Mechanism of NMDA Receptor Inhibition and Activation.

N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated, calcium-permeable ion channels that mediate synaptic transmission and underpin learning and memory. NMDAR dysfunction is directly implicated in diseases ranging from seizure to ischemia. Despite its fundamental importance, little is known about how the NMDAR transitions between inactive and active states and how small molecules inhibit or activate ion channel gating. Here, we report electron cryo-microscopy structures of the GluN1-GluN2B NMDA receptor in an ensemble of competitive antagonist-bound states, an agonist-bound form, and a state bound with agonists and the allosteric inhibitor Ro25-6981. Together with double electron-electron resonance experiments, we show how competitive antagonists rupture the ligand binding domain (LBD) gating "ring," how agonists retain the ring in a dimer-of-dimers configuration, and how allosteric inhibitors, acting within the amino terminal domain, further stabilize the LBD layer. These studies illuminate how the LBD gating ring is fundamental to signal transduction and gating in NMDARs.

Unfolding of a Temperature-Sensitive Domain Controls Voltage-Gated Channel Activation.

Voltage-gated ion channels (VGICs) are outfitted with diverse cytoplasmic domains that impact function. To examine how such elements may affect VGIC behavior, we addressed how the bacterial voltage-gated sodium channel (BacNa(V)) C-terminal cytoplasmic domain (CTD) affects function. Our studies show that the BacNa(V) CTD exerts a profound influence on gating through a temperature-dependent unfolding transition in a discrete cytoplasmic domain, the neck domain, proximal to the pore. Structural and functional studies establish that the BacNa(V) CTD comprises a bi-partite four-helix bundle that bears an unusual hydrophilic core whose integrity is central to the unfolding mechanism and that couples directly to the channel activation gate. Together, our findings define a general principle for how the widespread four-helix bundle cytoplasmic domain architecture can control VGIC responses, uncover a mechanism underlying the diverse BacNa(V) voltage dependencies, and demonstrate that a discrete domain can encode the temperature-dependent response of a channel.

Structure and dynamics of AMPA receptor GluA2 in resting, pre-open, and desensitized states.

Ionotropic glutamate receptors (iGluRs) mediate the majority of fast excitatory signaling in the nervous system. Despite the profound importance of iGluRs to neurotransmission, little is known about the structures and dynamics of intact receptors in distinct functional states. Here, we elucidate the structures of the intact GluA2 AMPA receptor in an apo resting/closed state, in an activated/pre-open state bound with partial agonists and a positive allosteric modulator, and in a desensitized/closed state in complex with fluorowilliardiine. To probe the conformational properties of these states, we carried out double electron-electron resonance experiments on cysteine mutants and cryoelectron microscopy studies. We show how agonist binding modulates the conformation of the ligand-binding domain "layer" of the intact receptors and how, upon desensitization, the receptor undergoes large conformational rearrangements of the amino-terminal and ligand-binding domains. We define mechanistic principles by which to understand antagonism, activation, and desensitization in AMPA iGluRs.

Integrated AlphaFold2 and DEER investigation of the conformational dynamics of a pH-dependent APC antiporter.

The Amino Acid-Polyamine-Organocation (APC) transporter GadC contributes to the survival of pathogenic bacteria under extreme acid stress by exchanging extracellular glutamate for intracellular γ-aminobutyric acid (GABA). Its structure, determined in an inward-facing conformation at alkaline pH, consists of the canonical LeuT-fold with a conserved five-helix inverted repeat, thereby resembling functionally divergent transporters such as the serotonin transporter SERT and the glucose-sodium symporter SGLT1. However, despite this structural similarity, it is unclear if the conformational dynamics of antiporters such as GadC follow the blueprint of these or other LeuT-fold transporters. Here, we used double electron-electron resonance (DEER) spectroscopy to monitor the conformational dynamics of GadC in lipid bilayers in response to acidification and substrate binding. To guide experimental design and facilitate the interpretation of the DEER data, we generated an ensemble of structural models in multiple conformations using a recently introduced modification of AlphaFold2 . Our experimental results reveal acid-induced conformational changes that dislodge the Cterminus from the permeation pathway coupled with rearrangement of helices that enables isomerization between inward- and outward-facing states. The substrate glutamate, but not GABA, modulates the dynamics of an extracellular thin gate without shifting the equilibrium between inward- and outward-facing conformations. In addition to introducing an integrated methodology for probing transporter conformational dynamics, the congruence of the DEER data with patterns of structural rearrangements deduced from ensembles of AlphaFold2 models illuminates the conformational cycle of GadC underpinning transport and exposes yet another example of the divergence between the dynamics of different families in the LeuT-fold.

