Biology subjects

Goldfarb, D.

Publications and source records attributed to Goldfarb, D..

At least 19 recordsLinked to original sources

bioRxiv2026

TMEM106B mediates ACE2-independent replication of the SARS-CoV-2 S-E484D variant in airway-derived cell models

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to cause significant respiratory disease, particularly in vulnerable populations. Although ACE2 is the primary receptor for viral entry, previous studies have identified a naturally occurring, ACE2-independent entry pathway in certain airway-derived cell lines. Utilization of this pathway depends on surface heparan sulfates and requires the E484D substitution within the receptor-binding domain of the viral Spike (S) protein. In this study, we expand the panel of airway-derived cell lines that support ACE2-independent, S-E484D-dependent replication and identify the host lysosomal transmembrane protein TMEM106B as a critical host factor for infection. Knockout of TMEM106B completely abolishes infection by SARS-CoV-2 SE484D in NCI-H522 and NCI-H661 cells. Moreover, ectopic expression of the luminal C-terminal domain (CTD) of TMEM106B-either alone or redirected to the plasma membrane - is sufficient to enable viral entry and infection in otherwise non-permissive cells. We further show that Fc-TMEM106B-CTD decoy protein blocks infection by SARS-CoV-2 SE484D, supporting a direct interaction between the S-E484D protein and TMEM106B-CTD. Finally, passaging experiments with a chimeric VSV-SARS-CoV-2 SE484D identify additional mutations within the heptad repeat 1 that enhance TMEM106B utilization and viral spread in the ACE2-independent cell models. Together, these findings demonstrate that TMEM106B is a key mediator of a naturally occurring ACE2-independent pathway in multiple airway-derived cells lines and suggest that variation in the Spike protein can expand receptor usage by SARS-CoV-2. IMPORTANCESevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to acquire mutations in the viral spike (S) protein as it circulates in humans. How these mutations influence the cell and tissue tropism of SARS-CoV-2 remains poorly understood. While ACE2 is the canonical host cell receptor for SARS-CoV-2, previous studies revealed the presence of a naturally occurring ACE2-independent entry pathway in multiple airway-derived cell lines that can be utilized only by a SARS-CoV-2 variant bearing the E484D substitution within the viral S protein. Here we expand the airway-derived cell line models that support ACE2-independent and S-E484D-dependent entry and show that the host lysosomal transmembrane protein, TMEM106B, is an essential viral entry factor. Although the E484D substitution is currently rare in human isolates, our findings raise the possibility that small mutations in SARS-CoV-2 S can allow the utilization of alternative entry pathways that broadens its cell and tissue tropism.

microbiology↗

bioRxiv2026

Pioneer and Altimeter: Fast Analysis of DIA Proteomics Data Optimized for Narrow Isolation Windows

Advances in protein mass spectrometry have enabled a single instrument to acquire data for hundreds of samples per day, but the speed of analysis software has not kept pace. Here we introduce Pioneer and Altimeter for rapid analysis of proteomics data acquired by data-independent acquisition (DIA). Altimeter generates in silico spectral libraries, which Pioneer uses to identify and quantify peptides and proteins. Pioneer exceeds peptide coverage by up to 10%, improves quantitative accuracy, and runs four to eight times faster compared with state-of-the-art tools. Pioneer and Altimeter also model isolation-window effects on fragment isotopes to take advantage of narrow isolation windows deployed on fast-scanning time-of-flight instruments. We benchmarked Pioneer and validated its false-discovery-rate and false-transfer-rate control across diverse instruments, sample types, and acquisition settings. Altimeter and Pioneer are open source and cross-platform, with a graphical interface for streamlined analysis on personal computers and a command-line interface for compute-cluster workflows.

bioinformatics↗

bioRxiv2024

Proximity proteomics provides a new resource for exploring the function of Afadin and the complexity of cell-cell adherens junctions

The network of proteins at the interface between cell-cell adherens junctions and the actomyosin cytoskeleton provides robust yet dynamic connections that facilitate cell shape change and motility. While this was initially thought to be a simple linear connection via classic cadherins and their associated catenins, we now have come to appreciate that many more proteins are involved, providing robustness and mechanosensitivity. Defining the full network of proteins in this network remains a key objective in our field. Proximity proteomics provides a means to define these networks. Mammalian Afadin and its Drosophila homolog Canoe are key parts of this protein network, facilitating diverse cell shape changes during gastrulation and other events of embryonic morphogenesis. Here we report results of several proximity proteomics screens, defining proteins in the neighborhood of both the N- and C-termini of mammalian Afadin in the premier epithelial model, MDCK cells. We compare our results with previous screens done in other cell types, and with proximity proteomics efforts with other junctional proteins. These reveal the value of multiple screens in defining the full network of neighbors and offer interesting insights into the overlap in protein composition between different epithelial cell junctions. Summary StatementAfadin BioID reveals new adherens junction proteins.

