PELDOR Spectroscopy Reveals Two Defined States of a Sialic Acid TRAP Transporter SBP in Solution

The tripartite ATP-independent periplasmic (TRAP) transporters are a widespread class of membrane transporters in bacteria and archaea. Typical substrates for TRAP transporters are organic acids including the sialic acid N-acetylneuraminic acid. The substrate binding proteins (SBP) of TRAP transporters are the best studied component and are responsible for initial high-affinity substrate binding. To better understand the dynamics of the ligand binding process, pulsed electron-electron double resonance (PELDOR, also known as DEER) spectroscopy was applied to study the conformational changes in the N-acetylneuraminic acid-specific SBP VcSiaP. The protein is the SBP of VcSiaPQM, a sialic acid TRAP transporter from Vibrio cholerae. Spin-labeled double-cysteine mutants of VcSiaP were analyzed in the substrate-bound and -free state and the measured distances were compared to available crystal structures. The data were compatible with two clear states only, which are consistent with the open and closed forms seen in TRAP SBP crystal structures. Substrate titration experiments demonstrated the transition of the population from one state to the other with no other observed forms. Mutants of key residues involved in ligand binding and/or proposed to be involved in domain closure were produced and the corresponding PELDOR experiments reveal important insights into the open-closed transition. The results are in excellent agreement with previous in vivo sialylation experiments. The structure of the spin-labeled Q54R1/L173R1 R125A mutant was solved at 2.1 Å resolution, revealing no significant changes in the protein structure. Thus, the loss of domain closure appears to be solely due to loss of binding. In conclusion, these data are consistent with TRAP SBPs undergoing a simple two-state transition from an open-unliganded to closed-liganded state during the transport cycle.     

A Conserved Motif in Intracellular Loop 1 Stabilizes the Outward-Facing Conformation of TmrAB

The ATP binding cassette (ABC) family of transporters moves small molecules (lipids, sugars, peptides, drugs, nutrients) across membranes in nearly all organisms. Transport activity requires conformational switching between inward-facing and outward-facing states driven by ATP-dependent dimerization of two nucleotide binding domains (NBDs). The mechanism that connects ATP binding and hydrolysis in the NBDs to conformational changes in a substrate binding site in the transmembrane domains (TMDs) is currently an outstanding question. Here we use sequence coevolution analyses together with biochemical characterization to investigate the role of a highly conserved region in intracellular loop 1 we define as the GRD motif in coordinating domain rearrangements in the heterodimeric peptide exporter from Thermus thermophilus, TmrAB. Mutations in the GRD motif alter ATPase activity as well as transport. Disulfide crosslinking, evolutionary trace, and evolutionary coupling analysis reveal that these effects are likely due to the destabilization of a network in which the GRD motif in TmrA bridges residues of the Q-loop, X-loop, and ABC motif in the NBDs to residues in the TmrAB peptide substrate binding site, thus providing an avenue for conformational coupling. We further find that disruption of this network in TmrA versus TmrB has different functional consequences, hinting at an intrinsic asymmetry in heterodimeric ABC transporters extending beyond that of the NBDs. These results support a mechanism in which the GRD motifs help coordinate a transition to an outward open conformation, and each half of the transporter likely plays a different role in the conformational cycle of TmrAB.     

Dissecting the Conformational Dynamics of the Bile Acid Transporter Homologue ASBTNM

