Structural insights into the inhibition of glycine reuptake.

The human glycine transporter 1 (GlyT1) regulates glycine-mediated neuronal excitation and inhibition through the sodium- and chloride-dependent reuptake of glycine1-3. Inhibition of GlyT1 prolongs neurotransmitter signalling, and has long been a key strategy in the development of therapies for a broad range of disorders of the central nervous system, including schizophrenia and cognitive impairments4. Here, using a synthetic single-domain antibody (sybody) and serial synchrotron crystallography, we have determined the structure of GlyT1 in complex with a benzoylpiperazine chemotype inhibitor at 3.4 Å resolution. We find that the inhibitor locks GlyT1 in an inward-open conformation and binds at the intracellular gate of the release pathway, overlapping with the glycine-release site. The inhibitor is likely to reach GlyT1 from the cytoplasmic leaflet of the plasma membrane. Our results define the mechanism of inhibition and enable the rational design of new, clinically efficacious GlyT1 inhibitors.

Biomolecules capturing live bacteria from clinical samples.

Rapid phenotypic antimicrobial susceptibility testing (AST) requires the enrichment of live bacteria from patient samples, which is particularly challenging in the context of life-threatening bloodstream infections (BSIs) due to low bacterial titers. Over two decades, an extensive array of pathogen-specific biomolecules has been identified to capture live bacteria. The prevailing biomolecules are immune proteins of the complement system, antibodies, aptamers, phage proteins, and antimicrobial peptides. These biomolecules differ by their binder generation technologies and exhibit highly variable specificities, ranging from bacterial strains to most pathogenic bacteria. Here, we summarize how these diverse biomolecules were identified, list examples of successfully reported capture assays, and provide an outlook on the use of nanobodies raised against conserved surface-accessible proteins as promising biomolecules for pathogen capture.

The ABC exporter IrtAB imports and reduces mycobacterial siderophores.

Intracellular replication of the deadly pathogen Mycobacterium tuberculosis relies on the production of small organic molecules called siderophores that scavenge iron from host proteins1. M. tuberculosis produces two classes of siderophore, lipid-bound mycobactin and water-soluble carboxymycobactin2,3. Functional studies have revealed that iron-loaded carboxymycobactin is imported into the cytoplasm by the ATP binding cassette (ABC) transporter IrtAB4, which features an additional cytoplasmic siderophore interaction domain5. However, the predicted ABC exporter fold of IrtAB is seemingly contradictory to its import function. Here we show that membrane-reconstituted IrtAB is sufficient to import mycobactins, which are then reduced by the siderophore interaction domain to facilitate iron release. Structure determination by X-ray crystallography and cryo-electron microscopy not only confirms that IrtAB has an ABC exporter fold, but also reveals structural peculiarities at the transmembrane region of IrtAB that result in a partially collapsed inward-facing substrate-binding cavity. The siderophore interaction domain is positioned in close proximity to the inner membrane leaflet, enabling the reduction of membrane-inserted mycobactin. Enzymatic ATPase activity and in vivo growth assays show that IrtAB has a preference for mycobactin over carboxymycobactin as its substrate. Our study provides insights into an unusual ABC exporter that evolved as highly specialized siderophore-import machinery in mycobacteria.

Deep mutational scan of a drug efflux pump reveals its structure-function landscape.

Drug efflux is a common resistance mechanism found in bacteria and cancer cells, but studies providing comprehensive functional insights are scarce. In this study, we performed deep mutational scanning (DMS) on the bacterial ABC transporter EfrCD to determine the drug efflux activity profile of more than 1,430 single variants. These systematic measurements revealed that the introduction of negative charges at different locations within the large substrate binding pocket results in strongly increased efflux activity toward positively charged ethidium, whereas additional aromatic residues did not display the same effect. Data analysis in the context of an inward-facing cryogenic electron microscopy structure of EfrCD uncovered a high-affinity binding site, which releases bound drugs through a peristaltic transport mechanism as the transporter transits to its outward-facing conformation. Finally, we identified substitutions resulting in rapid Hoechst influx without affecting the efflux activity for ethidium and daunorubicin. Hence, single mutations can convert EfrCD into a drug-specific ABC importer.