Structural analysis of the P132L disease mutation in caveolin-1 reveals its role in the assembly of oligomeric complexes.

Caveolin-1 (CAV1) is a membrane-sculpting protein that oligomerizes to generate flask-shaped invaginations of the plasma membrane known as caveolae. Mutations in CAV1 have been linked to multiple diseases in humans. Such mutations often interfere with oligomerization and the intracellular trafficking processes required for successful caveolae assembly, but the molecular mechanisms underlying these defects have not been structurally explained. Here, we investigate how a disease-associated mutation in one of the most highly conserved residues in CAV1, P132L, affects CAV1 structure and oligomerization. We show that P132 is positioned at a major site of protomer-protomer interactions within the CAV1 complex, providing a structural explanation for why the mutant protein fails to homo-oligomerize correctly. Using a combination of computational, structural, biochemical, and cell biological approaches, we find that despite its homo-oligomerization defects P132L is capable of forming mixed hetero-oligomeric complexes with WT CAV1 and that these complexes can be incorporated into caveolae. These findings provide insights into the fundamental mechanisms that control the formation of homo- and hetero-oligomers of caveolins that are essential for caveolae biogenesis, as well as how these processes are disrupted in human disease.

Generation and characterization of a zebrafish knockout model of abcb4, a homolog of the human multidrug efflux transporter P-glycoprotein.

The ATP-binding cassette subfamily B member 1 (ABCB1), encoding a multidrug transporter referred to as P-glycoprotein (Pgp), plays a critical role in the efflux of xenobiotics in humans and is implicated in cancer resistance to chemotherapy. Therefore, developing high-throughput animal models to screen for Pgp function and bioavailability of substrates and inhibitors is paramount. Here, we generated and validated a zebrafish knockout line of abcb4, a human Pgp transporter homolog. CRISPR/Cas9 genome editing technology was deployed to generate a frameshift mutation in exon 4 of zebrafish abcb4. The zebrafish abcb4 homozygous mutant exhibited elevated accumulation of fluorescent rhodamine 123, a substrate of human Pgp, in the intestine and brain area of embryos. Moreover, abcb4 knockout embryos were sensitized toward toxic compounds such as doxorubicin and vinblastine compared to the WT zebrafish. Immunostaining for zebrafish Abcb4 colocalized in the endothelial brain cells of adult zebrafish. Transcriptome profiling using Gene Set Enrichment Analysis uncovered that the 'cell cycle process,' 'mitotic cell cycles,' and 'microtubule-based process' were significantly downregulated in the abcb4 knockout brain with age. This study establishes and validates the abcb4 knockout zebrafish as an animal model to study Pgp function in vivo. Unexpectedly it reveals a potentially novel role for zebrafish abcb4 in age-related changes in the brain. The zebrafish lines generated here will provide a platform to aid in the discovery of modulators of Pgp function as well as the characterization of human mutants thereof.

Asymmetric drug binding in an ATP-loaded inward-facing state of an ABC transporter.

Substrate efflux by ATP-binding cassette (ABC) transporters, which play a major role in multidrug resistance, entails the ATP-powered interconversion between transporter intermediates. Despite recent progress in structure elucidation, a number of intermediates have yet to be visualized and mechanistically interpreted. Here, we combine cryogenic-electron microscopy (cryo-EM), double electron-electron resonance spectroscopy and molecular dynamics simulations to profile a previously unobserved intermediate of BmrCD, a heterodimeric multidrug ABC exporter from Bacillus subtilis. In our cryo-EM structure, ATP-bound BmrCD adopts an inward-facing architecture featuring two molecules of the substrate Hoechst-33342 in a striking asymmetric head-to-tail arrangement. Deletion of the extracellular domain capping the substrate-binding chamber or mutation of Hoechst-coordinating residues abrogates cooperative stimulation of ATP hydrolysis. Together, our findings support a mechanistic role for symmetry mismatch between the nucleotide binding and the transmembrane domains in the conformational cycle of ABC transporters and is of notable importance for rational design of molecules for targeted ABC transporter inhibition.

Mechanism of NMDA receptor channel block by MK-801 and memantine.