Apical sodium-dependent bile acid transporter (ASBT) catalyses uphill transport of bile acids using the electrochemical gradient of Na⁺ as the driving force. The crystal structures of two bacterial homologues ASBT_NM and ASBT_Yf have previously been determined, with the former showing an inward-facing conformation, and the latter adopting an outward-facing conformation accomplished by the substitution of the critical Na⁺-binding residue glutamate-254 with an alanine residue. While the two crystal structures suggested an elevator-like movement to afford alternating access to the substrate binding site, the mechanistic role of Na⁺ and substrate in the conformational isomerization remains unclear. In this study, we utilized site-directed alkylation monitored by in-gel fluorescence (SDAF) to probe the solvent accessibility of the residues lining the substrate permeation pathway of ASBT_NM under different Na⁺ and substrate conditions, and interpreted the conformational states inferred from the crystal structures. Unexpectedly, the crosslinking experiments demonstrated that ASBT_NM is a monomer protein, unlike the other elevator-type transporters, usually forming a homodimer or a homotrimer. The conformational dynamics observed by the biochemical experiments were further validated using DEER measuring the distance between the spin-labelled pairs. Our results revealed that Na⁺ ions shift the conformational equilibrium of ASBT_NM toward the inward-facing state thereby facilitating cytoplasmic uptake of substrate. The current findings provide a novel perspective on the conformational equilibrium of secondary active transporters.     

MlaC belongs to a unique class of non-canonical substrate-binding proteins and follows a novel phospholipid-binding mechanism

The outer membrane (OM) of Gram-negative bacteria acts as a formidable barrier against a plethora of detrimental compounds owing to its asymmetric nature. This is because the OM possesses lipopolysaccharides (LPSs) in the outer leaflet and phospholipids (PLs) in the inner leaflet. The maintenance of lipid asymmetry (Mla) system is involved in preserving the distribution of PLs in OM. The periplasmic component of the system MlaC serves as the substrate-binding protein (SBP) that shuttles PLs between the inner and outer membranes. However, an in-depth report highlighting its mechanism of ligand binding is still lacking. This study reports the crystal structure of MlaC from Escherichia coli (EcMlaC) at a resolution of 2.5 Å in a quasi-open state, complexed with PL. The structural analysis reveals that EcMlaC and orthologs comprise two major domains, viz. nuclear transport factor 2-like (NTF2-like) and phospholipid-binding protein (PBP). Each domain can be further divided into two subdomains arranged in a discontinuous fashion. This study further reveals that EcMlaC is polyspecific in nature and follows a reverse mechanism of the opening of the substrate-binding site during the ligand binding. Furthermore, MlaC can bind two PLs by forming subsites in the binding pocket. These findings, altogether, have led to the proposition of the unique “segmented domain movement” mechanism of PL binding, not reported for any known SBP to date. Further, unlike typical SBPs, MlaC has originated from a cystatin-like fold. Overall, this study establishes MlaC to be a non-canonical SBP with a unique ligand-binding mechanism.     

Structural Basis of the Allosteric Inhibition of Human ABCG2 by Nanobodies

ABCG2 is an ATP-binding cassette transporter that exports a wide range of xenobiotic compounds and has been recognized as a contributing factor for multidrug resistance in cancer cells. Substrate and inhibitor interactions with ABCG2 have been extensively studied and small molecule inhibitors have been developed that prevent the export of anticancer drugs from tumor cells. Here, we explore the potential for inhibitors that target sites other than the substrate binding pocket of ABCG2. We developed novel nanobodies against ABCG2 and used functional analyses to select three inhibitory nanobodies (Nb8, Nb17 and Nb96) for structural studies by single particle cryo-electron microscopy. Our results showed that these nanobodies allosterically bind to different regions of the nucleotide binding domains. Two copies of Nb8 bind to the apex of the NBDs preventing them from fully closing. Nb17 binds near the two-fold axis of the transporter and interacts with both NBDs. Nb96 binds to the side of the NBD and immobilizes a region connected to key motifs involved in ATP binding and hydrolysis. All three nanobodies prevent the transporter from undergoing conformational changes required for substrate transport. These findings advance our understanding of the molecular basis of modulation of ABCG2 by external binders, which may contribute to the development of a new generation of inhibitors. Furthermore, this is the first example of modulation of human multidrug resistance transporters by nanobodies.     