Biparatopic sybodies neutralize SARS-CoV-2 variants of concern and mitigate drug resistance.

The ongoing COVID-19 pandemic represents an unprecedented global health crisis. Here, we report the identification of a synthetic nanobody (sybody) pair, Sb#15 and Sb#68, that can bind simultaneously to the SARS-CoV-2 spike RBD and efficiently neutralize pseudotyped and live viruses by interfering with ACE2 interaction. Cryo-EM confirms that Sb#15 and Sb#68 engage two spatially discrete epitopes, influencing rational design of bispecific and tri-bispecific fusion constructs that exhibit up to 100- and 1,000-fold increase in neutralization potency, respectively. Cryo-EM of the sybody-spike complex additionally reveals a novel up-out RBD conformation. While resistant viruses emerge rapidly in the presence of single binders, no escape variants are observed in the presence of the bispecific sybody. The multivalent bispecific constructs further increase the neutralization potency against globally circulating SARS-CoV-2 variants of concern. Our study illustrates the power of multivalency and biparatopic nanobody fusions for the potential development of therapeutic strategies that mitigate the emergence of new SARS-CoV-2 escape mutants.

Spin-labeled nanobodies as protein conformational reporters for electron paramagnetic resonance in cellular membranes.

Nanobodies are emerging tools in a variety of fields such as structural biology, cell imaging, and drug discovery. Here we pioneer the use of their spin-labeled variants as reporters of conformational dynamics of membrane proteins using DEER spectroscopy. At the example of the bacterial ABC transporter TM287/288, we show that two gadolinium-labeled nanobodies allow us to quantify, via analysis of the modulation depth of DEER traces, the fraction of transporters adopting the outward-facing state under different experimental conditions. Additionally, we quantitatively follow the interconversion from the outward- to the inward-facing state in the conformational ensemble under ATP turnover conditions. We finally show that the specificity of the nanobodies for the target protein allows the direct attainment of structural information on the wild-type TM287/288 expressed in cellular membranes without the need to purify or label the investigated membrane protein.

Engineered peptide barcodes for in-depth analyses of binding protein libraries.

Binding protein generation typically relies on laborious screening cascades that process candidate molecules individually. We have developed NestLink, a binder selection and identification technology able to biophysically characterize thousands of library members at once without the need to handle individual clones at any stage of the process. NestLink uses genetically encoded barcoding peptides termed flycodes, which were designed for maximal detectability by mass spectrometry and support accurate deep sequencing. We demonstrate NestLink's capacity to overcome the current limitations of binder-generation methods in three applications. First, we show that hundreds of binder candidates can be simultaneously ranked according to kinetic parameters. Next, we demonstrate deep mining of a nanobody immune repertoire for membrane protein binders, carried out entirely in solution without target immobilization. Finally, we identify rare binders against an integral membrane protein directly in the cellular environment of a human pathogen. NestLink opens avenues for the selection of tailored binder characteristics directly in tissues or in living organisms.

The structure of the Legionella response regulator LqsR reveals amino acids critical for phosphorylation and dimerization.

The water-borne bacterium Legionella pneumophila replicates in environmental protozoa and upon inhalation destroys alveolar macrophages, thus causing a potentially fatal pneumonia termed 'Legionnaires' disease'. L. pneumophila employs the Legionella quorum sensing (Lqs) system to control its life cycle, pathogen-host cell interactions, motility and natural competence. Signaling through the Lqs system occurs through the α-hydroxyketone compound Legionella autoinducer-1 (LAI-1) and converges on the prototypic response regulator LqsR, which dimerizes upon phosphorylation of the conserved aspartate, D108 . In this study, we determine the high-resolution structure of monomeric LqsR. The structure reveals a receiver domain adopting a canonical (ßα)5 fold, which is connected through an additional sixth helix and an extended α5-helix to a novel output domain. The two domains delineate a mainly positively charged groove, and the output domain adopts a five-stranded antiparallel ß-sheet fold similar to nucleotide-binding proteins. Structure-based mutagenesis identified amino acids critical for LqsR phosphorylation and dimerization. Upon phosphorylation, the LqsRD172A and LqsRD302N/E303Q mutant proteins dimerized even more readily than wild-type LqsR, and no evidence for semi-phosphorylated heterodimers was obtained. Taken together, the high-resolution structure of LqsR reveals functionally relevant amino acid residues implicated in signal transduction of the prototypic response regulator.

