Structural basis for adhesin secretion by the outer-membrane usher in type 1 pili.

Gram-negative bacteria produce chaperone-usher pathway pili, which are extracellular protein fibers tipped with an adhesive protein that binds to a receptor with stereochemical specificity to determine host and tissue tropism. The outer-membrane usher protein, together with a periplasmic chaperone, assembles thousands of pilin subunits into a highly ordered pilus fiber. The tip adhesin in complex with its cognate chaperone activates the usher to allow extrusion across the outer membrane. The structural requirements to translocate the adhesin through the usher pore from the periplasm to the extracellular space remains incompletely understood. Here, we present a cryoelectron microscopy structure of a quaternary tip complex in the type 1 pilus system from Escherichia coli, which consists of the usher FimD, chaperone FimC, adhesin FimH, and the tip adapter FimF. In this structure, the usher FimD is caught in the act of secreting its cognate adhesin FimH. Comparison with previous structures depicting the adhesin either first entering or having completely exited the usher pore reveals remarkable structural plasticity of the two-domain adhesin during translocation. Moreover, a piliation assay demonstrated that the structural plasticity, enabled by a flexible linker between the two domains, is a prerequisite for adhesin translocation through the usher pore and thus pilus biogenesis. Overall, this study provides molecular details of adhesin translocation across the outer membrane and elucidates a unique conformational state adopted by the adhesin during stepwise secretion through the usher pore. This study elucidates fundamental aspects of FimH and usher dynamics critical in urinary tract infections and is leading to antibiotic-sparing therapeutics.

A Widespread Radical-Mediated Glycolysis Pathway.

Glycyl radical enzymes (GREs) catalyze mechanistically diverse radical-mediated reactions, playing important roles in the metabolism of anaerobic bacteria. The model bacterium Escherichia coli MG1655 contains two GREs of unknown function, YbiW and PflD, which are widespread among human intestinal bacteria. Here, we report that YbiW and PflD catalyze ring-opening C-O cleavage of 1,5-anhydroglucitol-6-phosphate (AG6P) and 1,5-anhydromannitol-6-phosphate (AM6P), respectively. The product of both enzymes, 1-deoxy-fructose-6-phosphate (DF6P), is then cleaved by the aldolases FsaA or FsaB to form glyceraldehyde-3-phosphate (G3P) and hydroxyacetone (HA), which are then reduced by the NADH-dependent dehydrogenase GldA to form 1,2-propanediol (1,2-PDO). Crystal structures of YbiW and PflD in complex with their substrates provided insights into the mechanism of radical-mediated C-O cleavage. This "anhydroglycolysis" pathway enables anaerobic growth of E. coli on 1,5-anhydroglucitol (AG) and 1,5-anhydromannitol (AM), and we probe the feasibility of harnessing this pathway for the production of 1,2-PDO, a highly demanded chiral chemical feedstock, from inexpensive starch. Discovery of the anhydroglycolysis pathway expands the known catalytic repertoire of GREs, clarifies the hitherto unknown physiological functions of the well-studied enzymes FsaA, FsaB, and GldA, and demonstrates how enzyme discovery efforts can cast light on prevalent yet overlooked metabolites in the microbiome.

Structural insights into polyamine spermidine uptake by the ABC transporter PotD-PotABC.

Polyamines, characterized by their polycationic nature, are ubiquitously present in all organisms and play numerous cellular functions. Among polyamines, spermidine stands out as the predominant type in both prokaryotic and eukaryotic cells. The PotD-PotABC protein complex in Escherichia coli, belonging to the adenosine triphosphate-binding cassette transporter family, is a spermidine-preferential uptake system. Here, we report structural details of the polyamine uptake system PotD-PotABC in various states. Our analyses reveal distinct "inward-facing" and "outward-facing" conformations of the PotD-PotABC transporter, as well as conformational changes in the "gating" residues (F222, Y223, D226, and K241 in PotB; Y219 and K223 in PotC) controlling spermidine uptake. Therefore, our structural analysis provides insights into how the PotD-PotABC importer recognizes the substrate-binding protein PotD and elucidates molecular insights into the spermidine uptake mechanism of bacteria.

Dual client binding sites in the ATP-independent chaperone SurA.