The NMDA (N-methyl-D-aspartate) receptor transduces the binding of glutamate and glycine, coupling it to the opening of a calcium-permeable ion channel 1 . Owing to the lack of high-resolution structural studies of the NMDA receptor, the mechanism by which ion-channel blockers occlude ion permeation is not well understood. Here we show that removal of the amino-terminal domains from the GluN1-GluN2B NMDA receptor yields a functional receptor and crystals with good diffraction properties, allowing us to map the binding site of the NMDA receptor blocker, MK-801. This crystal structure, together with long-timescale molecular dynamics simulations, shows how MK-801 and memantine (a drug approved for the treatment of Alzheimer's disease) bind within the vestibule of the ion channel, promote closure of the ion channel gate and lodge between the M3-helix-bundle crossing and the M2-pore loops, physically blocking ion permeation.

Template-free prediction of a new monotopic membrane protein fold and assembly by AlphaFold2.

AlphaFold2 (AF2) has revolutionized the field of protein structural prediction. Here, we test its ability to predict the tertiary and quaternary structure of a previously undescribed scaffold with new folds and unusual architecture, the monotopic membrane protein caveolin-1 (CAV1). CAV1 assembles into a disc-shaped oligomer composed of 11 symmetrically arranged protomers, each assuming an identical new fold, and contains the largest parallel ß-barrel known to exist in nature. Remarkably, AF2 predicts both the fold of the protomers and the interfaces between them. It also assembles between seven and 15 copies of CAV1 into disc-shaped complexes. However, the predicted multimers are energetically strained, especially the parallel ß-barrel. These findings highlight the ability of AF2 to correctly predict new protein folds and oligomeric assemblies at a granular level while missing some elements of higher-order complexes, thus positing a new direction for the continued development of deep-learning protein structure prediction approaches.

Biophysical and functional properties of purified glucose-6-phosphatase catalytic subunit 1.

Glucose-6-phosphatase catalytic subunit 1 (G6PC1) plays a critical role in hepatic glucose production during fasting by mediating the terminal step of the gluconeogenesis and glycogenolysis pathways. In concert with accessory transport proteins, this membrane-integrated enzyme catalyzes glucose production from glucose-6-phosphate (G6P) to support blood glucose homeostasis. Consistent with its metabolic function, dysregulation of G6PC1 gene expression contributes to diabetes, and mutations that impair phosphohydrolase activity form the clinical basis of glycogen storage disease type 1a. Despite its relevance to health and disease, a comprehensive view of G6PC1 structure and mechanism has been limited by the absence of expression and purification strategies that isolate the enzyme in a functional form. In this report, we apply a suite of biophysical and biochemical tools to fingerprint the in vitro attributes of catalytically active G6PC1 solubilized in lauryl maltose neopentyl glycol (LMNG) detergent micelles. When purified from Sf9 insect cell membranes, the glycosylated mouse ortholog (mG6PC1) recapitulated functional properties observed previously in intact hepatic microsomes and displayed the highest specific activity reported to date. Additionally, our results establish a direct correlation between the catalytic and structural stability of mG6PC1, which is underscored by the enhanced thermostability conferred by phosphatidylcholine and the cholesterol analog cholesteryl hemisuccinate. In contrast, the N96A variant, which blocks N-linked glycosylation, reduced thermostability. The methodologies described here overcome long-standing obstacles in the field and lay the necessary groundwork for a detailed analysis of the mechanistic structural biology of G6PC1 and its role in complex metabolic disorders.

Nonsynonymous single-nucleotide polymorphisms in the G6PC2 gene affect protein expression, enzyme activity, and fasting blood glucose.

G6PC2 encodes a glucose-6-phosphatase (G6Pase) catalytic subunit that modulates the sensitivity of insulin secretion to glucose and thereby regulates fasting blood glucose (FBG). A common single-nucleotide polymorphism (SNP) in G6PC2, rs560887 is an important determinant of human FBG variability. This SNP has a subtle effect on G6PC2 RNA splicing, which raises the question as to whether nonsynonymous SNPs with a major impact on G6PC2 stability or enzyme activity might have a broader disease/metabolic impact. Previous attempts to characterize such SNPs were limited by the very low inherent G6Pase activity and expression of G6PC2 protein in islet-derived cell lines. In this study, we describe the use of a plasmid vector that confers high G6PC2 protein expression in islet cells, allowing for a functional analysis of 22 nonsynonymous G6PC2 SNPs, 19 of which alter amino acids that are conserved in mouse G6PC2 and the human and mouse variants of the related G6PC1 isoform. We show that 16 of these SNPs markedly impair G6PC2 protein expression (>50% decrease). These SNPs have variable effects on the stability of human and mouse G6PC1, despite the high sequence homology between these isoforms. Four of the remaining six SNPs impaired G6PC2 enzyme activity. Electronic health record-derived phenotype analyses showed an association between high-impact SNPs and FBG, but not other diseases/metabolites. While homozygous G6pc2 deletion in mice increases the risk of hypoglycemia, these human data reveal no evidence that the beneficial use of partial G6PC2 inhibitors to lower FBG would be associated with unintended negative consequences.