cell biology↗

bioRxiv2024

Design of linked-domain protein inhibitors of UBE2D as tools to study cellular ubiquitination

Ubiquitin (Ub) is a post-translational modification that largely controls proteostasis through mechanisms spanning transcription, translation, and notably, protein degradation. Ub conjugation occurs through a hierarchical cascade of three enzyme classes (E1, E2, and E3s) involving >1000 proteins that regulate the ubiquitination of proteins. The E2 Ub-conjugating enzymes are the midpoint, yet their cellular roles remain under-characterized, partly due to a lack of inhibitors. For example, the cellular roles of the promiscuous E2 UBE2D/UBCH5 are not well described. Here, we develop a highly selective, multivalent, engineered protein inhibitor for the UBE2D family that simultaneously targets the RING- and backside-binding sites. In HeLa cells, these inhibitors phenocopy knockdown of UBE2D by reducing the IC50 to cisplatin and whole-cell proteomics reveal an increased abundance of [~]20% of the identified proteins, consistent with reduced Ub degradation and proteotoxic stress. These precision tools will enable new studies probing UBE2Ds central role in proteome management.

biochemistry↗

bioRxiv2024

Insulin-Independent Regulation of Type 1 Diabetes via Brown Adipocyte-Secreted Proteins and the Novel Glucagon Regulator Nidogen-2.

Current treatments for type 1 diabetes (T1D) focus on insulin replacement. We demonstrate the therapeutic potential of a secreted protein fraction from embryonic brown adipose tissue (BAT), independent of insulin. The large molecular weight secreted fraction mediates insulin receptor-dependent recovery of euglycemia in a T1D animal model, nonobese diabetic (NOD) mice, by suppressing glucagon secretion. This fraction also promotes white adipocyte differentiation and browning, maintains healthy BAT, and enhances glucose uptake in adipose tissue, skeletal muscle, and liver. From this fraction, we identify nidogen-2 as a critical BAT-secreted protein that reverses hyperglycemia in NOD mice, inhibits glucagon secretion from pancreatic -cells, and mimics other actions of the entire secreted fraction. These findings confirm that BAT transplants affect physiology and demonstrate that BAT-secreted peptides represent a novel therapeutic approach to diabetes management. Furthermore, our research reveals a novel signaling role for nidogen-2, beyond its traditional classification as an extracellular matrix protein. HIGHLIGHTSO_LIThe large molecular weight brown adipocyte-secreted protein fraction suppresses glucagon secretion and normalizes glycemia in mouse models of type 1 diabetes (T1D), independent of insulin, offering a novel therapeutic strategy for disease management. C_LIO_LINidogen-2, a critical component of this fraction, is identified as an inhibitor of glucagon secretion in pancreatic -cells by regulating intracellular messenger activities. C_LIO_LIThe large-secreted protein fraction prevents T1D-related whitening of brown adipose tissue, promotes adipocyte differentiation, and enhances browning of inguinal white adipose tissue. C_LIO_LIThis fraction enhances glucose uptake in adipose tissue, skeletal muscle, and liver through an insulin receptor-dependent pathway. C_LI

cell biology↗

bioRxiv2024

A basally active cGAS-STING pathway limits SARS-CoV-2 replication in a subset of ACE2 positive airway cell models

Host factors that define the cellular tropism of SARS-CoV-2 beyond the cognate ACE2 receptor are poorly defined. Here we report that SARS-CoV-2 replication is restricted at a post-entry step in a number of ACE2-positive airway-derived cell lines due to tonic activation of the cGAS-STING pathway mediated by mitochondrial DNA leakage and naturally occurring cGAS and STING variants. Genetic and pharmacological inhibition of the cGAS-STING and type I/III IFN pathways as well as ACE2 overexpression overcome these blocks. SARS-CoV-2 replication in STING knockout cell lines and primary airway cultures induces ISG expression but only in uninfected bystander cells, demonstrating efficient antagonism of the type I/III IFN-pathway in productively infected cells. Pharmacological inhibition of STING in primary airway cells enhances SARS-CoV-2 replication and reduces virus-induced innate immune activation. Together, our study highlights that tonic activation of the cGAS-STING and IFN pathways can impact SARS-CoV-2 cellular tropism in a manner dependent on ACE2 expression levels.