The ATP-independent chaperone SurA protects unfolded outer membrane proteins (OMPs) from aggregation in the periplasm of Gram-negative bacteria, and delivers them to the ß-barrel assembly machinery (BAM) for folding into the outer membrane (OM). Precisely how SurA recognises and binds its different OMP clients remains unclear. Escherichia coli SurA comprises three domains: a core and two PPIase domains (P1 and P2). Here, by combining methyl-TROSY NMR, single-molecule Förster resonance energy transfer (smFRET), and bioinformatics analyses we show that SurA client binding is mediated by two binding hotspots in the core and P1 domains. These interactions are driven by aromatic-rich motifs in the client proteins, leading to SurA core/P1 domain rearrangements and expansion of clients from collapsed, non-native states. We demonstrate that the core domain is key to OMP expansion by SurA, and uncover a role for SurA PPIase domains in limiting the extent of expansion. The results reveal insights into SurA-OMP recognition and the mechanism of activation for an ATP-independent chaperone, and suggest a route to targeting the functions of a chaperone key to bacterial virulence and OM integrity.

CsgA gatekeeper residues control nucleation but not stability of functional amyloid.

Functional amyloids, beneficial to the organism producing them, are found throughout life, from bacteria to humans. While disease-related amyloids form by uncontrolled aggregation, the fibrillation of functional amyloid is regulated by complex cellular machinery and optimized sequences, including so-called gatekeeper residues such as Asp. However, the molecular basis for this regulation remains unclear. Here we investigate how the introduction of additional gatekeeper residues affects fibril formation and stability in the functional amyloid CsgA from E. coli. Step-wise introduction of additional Asp gatekeepers gradually eliminated fibrillation unless preformed fibrils were added, illustrating that gatekeepers mainly affect nucleus formation. Once formed, the mutant CsgA fibrils were just as stable as wild-type CsgA. HSQC NMR spectra confirmed that CsgA is intrinsically disordered, and that the introduction of gatekeeper residues does not alter this ensemble. NMR-based Dark-state Exchange Saturation Transfer (DEST) experiments on the different CsgA variants, however, show a decrease in transient interactions between monomeric states and the fibrils, highlighting a critical role for these interactions in the fibrillation process. We conclude that gatekeeper residues affect fibrillation kinetics without compromising structural integrity, making them useful and selective modulators of fibril properties.

Characterization of the N5-dimethylallyl-FMN Intermediate in the Biosynthesis of Prenylated-FMN Catalyzed by UbiX.

Prenylated-FMN (prFMN) is the cofactor used by the UbiD-like family of decarboxylases that catalyzes the decarboxylation of various aromatic and unsaturated carboxylic acids. prFMN is synthesized from reduced FMN and dimethylallyl phosphate (DMAP) by a specialized prenyl transferase, UbiX. UbiX catalyzes the sequential formation of two bonds, the first between N5 of the flavin and C1 of DMAP, and the second between C6 of the flavin and C3 of DMAP. We have examined the reaction of UbiX with both FMN and riboflavin. Although UbiX converts FMN to prFMN, we show that significant amounts of the N5-dimethylallyl-FMN intermediate are released from the enzyme during catalysis. With riboflavin as the substrate, UbiX catalyzes only a partial reaction, resulting in only N5-dimethylallyl-riboflavin being formed. Purification of the N5-dimethylallyl-FMN adduct allowed its structure to be verified by 1H NMR spectroscopy and its reactivity to be investigated. Surprisingly, whereas reduced prFMN oxidizes in seconds to form the stable prFMN semiquinone radical when exposed to air, N5-dimethylallyl-FMN oxidizes much more slowly over several hours; in this case, oxidation is accompanied by spontaneous hydrolysis to regenerate FMN. These studies highlight the important contribution that cyclization of the prenyl-derived ring of prFMN makes to the cofactor's biological activity.

Structure and mechanism of a phosphotransferase system glucose transporter.

Glucose is the primary source of energy for many organisms and is efficiently taken up by bacteria through a dedicated transport system that exhibits high specificity. In Escherichia coli, the glucose-specific transporter IICBGlc serves as the major glucose transporter and functions as a component of the phosphoenolpyruvate-dependent phosphotransferase system. Here, we report cryo-electron microscopy (cryo-EM) structures of the glucose-bound IICBGlc protein. The dimeric transporter embedded in lipid nanodiscs was captured in the occluded, inward- and occluded, outward-facing conformations. Together with biochemical and biophysical analyses, and molecular dynamics (MD) simulations, we provide insights into the molecular basis and dynamics for substrate recognition and binding, including the gates regulating the binding sites and their accessibility. By combination of these findings, we present a mechanism for glucose transport across the plasma membrane. Overall, this work provides molecular insights into the structure, dynamics, and mechanism of the IICBGlc transporter in a native-like lipid environment.