Mycobacterium marinum produces distinct mycobactin and carboxymycobactin siderophores to promote growth in broth and phagocytes.

Mycobacterium marinum is a model organism for pathogenic Mycobacterium species, including Mycobacterium tuberculosis, the causative agent of tuberculosis. These pathogens enter phagocytes and replicate within the Mycobacterium-containing vacuole, possibly followed by vacuole exit and growth in the host cell cytosol. Mycobacteria release siderophores called mycobactins to scavenge iron, an essential yet poorly soluble and available micronutrient. To investigate the role of M. marinum mycobactins, we purified by organic solvent extraction and identified by mass spectrometry the lipid-bound mycobactin (MBT) and the water-soluble variant carboxymycobactin (cMBT). Moreover, we generated by specialised phage transduction a defined M. marinum ΔmbtB deletion mutant predicted to be defective for mycobactin production. The M. marinum ΔmbtB mutant strain showed a severe growth defect in broth and phagocytes, which was partially complemented by supplying the mbtB gene on a plasmid. Furthermore, purified Fe-MBT or Fe-cMBT improved the growth of wild type as well as ΔmbtB mutant bacteria on minimal plates, but only Fe-cMBT promoted the growth of wild-type M. marinum during phagocyte infection. Finally, the intracellular growth of M. marinum ΔmbtB in Acanthamoeba castellanii amoebae was restored by coinfection with wild-type bacteria. Our study identifies and characterises the M. marinum MBT and cMBT siderophores and reveals the requirement of mycobactins for extra- and intracellular growth of the pathogen.

Increased drug permeability of a stiffened mycobacterial outer membrane in cells lacking MFS transporter Rv1410 and lipoprotein LprG.

The major facilitator superfamily transporter Rv1410 and the lipoprotein LprG (Rv1411) are encoded by a conserved two-gene operon and contribute to virulence in Mycobacterium tuberculosis. Rv1410 was originally postulated to function as a drug efflux pump, but recent studies suggested that Rv1410 and LprG work in concert to insert triacylglycerides and lipoarabinomannans into the outer membrane. Here, we conducted microscopic analyses of Mycobacterium smegmatis lacking the operon and observed a cell separation defect, while surface rigidity measured by atomic force microscopy was found to be increased. Whereas Rv1410 expressed in Lactococcus lactis did not confer drug resistance, deletion of the operon in Mycobacterium abscessus and M. smegmatis resulted in increased susceptibility toward vancomycin, novobiocin and rifampicin. A homology model of Rv1410 revealed a periplasmic loop as well as a highly conserved aspartate, which were found to be essential for the operon's function. Interestingly, influx of the fluorescent dyes BCECF-AM and calcein-AM in de-energized M. smegmatis cells was faster in the deletion mutant. Our results unambiguously show that elevated drug susceptibility in the deletion mutant is caused by increased drug influx through a defective mycobacterial cell envelope and not by drug efflux mediated by Rv1410.

Identification of conformation-selective nanobodies against the membrane protein insertase BamA by an integrated structural biology approach.

The insertase BamA is an essential protein of the bacterial outer membrane. Its 16-stranded transmembrane ß-barrel contains a lateral gate as a key functional element. This gate is formed by the C-terminal half of the last ß-strand. The BamA barrel was previously found to sample different conformations in aqueous solution, as well as different gate-open, gate-closed, and collapsed conformations in X-ray crystallography and cryo-electron microscopy structures. Here, we report the successful identification of conformation-selective nanobodies that stabilize BamA in specific conformations. While the initial candidate generation and selection protocol was based on established alpaca immunization and phage display selection procedures, the final selection of nanobodies was enhanced by a solution NMR-based screening step to shortlist the targets for crystallization. In this way, three crystal structures of BamA-nanobody complexes were efficiently obtained, showing two types of nanobodies that indeed stabilized BamA in two different conformations, i.e., with open and closed lateral gate, respectively. Then, by correlating the structural data with high resolution NMR spectra, we could for the first time assign the BamA conformational solution ensemble to defined structural states. The new nanobodies will be valuable tools towards understanding the client insertion mechanism of BamA and towards developing improved antibiotics.