microbiology↗

bioRxiv2023

Poly-basic peptides and polymers as new drug candidate against Plasmodium falciparum

Plasmodium falciparum, the malaria-causing parasite, is a leading cause of infection-induced deaths worldwide. The preferred treatment approach is artemisinin-combination therapy, which couples fast-acting artemisinin derivatives with longer-acting drugs like lumefantrine, mefloquine, and amodiaquine. However, the urgency for new treatments has risen due to the parasites growing resistance to existing therapies. Our study shows that a common characteristic of the P. falciparum proteome - stretches of poly-lysine residues such as those found in proteins related to adhesion and pathogenicity - can serve as an effective peptide treatment for infected erythrocytes. A single dose of these poly-basic peptides can successfully diminish parasitemia in human erythrocytes in vitro with minimal toxicity. The effectiveness of the treatment correlates with the length of the poly-lysine peptide, with 30 lysine peptides supporting the eradication of erythrocytic parasites within 72 hours. PEG-ylation of the poly-lysine peptides or utilizing poly-lysine dendrimers and polymers further increases parasite clearance efficiency and bolsters the stability of these potential new therapeutics. Lastly, our affinity pull-downs and mass-spectrometry identify P. falciparums outer membrane proteins as likely targets for polybasic peptide medications. Since poly-lysine dendrimers are already FDA-approved for drug delivery, their adaptation as antimalarial drugs presents a promising new therapeutic strategy. One-Sentence SummaryOur study demonstrates that poly-lysine peptides, particularly those modified through PEG-ylation or in the form of poly-lysine dendrimers, can effectively reduce Plasmodium falciparum, the causative agent of malaria, in human erythrocytes in vitro, with potential for use as a promising new antimalarial therapy.

microbiology↗

bioRxiv2023

Biophysical Mechanism of Allosteric Regulation of Actin Capping Protein

Actin capping protein (CP) can be regulated by steric and allosteric mechanisms. The molecular mechanism of the allosteric regulation at a biophysical level includes linkage between the binding sites for three ligands: F-actin, Capping-Protein-Interacting (CPI) motifs, and V-1/myotrophin, based on biochemical functional studies and solvent accessibility experiments. Here, we investigated the mechanism of allosteric regulation at the atomic level using single-molecule Forster resonance energy transfer (FRET) and molecular dynamics (MD) to assess the conformational and structural dynamics of CP in response to linked-binding site ligands. In the absence of ligand, both single-molecule FRET and MD revealed two distinct conformations of CP in solution; previous crystallographic studies revealed only one. CPI-motif peptide association induced conformational changes within CP that propagate in one direction, while V-1 association induced conformational changes in the opposite direction. Comparing CPI-motif peptides from different proteins, we identified variations in CP conformations and dynamics that are specific to each CPI motif. MD simulations for CP alone and in complex with a CPI motif and V-1 reveal atomistic details of the conformational changes. Analysis of the interaction of CP with wildtype (wt) and chimeric CPI-motif peptides using single-molecule FRET, isothermal calorimetry (ITC) and MD simulation indicated that conformational and affinity differences are intrinsic to the C-terminal portion of the CPI-motif. We conclude that allosteric regulation of CP involves changes in conformation that disseminate across the protein to link distinct binding-site functions. Our results provide novel insights into the biophysical mechanism of the allosteric regulation of CP.

biochemistry↗

bioRxiv2023

Targeted proteomic quantitation of NRF2 signaling and predictive biomarkers in HNSCC

The NFE2L2/NRF2 oncogene and transcription factor drives a gene expression program that promotes cancer progression, metabolic reprogramming, immune evasion and chemoradiation resistance. Patient stratification by NRF2 activity may guide treatment decisions to improve outcome. Here, we developed a mass spectrometry (MS)-based targeted proteomics assay based on internal standard triggered parallel reaction monitoring (IS-PRM) to quantify 69 NRF2 pathway components and targets as well as 21 proteins of broad clinical significance in head and neck squamous cell carcinoma (HNSCC). We improved the existing IS-PRM acquisition algorithm, called SureQuant, to increase throughput, sensitivity, and precision. Testing the optimized platform on 27 lung and upper aerodigestive cancer cell models revealed 35 NRF2 responsive proteins. In formalin-fixed paraffin-embedded (FFPE) HNSCCs, NRF2 signaling intensity positively correlated with NRF2 activating mutations and with SOX2 protein expression. PD-L2/CD273 and protein markers of T-cell infiltration correlated positively with one another and with human papilloma virus (HPV) infection status. p16/CDKN2A protein expression positively correlated with the HPV oncogenic E7 protein, and confirmed the presence of translationally active virus. This work establishes a clinically actionable HNSCC protein biomarker assay capable of quantifying over 600 peptides from frozen or FFPE archived tissues in under 90 minutes.