Outer membrane protein assembly mediated by BAM-SurA complexes.

The outer membrane is a formidable barrier that protects Gram-negative bacteria against environmental threats. Its integrity requires the correct folding and insertion of outer membrane proteins (OMPs) by the membrane-embedded ß-barrel assembly machinery (BAM). Unfolded OMPs are delivered to BAM by the periplasmic chaperone SurA, but how SurA and BAM work together to ensure successful OMP delivery and folding remains unclear. Here, guided by AlphaFold2 models, we use disulphide bond engineering in an attempt to trap SurA in the act of OMP delivery to BAM, and solve cryoEM structures of a series of complexes. The results suggest that SurA binds BAM at its soluble POTRA-1 domain, which may trigger conformational changes in both BAM and SurA that enable transfer of the unfolded OMP to the BAM lateral gate for insertion into the outer membrane. Mutations that disrupt the interaction between BAM and SurA result in outer membrane assembly defects, supporting the key role of SurA in outer membrane biogenesis.

Structure-Based Design and Development of Phosphine Oxides as a Novel Chemotype for Antibiotics that Dysregulate Bacterial ClpP Proteases.

A series of arylsulfones and heteroarylsulfones have previously been demonstrated to dysregulate the conserved bacterial ClpP protease, causing the unspecific degradation of essential cellular housekeeping proteins and ultimately resulting in cell death. A cocrystal structure of a 2-ß-sulfonylamide analog, ACP1-06, with Escherichia coli ClpP showed that its 2-pyridyl sulfonyl substituent adopts two orientations in the binding site related through a sulfone bond rotation. From this, a new bis-aryl phosphine oxide scaffold, designated as ACP6, was designed based on a "conformation merging" approach of the dual orientation of the ACP1-06 sulfone. One analog, ACP6-12, exhibited over a 10-fold increase in activity over the parent ACP1-06 compound, and a cocrystal X-ray structure with ClpP confirmed its predicted binding conformation. This allowed for a comparative analysis of how different ligand classes bind to the hydrophobic binding site. The study highlights the successful application of structure-based rational design of novel phosphine oxide-based antibiotics.

The Assembly of the Inverse Autotransporter Protein YeeJ is Driven by its C-terminal ß-strand.

Autotransporter proteins are bacterial outer membrane proteins that display passenger domains with various functions through a ß-barrel shaped translocation domain. YeeJ is an autotransporter protein from E. coli MG1655. In contrast to most other autotransporter proteins, its passenger domain is located at the C-terminus of the translocation domain. Due to this inverted domain organization, YeeJ belongs to autotransporter proteins of type Ve. To investigate the assembly of YeeJ, the fluorescence of a heterologous mCherry passenger domain was measured to quantify its assembly. Based on AlphaFold2 models of 119 sequences similar to YeeJ, a sequence conservation logo for the ß1- and the ß12-strand of type Ve autotransporter proteins was generated. Then, the effect of mutations in these strands on the assembly of YeeJ were analyzed. Mutations of the N-terminal aromatic amino acid of the ß1-strand did not affect the assembly of the translocation domain and the display of the passenger domain. Likewise, exchange of the ß1-strand with the ß3-strand did not impair the assembly of the autotransporter fusion protein. Mutation of the C-terminal aromatic amino acid of the ß12-strand strongly impaired surface display of the mCherry passenger domain. This amino acid has been shown before as an essential feature of the ß-signals of classical autotransporter proteins and outer membrane ß-barrel proteins in general. We therefore propose that the ß12-strand of YeeJ acts as its ß-signal and that the assembly of the YeeJ ß-barrel is driven by its C-terminal ß-strand, like in most other autotransporter proteins, despite its inverted domain organization.

Structural insights into RNA cleavage by a novel family of bacterial RNases.