Atomistic Mechanism of Large-Scale Conformational Transition in a Heterodimeric ABC Exporter.

ATP-binding cassette (ABC) transporters are ATP-driven molecular machines, in which ATP binding and hydrolysis in the nucleotide-binding domains (NBDs) is chemomechanically coupled to large-scale, alternating access conformational changes in the transmembrane domains (TMDs), ultimately leading to the translocation of substrates across biological membranes. The precise nature of the structural dynamics behind the large-scale conformational transition as well as the coupling of NBD and TMD motions is still unresolved. In this work, we combine all-atom molecular dynamics (MD) simulations with electron paramagnetic resonance (EPR) spectroscopy to unravel the atomic-level mechanism of the dynamic conformational transitions underlying the functional working cycle of the heterodimeric ABC exporter TM287/288. Extensive multimicrosecond simulations in an explicit membrane/water environment show how in response to ATP binding, TM287/288 undergoes spontaneous conformational transitions from the inward-facing (IF) state via an occluded (Occ) intermediate to an outward-facing (OF) state. The latter two states have thus far not been characterized at atomic level. ATP-induced tightening of the NBD dimer involves closing and reorientation of the two NBD monomers concomitant with a closure of the intracellular TMD gate, which leads to the occluded state. Subsequently, opening at the extracellular TMD gate yields the OF conformer. The obtained mechanism imposes NBD-TMD coupling via a tight orchestration of conformational transitions, between both the two domains and also within the TMDs, ensuring that the cytoplasmic and periplasmic gate regions are never open simultaneously.

Membrane transporter research in times of countless structures.

Structural biology has advanced our understanding of membrane proteins like no other scientific discipline in the past two decades and the number of high resolution membrane transporter structures solved by X-ray crystallography has increased exponentially over this time period. Currently, single particle cryo-EM is in full swing due to a recent resolution revolution and permits for structural insights of proteins that were refractory to crystallization. It is foreseeable that multiple structures of many human transporters will be solved in the coming five years. Nevertheless, many scientifically important questions remain unanswered despite of available structures, as is illustrated in this article at the example of multidrug efflux pumps and ABC transporters. Structure-function studies likely continue to be a supporting pillar of membrane transporter research. However, there is a trend towards the "integrated structural biologist", whose research focusses on a biological question and who closely collaborates with other research groups specialized in spectroscopy techniques or molecular dynamics simulation. Future membrane protein research requires joint efforts from specialists of various disciplines to finally work towards a molecular understanding of membrane transport in the context of the living cell. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.

Structural basis for allosteric cross-talk between the asymmetric nucleotide binding sites of a heterodimeric ABC exporter.

ATP binding cassette (ABC) transporters mediate vital transport processes in every living cell. ATP hydrolysis, which fuels transport, displays positive cooperativity in numerous ABC transporters. In particular, heterodimeric ABC exporters exhibit pronounced allosteric coupling between a catalytically impaired degenerate site, where nucleotides bind tightly, and a consensus site, at which ATP is hydrolyzed in every transport cycle. Whereas the functional phenomenon of cooperativity is well described, its structural basis remains poorly understood. Here, we present the apo structure of the heterodimeric ABC exporter TM287/288 and compare it to the previously solved structure with adenosine 5'-(ß,γ-imido)triphosphate (AMP-PNP) bound at the degenerate site. In contrast to other ABC exporter structures, the nucleotide binding domains (NBDs) of TM287/288 remain in molecular contact even in the absence of nucleotides, and the arrangement of the transmembrane domains (TMDs) is not influenced by AMP-PNP binding, a notion confirmed by double electron-electron resonance (DEER) measurements. Nucleotide binding at the degenerate site results in structural rearrangements, which are transmitted to the consensus site via two D-loops located at the NBD interface. These loops owe their name from a highly conserved aspartate and are directly connected to the catalytically important Walker B motif. The D-loop at the degenerate site ties the NBDs together even in the absence of nucleotides and substitution of its aspartate by alanine is well-tolerated. By contrast, the D-loop of the consensus site is flexible and the aspartate to alanine mutation and conformational restriction by cross-linking strongly reduces ATP hydrolysis and substrate transport.