Validation of an Allosteric Binding Site of Src Kinase Identified by Unbiased Ligand Binding Simulations

Allostery plays a primary role in regulating protein activity, making it an important mechanism in human disease and drug discovery. Identifying allosteric regulatory sites to explore their biological significance and therapeutic potential is invaluable to drug discovery; however, identification remains a challenge. Allosteric sites are often “cryptic” without clear geometric or chemical features. Since allosteric regulatory sites are often less conserved in protein kinases than the orthosteric ATP binding site, allosteric ligands are commonly more specific than ATP competitive inhibitors. We present a generalizable computational protocol to predict allosteric ligand binding sites based on unbiased ligand binding simulation trajectories. We demonstrate the feasibility of this protocol by revisiting our previously published ligand binding simulations using the first identified viral proto-oncogene, Src kinase, as a model system. The binding paths for kinase inhibitor PP1 uncovered three metastable intermediate states before binding the high-affinity ATP-binding pocket, revealing two previously known allosteric sites and one novel site. Herein, we validate the novel site using a combination of virtual screening and experimental assays to identify a V-type allosteric small-molecule inhibitor that targets this novel site with specificity for Src over closely related kinases. This study provides a proof-of-concept for employing unbiased ligand binding simulations to identify cryptic allosteric binding sites and is widely applicable to other protein–ligand systems.     

Structural Analysis of Cholesterol Binding and Sterol Selectivity by ABCG5/G8

The ATP-binding cassette (ABC) sterol transporters are responsible for maintaining cholesterol homeostasis in mammals by participating in reverse cholesterol transport (RCT) or transintestinal cholesterol efflux (TICE). The heterodimeric ABCG5/G8 carries out selective sterol excretion, preventing the abnormal accumulation of plant sterols in human bodies, while homodimeric ABCG1 contributes to the biogenesis and metabolism of high-density lipoproteins. A sterol-binding site on ABCG5/G8 was proposed at the interface of the transmembrane domain and the core of lipid bilayers. In this study, we have determined the crystal structure of ABCG5/G8 in a cholesterol-bound state. The structure combined with amino acid sequence analysis shows that in the proximity of the sterol-binding site, a highly conserved phenylalanine array supports functional implications for ABCG cholesterol/sterol transporters. Lastly, in silico docking analysis of cholesterol and stigmasterol (a plant sterol) suggests sterol-binding selectivity on ABCG5/G8, but not ABCG1. Together, our results provide a structural basis for cholesterol binding on ABCG5/G8 and the sterol selectivity by ABCG transporters.     

A Complex Connection Between the Diversity of Human Gastric Mucin O-Glycans,Helicobacter pylori Binding,Helicobacter Infection and Fucosylation

Helicobacter pylori colonizes the stomach of half of the human population. Most H. pylori are located in the mucus layer, which is mainly comprised by glycosylated mucins. Using mass spectrometry, we identified 631 glycans (whereof 145 were fully characterized and the remainder assigned as compositions) on mucins isolated from 14 Helicobacter spp.-infected and 14 Helicobacter spp.-noninfected stomachs. Only six identified glycans were common to all individuals, from a total of 60 to 189 glycans in each individual. An increased number of unique glycan structures together with an increased intraindividual diversity and larger interindividual variation were identified among O-glycans from Helicobacter spp.-infected stomachs compared with noninfected stomachs. H. pylori strain J99, which carries the blood group antigen–binding adhesin (BabA), the sialic acid–binding adhesin (SabA), and the LacdiNAc-binding adhesin, bound both to Lewis b (Leb)-positive and Leb-negative mucins. Among Leb-positive mucins, H. pylori J99 binding was higher to mucins from Helicobacter spp.-infected individuals than noninfected individuals. Statistical correlation analysis, binding experiments with J99 wt, and J99ΔbabAΔsabA and inhibition experiments using synthetic glycoconjugates demonstrated that the differences in H. pylori-binding ability among these four groups were governed by BabA-dependent binding to fucosylated structures. LacdiNAc levels were lower in mucins that bound to J99 lacking BabA and SabA than in mucins that did not, suggesting that LacdiNAc did not significantly contribute to the binding. We identified 24 O-glycans from Leb-negative mucins that correlated well with H. pylori binding whereof 23 contained α1,2-linked fucosylation. The large and diverse gastric glycan library identified, including structures that correlated with H. pylori binding, could be used to select glycodeterminants to experimentally investigate further for their importance in host–pathogen interactions and as candidates to develop glycan-based therapies.     