Processing of RNA is a key regulatory mechanism for all living systems. Escherichia coli protein YicC belongs to the well-conserved YicC family and has been identified as a novel ribonuclease. Here, we report a 2.8-Å-resolution crystal structure of the E. coli YicC apo protein and a 3.2-Å-cryo-EM structure of YicC bound to an RNA substrate. The apo YicC forms a dimer of trimers with a large open channel. In the RNA-bound form, the top trimer of YicC rotates nearly 70° and closes the RNA substrate inside the cavity to form a clamshell-pearl conformation that resembles no other known RNases. The structural information combined with mass spectrometry and biochemical data identified cleavage on the upstream side of an RNA hairpin. Mutagenesis studies demonstrated that the previously uncharacterized domain, DUF1732, is critical in both RNA binding and catalysis. These studies shed light on the mechanism of the previously unexplored YicC RNase family.

Conservation of kinetic stability, but not the unfolding mechanism, between human transthyretin and a transthyretin-related enzyme.

Kinetic stability is thought to be an attribute of proteins that require a long lifetime, such as the transporter of thyroxine and holo retinol-binding protein or transthyretin (TTR) functioning in the bloodstream, cerebrospinal fluid, and vitreous humor. TTR evolved from ancestral enzymes known as TTR-related proteins (TRPs). Here, we develop a rate-expansion approach that allows unfolding rates to be measured directly at low denaturant concentration, revealing that kinetic stability exists in the Escherichia coli TRP (EcTRP), even though the enzyme structure is more energetically frustrated and has a more mutation-sensitive folding mechanism than human TTR. Thus, the ancient tetrameric enzyme may already have been poised to mutate into a kinetically stable human transporter. An extensive mutational study that exchanges residues at key sites within the TTR and EcTRP dimer-dimer interface shows that tyrosine 111, replaced by a threonine in TTR, is the gatekeeper of frustration in EcTRP because it is critical for function. Frustration, virtually absent in TTR, occurs at multiple sites in EcTRP and even cooperatively for certain pairs of mutations. We present evidence that evolution at the C terminus of TTR was a compensatory event to maintain the preexisting kinetic stability while reducing frustration and sensitivity to mutation. We propose an "overcompensation" pathway from EcTRPs to functional hybrids to modern TTRs that is consistent with the biophysics discussed here. An alternative plausible pathway is also presented.

The importance of the location of the N-terminus in successful protein folding in vivo and in vitro.

Protein folding in the cell often begins during translation. Many proteins fold more efficiently cotranslationally than when refolding from a denatured state. Changing the vectorial synthesis of the polypeptide chain through circular permutation could impact functional, soluble protein expression and interactions with cellular proteostasis factors. Here, we measure the solubility and function of every possible circular permutant (CP) of HaloTag in Escherichia coli cell lysate using a gel-based assay, and in living E. coli cells via FACS-seq. We find that 78% of HaloTag CPs retain protein function, though a subset of these proteins are also highly aggregation-prone. We examine the function of each CP in E. coli cells lacking the cotranslational chaperone trigger factor and the intracellular protease Lon and find no significant changes in function as a result of modifying the cellular proteostasis network. Finally, we biophysically characterize two topologically interesting CPs in vitro via circular dichroism and hydrogen-deuterium exchange coupled with mass spectrometry to reveal changes in global stability and folding kinetics with circular permutation. For CP33, we identify a change in the refolding intermediate as compared to wild-type (WT) HaloTag. Finally, we show that the strongest predictor of aggregation-prone expression in cells is the introduction of termini within the refolding intermediate. These results, in addition to our finding that termini insertion within the conformationally restrained core is most disruptive to protein function, indicate that successful folding of circular permutants may depend more on changes in folding pathway and termini insertion in flexible regions than on the availability of proteostasis factors.

Genetically engineered bacterial biofilm materials enhances portable whole cell sensing.