Cucurbit[7]uril Enhances Distance Measurements of Spin-Labeled Proteins.

We report complex formation between the chloroacetamide 2,6-diazaadamantane nitroxide radical (ClA-DZD) and cucurbit[7]uril (CB-7), for which the association constant in water, Ka = 1.9 × 106 M-1, is at least 1 order of magnitude higher than the previously studied organic radicals. The radical is highly immobilized by CB-7, as indicated by the increase in the rotational correlation time, τrot, by a factor of 36, relative to that in the buffer solution. The X-ray structure of ClA-DZD@CB-7 shows the encapsulated DZD guest inside the undistorted CB-7 host, with the pendant group protruding outside. Upon addition of CB-7 to T4 Lysozyme (T4L) doubly spin-labeled with the iodoacetamide derivative of DZD, we observe the increase in τrot and electron spin coherence time, Tm, along with the narrowing of interspin distance distributions. Sensitivity of the DEER measurements at 83 K increases by a factor 4-9, compared to the common spin label such as MTSL, which is not affected by CB-7. Interspin distances of 3 nm could be reliably measured in water/glycerol up to temperatures near the glass transition/melting temperature of the matrix at 200 K, thus bringing us closer to the goal of supramolecular recognition-enabled long-distance DEER measurements at near physiological temperatures. The X-ray structure of DZD-T4L 65 at 1.12 Å resolution allows for unambiguous modeling of the DZD label (0.88 occupancy), indicating an undisturbed structure and conformation of the protein.

SPEACH_AF: Sampling protein ensembles and conformational heterogeneity with Alphafold2.

The unprecedented performance of Deepmind's Alphafold2 in predicting protein structure in CASP XIV and the creation of a database of structures for multiple proteomes and protein sequence repositories is reshaping structural biology. However, because this database returns a single structure, it brought into question Alphafold's ability to capture the intrinsic conformational flexibility of proteins. Here we present a general approach to drive Alphafold2 to model alternate protein conformations through simple manipulation of the multiple sequence alignment via in silico mutagenesis. The approach is grounded in the hypothesis that the multiple sequence alignment must also encode for protein structural heterogeneity, thus its rational manipulation will enable Alphafold2 to sample alternate conformations. A systematic modeling pipeline is benchmarked against canonical examples of protein conformational flexibility and applied to interrogate the conformational landscape of membrane proteins. This work broadens the applicability of Alphafold2 by generating multiple protein conformations to be tested biologically, biochemically, biophysically, and for use in structure-based drug design.

Energy transduction and alternating access of the mammalian ABC transporter P-glycoprotein.

ATP binding cassette (ABC) transporters of the exporter class harness the energy of ATP hydrolysis in the nucleotide-binding domains (NBDs) to power the energetically uphill efflux of substrates by a dedicated transmembrane domain (TMD). Although numerous investigations have described the mechanism of ATP hydrolysis and defined the architecture of ABC exporters, a detailed structural dynamic understanding of the transduction of ATP energy to the work of substrate translocation remains elusive. Here we used double electron-electron resonance and molecular dynamics simulations to describe the ATP- and substrate-coupled conformational cycle of the mouse ABC efflux transporter P-glycoprotein (Pgp; also known as ABCB1), which has a central role in the clearance of xenobiotics and in cancer resistance to chemotherapy. Pairs of spin labels were introduced at residues selected to track the putative inward-facing to outward-facing transition. Our findings illuminate how ATP energy is harnessed in the NBDs in a two-stroke cycle and elucidate the consequent conformational motion that reconfigures the TMD, two critical aspects of Pgp transport mechanism. Along with a fully atomistic model of the outward-facing conformation in membranes, the insight into Pgp conformational dynamics harmonizes mechanistic and structural data into a novel perspective on ATP-coupled transport and reveals mechanistic divergence within the efflux class of ABC transporters.