cancer biology↗

bioRxiv2022

Peptide-RNA Coacervates as a Cradle for the Evolution of Folded Domains

Peptide-RNA coacervates can result in the concentration and compartmentalization of simple biopolymers. Given their primordial relevance, peptide-RNA coacervates may have also been a key site of early protein evolution. However, the extent to which such coacervates might promote or suppress the exploration of novel peptide conformations is fundamentally unknown. To this end, we used electron paramagnetic resonance (EPR) spectroscopy to characterize the structure and dynamics of an ancient and ubiquitous nucleic acid binding element, the helix-hairpin-helix (HhH) motif, alone and in the presence of RNA, with which it forms coacervates. Double electron-electron resonance (DEER) spectroscopy applied to singly labeled peptides containing one HhH motif reveals the presence of dimers, even in the absence of RNA, and transient -helical character. Moreover, dimer formation is promoted upon RNA binding and was detectable within peptide-RNA coacervates. The distance distributions between spin labels are consistent with the symmetric (HhH)2-Fold, which is generated upon duplication and fusion of a single HhH motif and traditionally associated with dsDNA binding. These results support the hypothesis that coacervates are a unique testing ground for peptide oligomerization and that phase-separating peptides could have been a resource for the construction of complex protein structures via common evolutionary processes, such as duplication and fusion.

biophysics↗

bioRxiv2021

Nanotrap Particles Improve Nanopore Sequencing of SARS-CoV-2 and Other Respiratory Viruses

Presented here is a magnetic hydrogel particle enabled workflow for capturing and concentrating SARS-CoV-2 from diagnostic remnant swab samples that significantly improves sequencing results using the Oxford Nanopore Technologies MinION sequencing platform. Our approach utilizes a novel affinity-based magnetic hydrogel particle, circumventing low input sample volumes and allowing for both rapid manual and automated high throughput workflows that are compatible with nanopore sequencing. This approach enhances standard RNA extraction protocols, providing up to 40x improvements in viral mapped reads, and improves sequencing coverage by 20-80% from lower titer diagnostic remnant samples. Furthermore, we demonstrate that this approach works for contrived influenza virus and respiratory syncytial virus samples, suggesting that it can be used to identify and improve sequencing results of multiple viruses in VTM samples. These methods can be performed manually or on a KingFisher Apex system.

microbiology↗

bioRxiv2021

Protein proximity networks and functional evaluation of the Casein Kinase 1 γ family reveals unique roles for CK1γ3 in WNT signaling

The WNT/{beta}-catenin signaling pathway is evolutionarily conserved and controls normal embryonic development, adult tissue homeostasis and regeneration. Aberrant activation or suppression of WNT signaling contributes to cancer initiation and progression, developmental disorders, neurodegeneration, and bone disease. Despite great need and more than 40 years of research, targeted therapies for the WNT pathway have yet to be fully realized. Kinases are exceptionally druggable and occupy key nodes within the WNT signaling network, but several pathway-relevant kinases remain understudied and dark. Here we studied the function of the CSNK1{gamma} subfamily of human kinases. miniTurbo-based proximity biotinylation and mass spectrometry analysis of CSNK1{gamma}1, CSNK1{gamma}2, and CSNK1{gamma}3 revealed numerous established components of the {beta}-catenin- dependent and independent WNT signaling pathway, as well as novel interactors. In gain-of- function experiments leveraging a panel of transcriptional reporters, CSNK1{gamma}3 but not CSNK1{gamma}1 or CSNK1{gamma}2 activated {beta}-catenin-dependent WNT signaling and the Notch pathway. Within the family, CSNK1{gamma}3 expression uniquely induced LRP6 phosphorylation. Conversely, siRNA- mediated silencing of CSNK1{gamma}3 alone had no impact on WNT signaling, though co-silencing of all three family members decreased WNT pathway activity. We characterized two moderately selective and potent small molecule inhibitors of the CSNK1{gamma} family. These inhibitors and a CSNK1{gamma}3 kinase dead mutant suppressed but did not eliminate WNT-driven LRP6 phosphorylation and {beta}-catenin stabilization. Our data suggest that while CSNK1{gamma}3 expression uniquely drives pathway activity, potential functional redundancy within the family necessitates loss of all three family members to suppress the WNT signaling pathway.