In recent years, whole-cell biosensors (WCBs) have emerged as a potent approach for environmental monitoring and on-site analyte detection. These biosensors harness the biological apparatus of microorganisms to identify specific analytes, offering advantages in sensitivity, specificity, and real-time monitoring capabilities. A critical hurdle in biosensor development lies in ensuring the robust attachment of cells to surfaces, a crucial step for practical utility. In this study, we present a comprehensive approach to tackle this challenge via engineering Escherichia coli cells for immobilization on paper through the Curli biofilm pathway. Furthermore, incorporating a cellulose-binding peptide domain to the CsgA biofilm protein enhances cell adhesion to paper surfaces, consequently boosting biosensor efficacy. To demonstrate the versatility of this platform, we developed a WCB for copper, optimized to exhibit a discernible response, even with the naked eye. To confirm its suitability for practical field use, we characterized our copper sensor under various environmental conditions-temperature, salinity, and pH-to mimic real-world scenarios. The biosensor-equipped paper discs can be freeze-dried for deployment in on-site applications, providing a practical method for long-term storage without loss of sensitivity paper discs demonstrate sustained functionality and viability even after months of storage with 5 µM limit of detection for copper with visible-to-naked-eye signal levels. Biofilm-mediated surface attachment and analyte sensing can be independently engineered, allowing for flexible utilization of this platform as required. With the implementation of copper sensing as a proof-of-concept study, we underscore the potential of WCBs as a promising avenue for the on-site detection of a multitude of analytes.

Metadynamics Study of Lipid-Mediated Antibacterial Toxin Binding to the EmrE Multiefflux Protein.

EmrE is a bacterial efflux protein in the small multidrug-resistant (SMR) family present in Escherichia coli. Due to its small size, 110 residues in each dimer subunit, it is an ideal model system to study ligand-protein-membrane interactions. Here in our work, we have calculated the free energy landscape of benzyltrimetylammonium (BTMA) and tetraphenyl phosphonium (TPP) binding to EmrE using the enhanced sampling method-multiple walker metadynamics. We estimate that the free energy of BTMA binding to EmrE is -21.2 ± 3.3 kJ/mol and for TPP is -43.6 ± 3.8 kJ/mol. BTMA passes through two metastable states to reach the binding pocket, while TPP has a more complex binding landscape with four metastable states and one main binding site. Our simulations show that the ligands interact with the membrane lipids at a distance 1 nm away from the binding site which forms a broad local minimum, consistent for both BTMA and TPP. This site can be an alternate entry point for ligands to partition from the membrane into the protein, especially for bulky and/or branched ligands. We also observed the membrane lipid and C-terminal 110HisA form salt-bridge interactions with the helix-1 residue 22LysB. Our free energy estimates and clusters are in close agreement with experimental data and give us an atomistic view of the ligand-protein-lipid interactions. Understanding the binding pathway of these ligands can guide us in future design of ligands that can alter or halt the function of EmrE.

AlphaFold 2-based stacking model for protein solubility prediction and its transferability on seed storage proteins.

Accurate protein solubility prediction is crucial in screening suitable candidates for food application. Existing models often rely only on sequences, overlooking important structural details. In this study, a regression model for protein solubility was developed using both the sequences and predicted structures of 2983 E. coli proteins. The sequence and structural level properties of the proteins were bioinformatically extracted and subjected to multilayer perceptron (MLP). Moreover, residue level features and contact maps were utilized to construct a graph convolutional network (GCN). The out-of-fold predictions of the two models were combined and fed into multiple meta-regressors to create a stacking model. The stacking model with support vector regressor (SVR) achieved R2 of 0.502 and 0.468 on test and external validation datasets, respectively, displaying higher performance compared to existing regression models. Based on the improved performance compared to its based models, the stacking model effectively captured the strength of its base models as well as the significance of the different features used. Furthermore, the model's transferability was indirectly validated on a dataset of seed storage proteins using Osborne definition as well as on a case study using molecular dynamic simulation, showing potential for application beyond microbial proteins to food and agriculture-related ones.

PLM_Sol: predicting protein solubility by benchmarking multiple protein language models with the updated Escherichia coli protein solubility dataset.

Protein solubility plays a crucial role in various biotechnological, industrial, and biomedical applications. With the reduction in sequencing and gene synthesis costs, the adoption of high-throughput experimental screening coupled with tailored bioinformatic prediction has witnessed a rapidly growing trend for the development of novel functional enzymes of interest (EOI). High protein solubility rates are essential in this process and accurate prediction of solubility is a challenging task. As deep learning technology continues to evolve, attention-based protein language models (PLMs) can extract intrinsic information from protein sequences to a greater extent. Leveraging these models along with the increasing availability of protein solubility data inferred from structural database like the Protein Data Bank holds great potential to enhance the prediction of protein solubility. In this study, we curated an Updated Escherichia coli protein Solubility DataSet (UESolDS) and employed a combination of multiple PLMs and classification layers to predict protein solubility. The resulting best-performing model, named Protein Language Model-based protein Solubility prediction model (PLM_Sol), demonstrated significant improvements over previous reported models, achieving a notable 6.4% increase in accuracy, 9.0% increase in F1_score, and 11.1% increase in Matthews correlation coefficient score on the independent test set. Moreover, additional evaluation utilizing our in-house synthesized protein resource as test data, encompassing diverse types of enzymes, also showcased the good performance of PLM_Sol. Overall, PLM_Sol exhibited consistent and promising performance across both independent test set and experimental set, thereby making it well suited for facilitating large-scale EOI studies. PLM_Sol is available as a standalone program and as an easy-to-use model at https://zenodo.org/doi/10.5281/zenodo.10675340.