Sequence and structural determinants of ligand-dependent alternating access of a MATE transporter.

Multidrug and toxic compound extrusion (MATE) transporters are ubiquitous ion-coupled antiporters that extrude structurally and chemically dissimilar cytotoxic compounds and have been implicated in conferring multidrug resistance. Here, we integrate double electron-electron resonance (DEER) with functional assays and site-directed mutagenesis of conserved residues to illuminate principles of ligand-dependent alternating access of PfMATE, a proton-coupled MATE from the hyperthermophilic archaeon Pyrococcus furiosus Pairs of spin labels monitoring the two sides of the transporter reconstituted into nanodiscs reveal large-amplitude movement of helices that alter the orientation of a putative substrate binding cavity. We found that acidic pH favors formation of an inward-facing (IF) conformation, whereas elevated pH (>7) and the substrate rhodamine 6G stabilizes an outward-facing (OF) conformation. The lipid-dependent PfMATE isomerization between OF and IF conformation is driven by protonation of a previously unidentified intracellular glutamate residue that is critical for drug resistance. Our results can be framed in a mechanistic model of transport that addresses central aspects of ligand coupling and alternating access.

Conserved binding site in the N-lobe of prokaryotic MATE transporters suggests a role for Na+ in ion-coupled drug efflux.

In both prokaryotes and eukaryotes, multidrug and toxic-compound extrusion (MATE) transporters catalyze the efflux of a broad range of cytotoxic compounds, including human-made antibiotics and anticancer drugs. MATEs are secondary-active antiporters, i.e. their drug-efflux activity is coupled to, and powered by, the uptake of ions down a pre-existing transmembrane electrochemical gradient. Key aspects of this mechanism, however, remain to be delineated, such as its ion specificity and stoichiometry. We previously revealed the existence of a Na+-binding site in a MATE transporter from Pyroccocus furiosus (PfMATE) and hypothesized that this site might be broadly conserved among prokaryotic MATEs. Here, we evaluate this hypothesis by analyzing VcmN and ClbM, which along with PfMATE are the only three prokaryotic MATEs whose molecular structures have been determined at resolutions better than 3 Å. Analysis of available crystallographic data and molecular dynamics simulations indeed reveal an occupied Na+-binding site in the N-terminal lobe of both structures, analogous to that identified in PfMATE. We likewise find this site to be strongly selective against K+, suggesting it is mechanistically significant. Consistent with these computational results, DEER spectroscopy measurements for multiple doubly-spin-labeled VcmN constructs demonstrate Na+-dependent changes in protein conformation. The existence of this binding site in three MATE orthologs implicates Na+ in the ion-coupled drug-efflux mechanisms of this class of transporters. These results also imply that observations of H+-dependent activity stem either from a site elsewhere in the structure, or from H+ displacing Na+ under certain laboratory conditions, as has been noted for other Na+-driven transport systems.

Conserved binding site in the N-lobe of prokaryotic MATE transporters suggests a role for Na+ in ion-coupled drug efflux.

In both prokaryotes and eukaryotes, multidrug and toxic-compound extrusion (MATE) transporters catalyze the efflux of a broad range of cytotoxic compounds, including human-made antibiotics and anticancer drugs. MATEs are secondary-active antiporters, i.e., their drug-efflux activity is coupled to, and powered by, the uptake of ions down a preexisting transmembrane electrochemical gradient. Key aspects of this mechanism, however, remain to be delineated, such as its ion specificity and stoichiometry. We previously revealed the existence of a Na+-binding site in a MATE transporter from Pyroccocus furiosus (PfMATE) and hypothesized that this site might be broadly conserved among prokaryotic MATEs. Here, we evaluate this hypothesis by analyzing VcmN and ClbM, which along with PfMATE are the only three prokaryotic MATEs whose molecular structures have been determined at atomic resolution, i.e. better than 3 Å. Reinterpretation of existing crystallographic data and molecular dynamics simulations indeed reveal an occupied Na+-binding site in the N-terminal lobe of both structures, analogous to that identified in PfMATE. We likewise find this site to be strongly selective against K+, suggesting it is mechanistically significant. Consistent with these computational results, DEER spectroscopy measurements for multiple doubly-spin-labeled VcmN constructs demonstrate Na+-dependent changes in protein conformation. The existence of this binding site in three MATE orthologs implicates Na+ in the ion-coupled drug-efflux mechanisms of this class of transporters. These results also imply that observations of H+-dependent activity likely stem either from a site elsewhere in the structure, or from H+ displacing Na+ under certain laboratory conditions, as has been noted for other Na+-driven transport systems.