cell biology↗

bioRxiv2021

Generation of chimeric respiratory viruses with altered tropism by coinfection of influenza A virus and respiratory syncytial virus

Interactions between respiratory viruses impact viral transmission dynamics and clinical outcomes. To identify and characterize virus-virus interactions at the cellular level, we coinfected human lung cells with influenza A virus (IAV) and respiratory syncytial virus (RSV). Super-resolution microscopy, live-cell imaging, scanning electron microscopy, and cryo-electron tomography revealed extracellular and membrane-associated filamentous structures consistent with hybrid viral particles (HVPs). We show that HVPs harbor surface glycoproteins and ribonucleoproteins of IAV and RSV, and use the RSV fusion glycoprotein to evade anti-IAV neutralising antibodies and to infect and spread among cells lacking IAV receptors. Finally, we show evidence of IAV and RSV coinfection within cells of the bronchial epithelium, with viral proteins from both viruses co-localising at the apical surface. Our observations have profound implications for infection biology as they define a previously unknown interaction between respiratory viruses that might affect virus pathogenesis by expanding virus tropism and facilitating immune evasion.

microbiology↗

bioRxiv2021

Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell

Established in vitro models for SARS-CoV-2 infection are limited and include cell lines of non-human origin and those engineered to overexpress ACE2, the cognate host cell receptor. We identified human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of ACE2. Infection of H522 cells required the SARS-CoV-2 spike protein, though in contrast to ACE2-dependent models, spike alone was not sufficient for H522 infection. Temporally resolved transcriptomic and proteomic profiling revealed alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type-I interferon signaling. Focused chemical screens point to important roles for clathrin-mediated endocytosis and endosomal cathepsins in SARS-CoV-2 infection of H522 cells. These findings imply the utilization of an alternative SARS-CoV-2 host cell receptor which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.

microbiology↗

bioRxiv2020

Elevated temperature inhibits SARS-CoV-2 replication in respiratory epithelium independently of the induction of IFN-mediated innate immune defences

The pandemic spread of SARS-CoV-2, the etiological agent of COVID-19, represents a significant and ongoing international health crisis. A key symptom of SARS-CoV-2 infection is the onset of fever, with a hyperthermic temperature range of 38 to 41{degrees}C. Fever is an evolutionarily conserved host response to microbial infection and inflammation that can influence the outcome of viral pathogenicity and regulation of host innate and adaptive immune responses. However, it remains to be determined what effect elevated temperature has on SARS-CoV-2 tropism and replication. Utilizing a 3D air-liquid interface (ALI) model that closely mimics the natural tissue physiology and cellular tropism of SARS-CoV-2 infection in the respiratory airway, we identify tissue temperature to play an important role in the regulation of SARS-CoV-2 infection. We show that temperature elevation induces wide-spread transcriptome changes that impact upon the regulation of multiple pathways, including epigenetic regulation and lncRNA expression, without disruption of general cellular transcription or the induction of interferon (IFN)-mediated antiviral immune defences. Respiratory tissue incubated at temperatures >37{degrees}C remained permissive to SARS-CoV-2 infection but severely restricted the initiation of viral transcription, leading to significantly reduced levels of intraepithelial viral RNA accumulation and apical shedding of infectious virus. To our knowledge, we present the first evidence that febrile temperatures associated with COVID-19 inhibit SARS-CoV-2 replication. Our data identify an important role for temperature elevation in the epithelial restriction of SARS-CoV-2 that occurs independently of the induction of canonical IFN-mediated antiviral immune defences and interferon-stimulated gene (ISG) expression.

microbiology↗

bioRxiv2020

The ubiquitylome of developing cortical neurons

TRIM9 and TRIM67 are neuronally-enriched E3 ubiquitin. Both genes are required for neuronal morphological responses to the axon guidance cue netrin-1. For example, our previously published work demonstrated that the actin polymerase VASP and the netrin receptor DCC exhibit TRIM9 dependent ubiquitylation that is lost upon netrin stimulation. Deletion of either gene in the mouse results in subtle neuroanatomical anomalies yet overt deficits in spatial learning and memory. Despite their role in neuronal form and function, the identity of few TRIM9 or TRIM67 substrates are known. Here we performed ubiquitin remnant profiling approach in cultured wildtype and knockout murine embryonic cortical neurons to identify ubiquitylated peptides and proteins, with the ultimate goal of identifying substrates of TRIM9 and TRIM67 that exhibited reduced ubiquitylation in the absence of the ligase. This work reveals the ubiquitylome of developing cortical neurons.