The Heterodimeric ABC Transporter EfrCD Mediates Multidrug Efflux in Enterococcus faecalis.

Nosocomial infections with Enterococcus faecalis are an emerging health problem. However, drug efflux pumps contributing to intrinsic drug resistance are poorly studied in this Gram-positive pathogen. In this study, we functionally investigated seven heterodimeric ABC transporters of E. faecalis that are annotated as drug efflux pumps. Deletion of ef0789-ef0790 on the chromosome of E. faecalis resulted in increased susceptibility to daunorubicin, doxorubicin, ethidium, and Hoechst 33342, and the corresponding transporter was named EfrCD. Unexpectedly, the previously described heterodimeric multidrug ABC transporter EfrAB contributes marginally to drug efflux in the endogenous context of E. faecalis In contrast, heterologous expression in Lactococcus lactis revealed that EfrAB, EfrCD, and the product of ef2226-ef2227 (EfrEF) mediate the efflux of fluorescent substrates and confer resistance to multiple dyes and drugs, including fluoroquinolones. Four of seven transporters failed to exhibit drug efflux activity for the set of drugs and dyes tested, even upon overexpression in L. lactis Since all seven transporters were purified as heterodimers after overexpression in L. lactis, a lack of drug efflux activity is not attributed to poor expression or protein aggregation. Reconstitution of the purified multidrug transporters EfrAB, EfrCD, and EfrEF in proteoliposomes revealed functional coupling between ATP hydrolysis and drug binding. Our analysis creates an experimental basis for the accurate prediction of drug efflux transporters and indicates that many annotated multidrug efflux pumps might be incapable of drug transport and thus might fulfill other physiological functions in the cell.

A Transporter Motor Taken Apart: Flexibility in the Nucleotide Binding Domains of a Heterodimeric ABC Exporter.

ABC exporters are ubiquitous multidomain transport proteins that couple ATP hydrolysis at a pair of nucleotide binding domains to substrate transport across the lipid bilayer mediated by two transmembrane domains. Recently, the crystal structure of the heterodimeric ABC exporter TM287/288 was determined. One of its asymmetric ATP binding sites is called the degenerate site; it binds nucleotides tightly but is impaired in terms of ATP hydrolysis. Here we report the crystal structures of both isolated motor domains of TM287/288. Unexpectedly, structural elements constituting the degenerate ATP binding site are disordered in these crystals and become structured only in the context of the full-length transporter. In addition, hydrogen bonding patterns of key residues, including those of the catalytically important Walker B and the switch loop motifs, are fundamentally different in the solitary NBDs compared to those in the intact transport protein. The structures reveal crucial interdomain contacts that need to be established for the proper assembly of the functional transporter complex.

Transport of drugs by the multidrug transporter AcrB involves an access and a deep binding pocket that are separated by a switch-loop.

AcrAB-TolC is the major efflux protein complex in Escherichia coli extruding a vast variety of antimicrobial agents from the cell. The inner membrane component AcrB is a homotrimer, and it has been postulated that the monomers cycle consecutively through three conformational stages designated loose (L), tight (T), and open (O) in a concerted fashion. Binding of drugs has been shown at a periplasmic deep binding pocket in the T conformation. The initial drug-binding step and transport toward this drug-binding site has been elusive thus far. Here we report high resolution structures (1.9-2.25 Å) of AcrB/designed ankyrin repeat protein (DARPin) complexes with bound minocycline or doxorubicin. In the AcrB/doxorubicin cocrystal structure, binding of three doxorubicin molecules is apparent, with one doxorubicin molecule bound in the deep binding pocket of the T monomer and two doxorubicin molecules in a stacked sandwich arrangement in an access pocket at the lateral periplasmic cleft of the L monomer. This access pocket is separated from the deep binding pocket apparent in the T monomer by a switch-loop. The localization and conformational flexibility of this loop seems to be important for large substrates, because a G616N AcrB variant deficient in macrolide transport exhibits an altered conformation within this loop region. Transport seems to be a stepwise process of initial drug uptake in the access pocket of the L monomer and subsequent accommodation of the drug in the deep binding pocket during the L to T transition to the internal deep binding pocket of the T monomer.