Methodology for rigorous modeling of protein conformational changes by Rosetta using DEER distance restraints.

We describe an approach for integrating distance restraints from Double Electron-Electron Resonance (DEER) spectroscopy into Rosetta with the purpose of modeling alternative protein conformations from an initial experimental structure. Fundamental to this approach is a multilateration algorithm that harnesses sets of interconnected spin label pairs to identify optimal rotamer ensembles at each residue that fit the DEER decay in the time domain. Benchmarked relative to data analysis packages, the algorithm yields comparable distance distributions with the advantage that fitting the DEER decay and rotamer ensemble optimization are coupled. We demonstrate this approach by modeling the protonation-dependent transition of the multidrug transporter PfMATE to an inward facing conformation with a deviation to the experimental structure of less than 2Å Cα RMSD. By decreasing spin label rotamer entropy, this approach engenders more accurate Rosetta models that are also more closely clustered, thus setting the stage for more robust modeling of protein conformational changes.

Structural, functional, and behavioral insights of dopamine dysfunction revealed by a deletion in SLC6A3.

The human dopamine (DA) transporter (hDAT) mediates clearance of DA. Genetic variants in hDAT have been associated with DA dysfunction, a complication associated with several brain disorders, including autism spectrum disorder (ASD). Here, we investigated the structural and behavioral bases of an ASD-associated in-frame deletion in hDAT at N336 (∆N336). We uncovered that the deletion promoted a previously unobserved conformation of the intracellular gate of the transporter, likely representing the rate-limiting step of the transport process. It is defined by a "half-open and inward-facing" state (HOIF) of the intracellular gate that is stabilized by a network of interactions conserved phylogenetically, as we demonstrated in hDAT by Rosetta molecular modeling and fine-grained simulations, as well as in its bacterial homolog leucine transporter by electron paramagnetic resonance analysis and X-ray crystallography. The stabilization of the HOIF state is associated both with DA dysfunctions demonstrated in isolated brains of Drosophila melanogaster expressing hDAT ∆N336 and with abnormal behaviors observed at high-time resolution. These flies display increased fear, impaired social interactions, and locomotion traits we associate with DA dysfunction and the HOIF state. Together, our results describe how a genetic variation causes DA dysfunction and abnormal behaviors by stabilizing a HOIF state of the transporter.

ATP-dependent interactions of a cargo protein with the transmembrane domain of a polypeptide processing and secretion ABC transporter.

Powered by the energy of ATP binding and hydrolysis, protease-containing ABC transporters (PCATs) export amphipathic and hydrophilic bacteriocin and quorum-sensing proteins across the membrane hydrophobic barrier. The cargo proteins have N-terminal leader peptides that are cleaved off by the cysteine protease domain, referred to as the C39 domain, or referred to as the peptidase (PEP) domain. The sequence and structural determinants of the interaction between PCATs and cargo proteins are poorly understood, yet this interaction is a central aspect of the transport mechanism. Here, we demonstrate the ATP-dependent, equilibrium binding of the cargo protein to the transmembrane domain (TMD) of a PCAT subsequent to the removal of the leader peptide by the PEP domain. Binding of the cargo protein to PCAT1 variants devoid of the PEP domain is detected through changes in the spectroscopic properties of fluorescent or spin label. Moreover, we find similar energetics of binding regardless of the presence of the leader peptide, suggesting that although the PEP domain serves for recognition and orientation, interaction with the TMD is the main contributor to the affinity. These findings are in direct contradiction with a recent study claiming that the TMD does not interact with the cargo protein; rather acting as a "Teflon-like" conduit across the bilayer (Kieuvongngam, V., Olinares, P. D. B., Palillo, A., Oldham, M. L., Chait, B. T., and Chen, J. (2020) Structural basis of substrate recognition by a polypeptide processing and secretion transporter. eLife 9, e51492). A distinctive feature of the transport model emerging from our data invokes a stable complex between PCATs and their cargo proteins following processing of the leader peptide and prior to ATP-dependent alternating access that translocates the cargo protein to the extracellular side.