cell biology↗

bioRxiv2020

The TRIM9/TRIM67 neuronal interactome reveals novel activators of morphogenesis

TRIM9 and TRIM67 are neuronally-enriched E3 ubiquitin ligases essential for appropriate morphogenesis of cortical and hippocampal neurons and fidelitous responses to the axon guidance cue netrin-1. Deletion of murine Trim9 or Trim67 results in neuroanatomical defects and striking behavioral deficits, particularly in spatial learning and memory. TRIM9 and TRIM67 interact with cytoskeletal and exocytic proteins, but the full interactome is not known. Here we performed the unbiased proximity-dependent biotin identification (BioID) approach to define TRIM9 and TRIM67 protein-protein proximity network in developing cortical neurons and identified neuronal putative TRIM interaction partners. Candidates included cytoskeletal regulators, cytosolic protein transporters, exocytosis and endocytosis regulators, and proteins necessary for synaptic regulation. A subset of high priority candidates was validated, including Myo16, Coro1A, SNAP47, ExoC1, GRIP1, PRG-1, and KIF1A. For a subset of validated candidates, we utilized TIRF microscopy to demonstrate dynamic colocalization with TRIM proteins at the axonal periphery, including at the tips of filopodia. Further analysis demonstrated the RNAi-based knockdown of the unconventional myosin Myo16 in cortical neurons altered axonal branching patterns in a TRIM9 and netrin-1 dependent manner. Future analysis of other validated candidates will likely identify novel proteins and mechanisms by which TRIM9 and TRIM67 regulate neuronal form and function.

cell biology↗

bioRxiv2020

Drug binding sites in the multidrug transporter MdfA in detergent solution and in lipid nanodiscs

MdfA, a member of the major facilitator superfamily (MFS), is a multidrug/proton antiporter from E. coli that has been considered a model for secondary multidrug (Mdr) transporters. Its transport mechanism, driven by a proton gradient, is associated with conformational changes, which accompany the recruitment of drugs and their release. In this work, we applied double-electron electron resonance (DEER) spectroscopy to locate the binding site of one of its substrates, tetraphenylphosphonium (TPP) within available crystal structures. We carried out Gd(III)-nitroxide distance measurements between MdfA labeled with a Gd(III) tag and the TPP analog mito-TEMPO (bearing the nitroxide moiety). Data were obtained both for MdfA solubilized in detergent micelles (n-dodecyl-{beta}-D-maltopyranoside (DDM)), and reconstituted into lipid nanodiscs (ND). For both DDM and ND, the average position of the substrate at a neutral pH was found to be close to the ligand position in the If (inward facing) crystal structure, with the DDM environment exhibiting a somewhat better agreement than the ND environment. We therefore conclude that the If structure provides a good description for substrate-bound MdfA in DDM solution, while in ND the structure is slightly modified. A second binding site was found for the ND sample situated at the cytoplasmic side, towards the end of transmembrane helix 7 (TM7). In addition, we used DEER distance measurements on Gd(III) doubly labeled MdfA to track conformational changes within the periplasmic and cytoplasmic sides associated with substrate binding. We detected significant differences in the periplasmic side of MdfA, with the ND featuring a more closed conformation than in DDM, in agreement with earlier reports. The addition of TPP led to a noticeable conformational change in the periplasmic face in ND, attributed to a movement of TM10. This change was not observed in DDM. Statement of SignificanceMdfA is multidrug transporter from E. coli, which exhibits multidrug efflux activities with an unusually broad spectrum of drug specificities. While it has been established that solute transport by similar transporters is coupled to significant conformational changes, previous studies raised the possibility that this is not the case for MdfA. Moreover, it is not clear how MdfA functionally accommodates chemically dissimilar substrates. Towards resolving these open questions, we used double-electron electron resonance distance measurements to determine the binding site of a spin labeled drug analog within available crystal structures of MdfA and to examine how MdfA responds conformationally to drug binding. Moreover, we explored how these two are affected by the media, detergent micelles vs lipid nanodiscs.

biophysics↗