Interdomain-linkers control conformational transitions in the SLC23 elevator transporter UraA.

Uptake of nucleobases and ascorbate is an essential process in all living organisms mediated by SLC23 transport proteins. These transmembrane carriers operate via the elevator alternating-access mechanism, and are composed of two rigid domains whose relative motion drives transport. The lack of large conformational changes within these domains suggests that the interdomain-linkers act as flexible tethers. Here, we show that interdomain-linkers are not mere tethers, but have a key regulatory role in dictating the conformational space of the transporter and defining the rotation axis of the mobile transport domain. By resolving a wide inward-open conformation of the SLC23 elevator transporter UraA and combining biochemical studies using a synthetic nanobody as conformational probe with hydrogen-deuterium exchange mass spectrometry, we demonstrate that interdomain-linkers control the function of transport proteins by influencing substrate affinity and transport rate. These findings open the possibility to allosterically modulate the activity of elevator proteins by targeting their linkers.

Doa10/MARCH6 architecture interconnects E3 ligase activity with lipid-binding transmembrane channel to regulate SQLE.

Transmembrane E3 ligases play crucial roles in homeostasis. Much protein and organelle quality control, and metabolic regulation, are determined by ER-resident MARCH6 E3 ligases, including Doa10 in yeast. Here, we present Doa10/MARCH6 structural analysis by cryo-EM and AlphaFold predictions, and a structure-based mutagenesis campaign. The majority of Doa10/MARCH6 adopts a unique circular structure within the membrane. This channel is established by a lipid-binding scaffold, and gated by a flexible helical bundle. The ubiquitylation active site is positioned over the channel by connections between the cytosolic E3 ligase RING domain and the membrane-spanning scaffold and gate. Here, by assaying 95 MARCH6 variants for effects on stability of the well-characterized substrate SQLE, which regulates cholesterol levels, we reveal crucial roles of the gated channel and RING domain consistent with AlphaFold-models of substrate-engaged and ubiquitylation complexes. SQLE degradation further depends on connections between the channel and RING domain, and lipid binding sites, revealing how interconnected Doa10/MARCH6 elements could orchestrate metabolic signals, substrate binding, and E3 ligase activity.

Asymmetry in the homodimeric ABC transporter MsbA recognized by a DARPin.

ABC transporters harness the energy from ATP binding and hydrolysis to translocate substrates across the membrane. Binding of two ATP molecules at the nucleotide binding domains (NBDs) leads to the formation of an outward-facing state. The conformational changes required to reset the transporter to the inward-facing state are initiated by sequential hydrolysis of the bound nucleotides. In a homodimeric ABC exporter such as MsbA responsible for lipid A transport in Escherichia coli, sequential ATP hydrolysis implies the existence of an asymmetric conformation. Here we report the in vitro selection of a designed ankyrin repeat protein (DARPin) specifically binding to detergent-solubilized MsbA. Only one DARPin binds to the homodimeric transporter in the absence as well as in the presence of nucleotides, suggesting that it recognizes asymmetries in MsbA. DARPin binding increases the rate of ATP hydrolysis by a factor of two independent of the substrate-induced ATPase stimulation. Electron paramagnetic resonance (EPR) measurements are found to be in good agreement with the available crystal structures and reveal that DARPin binding does not affect the large nucleotide-driven conformational changes of MsbA. The binding epitope was mapped by cross-linking and EPR to the membrane-spanning part of the transmembrane domain (TMD). Using cross-linked DARPin-MsbA complexes, 8-azido-ATP was found to preferentially photolabel one chain of the homodimer, suggesting that the asymmetries captured by DARPin binding at the TMDs are propagated to the NBDs. This work demonstrates that in vitro selected binders are useful tools to study the mechanism of membrane proteins.