Identification of echinoderm metabolites as potential inhibitors targeting wild-type and mutant forms of Escherichia coli RNA polymerase (RpoB) for tuberculosis treatment.

Tuberculosis (TB) remains a critical global health challenge, with the emergence of drug-resistant strains heightening concerns. The development of effective drugs targeting both wild-type (WT) and mutant Escherichia coli RNA polymerase ß subunit (RpoB) is crucial for global TB control, aiming to alleviate TB incidence, mortality, and transmission. This study employs molecular docking and ADMET analyses to screen echinoderm metabolites for their potential inhibition of Escherichia coli RNA polymerase, focusing on wild-type and mutant RpoB variants associated with TB drug resistance. The evaluation of docking results using the glide gscore led to the selection of the top 10 compounds for each protein receptor. Notably, CMNPD2176 demonstrated the highest binding affinity against wild-type RpoB, CMNPD13873 against RpoB D516V mutant, CMNPD2177 against RpoB H526Y mutant, and CMNPD11620 against RpoB S531L mutant. ADMET screening confirmed the therapeutic potential of these selected compounds. Additionally, MM-GBSA binding free energy calculations and molecular dynamics simulations provided further support for the docking investigations. While the results suggest these compounds could be viable for tuberculosis treatment, it is crucial to note that further in-vitro research is essential for the transition from prospective inhibitors to clinical drugs.

Visualizing chaperonin function in situ by cryo-electron tomography.

Chaperonins are large barrel-shaped complexes that mediate ATP-dependent protein folding1-3. The bacterial chaperonin GroEL forms juxtaposed rings that bind unfolded protein and the lid-shaped cofactor GroES at their apertures. In vitro analyses of the chaperonin reaction have shown that substrate protein folds, unimpaired by aggregation, while transiently encapsulated in the GroEL central cavity by GroES4-6. To determine the functional stoichiometry of GroEL, GroES and client protein in situ, here we visualized chaperonin complexes in their natural cellular environment using cryo-electron tomography. We find that, under various growth conditions, around 55-70% of GroEL binds GroES asymmetrically on one ring, with the remainder populating symmetrical complexes. Bound substrate protein is detected on the free ring of the asymmetrical complex, defining the substrate acceptor state. In situ analysis of GroEL-GroES chambers, validated by high-resolution structures obtained in vitro, showed the presence of encapsulated substrate protein in a folded state before release into the cytosol. Based on a comprehensive quantification and conformational analysis of chaperonin complexes, we propose a GroEL-GroES reaction cycle that consists of linked asymmetrical and symmetrical subreactions mediating protein folding. Our findings illuminate the native conformational and functional chaperonin cycle directly within cells.

Structural shifts in TolC facilitate Efflux-Mediated ß-lactam resistance.

Efflux-mediated ß-lactam resistance is a major public health concern, reducing the effectiveness of ß-lactam antibiotics against many bacteria. Structural analyses show the efflux protein TolC in Gram-negative bacteria acts as a channel for antibiotics, impacting bacterial susceptibility and virulence. This study examines ß-lactam drug efflux mediated by TolC using experimental and computational methods. Molecular dynamics simulations of drug-free TolC reveal essential movements and key residues involved in TolC opening. A whole-gene-saturation mutagenesis assay, mutating each TolC residue and measuring fitness effects under ß-lactam selection, is performed. Here we show the TolC-mediated efflux of three antibiotics: oxacillin, piperacillin, and carbenicillin. Steered molecular dynamics simulations identify general and drug-specific efflux mechanisms, revealing key positions at TolC's periplasmic entry affecting efflux motions. Our findings provide insights into TolC's structural dynamics, aiding the design of new antibiotics to overcome bacterial efflux mechanisms.