the EA proteins has been well characterised structurally, little is known about the structure of the membrane domain of the largest subunit, FlhA in flagella, SctV in injectisomes. Furthermore, the biologically relevant nonameric assembly of FlhA/SctV has been infrequently observed and differences in conformation of the cytoplasmic portion of FlhA/SctV between open and closed states have been suggested to reflect secretion system specific differences. FlhA has been shown to bind to chaperone-substrate complexes in an open state, but in previous assembled ring structures, SctV is in a closed state. Here, we identify FlhA and SctV homologues that can be recombinantly produced in the oligomeric state and study them using cryo-electron microscopy. The structures of the cytoplasmic domains from both FlhA and SctV are in the open state and we observe a conserved interaction between a short stretch of residues at the N-terminus of the cytoplasmic domain, known as FlhAL/SctVL, with a groove on the adjacent protomer's cytoplasmic domain, which stabilises the nonameric ring assembly.

Molecular basis for redox control by the human cystine/glutamate antiporter system xc.

Cysteine plays an essential role in cellular redox homoeostasis as a key constituent of the tripeptide glutathione (GSH). A rate limiting step in cellular GSH synthesis is the availability of cysteine. However, circulating cysteine exists in the blood as the oxidised di-peptide cystine, requiring specialised transport systems for its import into the cell. System xc- is a dedicated cystine transporter, importing cystine in exchange for intracellular glutamate. To counteract elevated levels of reactive oxygen species in cancerous cells system xc- is frequently upregulated, making it an attractive target for anticancer therapies. However, the molecular basis for ligand recognition remains elusive, hampering efforts to specifically target this transport system. Here we present the cryo-EM structure of system xc- in both the apo and glutamate bound states. Structural comparisons reveal an allosteric mechanism for ligand discrimination, supported by molecular dynamics and cell-based assays, establishing a mechanism for cystine transport in human cells.

Molecular Basis for Bordetella pertussis Interference with Complement, Coagulation, Fibrinolytic, and Contact Activation Systems: the Cryo-EM Structure of the Vag8-C1 Inhibitor Complex.

Complement, contact activation, coagulation, and fibrinolysis are serum protein cascades that need strict regulation to maintain human health. Serum glycoprotein, a C1 inhibitor (C1-INH), is a key regulator (inhibitor) of serine proteases of all the above-mentioned pathways. Recently, an autotransporter protein, virulence-associated gene 8 (Vag8), produced by the whooping cough pathogen, Bordetella pertussis, was shown to bind to C1-INH and interfere with its function. Here, we present the structure of the Vag8-C1-INH complex determined using cryo-electron microscopy at a 3.6-Å resolution. The structure shows a unique mechanism of C1-INH inhibition not employed by other pathogens, where Vag8 sequesters the reactive center loop of C1-INH, preventing its interaction with the target proteases.IMPORTANCE The structure of a 10-kDa protein complex is one of the smallest to be determined using cryo-electron microscopy at high resolution. The structure reveals that C1-INH is sequestered in an inactivated state by burial of the reactive center loop in Vag8. By so doing, the bacterium is able to simultaneously perturb the many pathways regulated by C1-INH. Virulence mechanisms such as the one described here assume more importance given the emerging evidence about dysregulation of contact activation, coagulation, and fibrinolysis leading to COVID-19 pneumonia.

Cryo-EM structure and resistance landscape of M. tuberculosis MmpL3: An emergent therapeutic target.

Tuberculosis (TB) is the leading cause of death from a single infectious agent and in 2019 an estimated 10 million people worldwide contracted the disease. Although treatments for TB exist, continual emergence of drug-resistant variants necessitates urgent development of novel antituberculars. An important new target is the lipid transporter MmpL3, which is required for construction of the unique cell envelope that shields Mycobacterium tuberculosis (Mtb) from the immune system. However, a structural understanding of the mutations in Mtb MmpL3 that confer resistance to the many preclinical leads is lacking, hampering efforts to circumvent resistance mechanisms. Here, we present the cryoelectron microscopy structure of Mtb MmpL3 and use it to comprehensively analyze the mutational landscape of drug resistance. Our data provide a rational explanation for resistance variants local to the central drug binding site, and also highlight a potential alternative route to resistance operating within the periplasmic domain.