Protein Allostery and Ligand Design: Computational Design Meets Experiments to Discover Novel Chemical Probes

Herein we examine the determinants of the allosteric inhibition of the mitochondrial chaperone TRAP1 by a small molecule ligand. The knowledge generated is harnessed into the design of novel derivatives with interesting biological properties.TRAP1 is a member of the Hsp90 family of proteins, which work through sequential steps of ATP processing coupled to client-protein remodeling. Isoform selective inhibition of TRAP1 can provide novel information on the biomolecular mechanisms of molecular chaperones, as well as new insights into the development of small molecules with therapeutic potential.Our analysis of the interactions between an active first-generation allosteric ligand and TRAP1 shows how the small molecule induces long-range perturbations that influence the attainment of reactive poses in the active site. At the same time, the dynamic adaptation of the allosteric binding pocket to the presence of the first-generation compound sets the stage for the design of a set of second-generation ligands: the characterization of the formation/disappearance of pockets around the allosteric site that is used to guide optimize the ligands’ fit for the allosteric site and improve inhibitory activities. The effects of the newly designed molecules are validated experimentally in vitro and in vivo. We discuss the implications of our approach as a promising strategy towards understanding the molecular determinants of allosteric regulation in chemical and molecular biology, and towards speeding up the design of allosteric small molecule modulators.     

Sialic acid diversity in the human gut: Molecular impacts and tools for future discovery

Sialic acids are a family of structurally related sugars that are prevalent in mucosal surfaces, including the human intestine. In the gut, sialic acids have diverse biological roles at the interface of the host epithelium and the microbiota. N-acetylneuraminic acid (Neu5Ac), the best studied sialic acid, is a nutrient source for bacteria and, when displayed on the cell surface, a binding site for host immune factors, viruses, and bacterial toxins. Neu5Ac is extensively modified by host and microbial enzymes, and the impacts of Neu5Ac derivatives on host–microbe interactions, and generally on human and microbial biology, remain underexplored. In this mini-review, we highlight recent reports describing how host and microbial proteins differentiate Neu5Ac and its derivatives, draw attention to gaps in knowledge related to sialic acid biology, and suggest cutting-edge methodologies that may expand our appreciation and understanding of Neu5Ac in health and disease.     

High-resolution structure of phosphoketolase from Bifidobacterium longum determined by cryo-EM single-particle analysis

In bifidobacteria, phosphoketolase (PKT) plays a key role in the central hexose fermentation pathway called “bifid shunt.” The three-dimensional structure of PKT from Bifidobacterium longum with co-enzyme thiamine diphosphate (ThDpp) was determined at 2.1 Å resolution by cryo-EM single-particle analysis using 196,147 particles to build up the structural model of a PKT octamer related by D₄ symmetry. Although the cryo-EM structure of PKT was almost identical to the X-ray crystal structure previously determined at 2.2 Å resolution, several interesting structural features were observed in the cryo-EM structure. Because this structure was solved at relatively high resolution, it was observed that several amino acid residues adopt multiple conformations. Among them, Q546–D547–H548–N549 (the QN-loop) demonstrate the largest structural change, which seems to be related to the enzymatic function of PKT. The QN-loop is at the entrance to the substrate binding pocket. The minor conformer of the QN-loop is similar to the conformation of the QN-loop in the crystal structure. The major conformer is located further from ThDpp than the minor conformer. Interestingly, the major conformer in the cryo-EM structure of PKT resembles the corresponding loop structure of substrate-bound Escherichia coli transketolase. That is, the minor and major conformers may correspond to “closed” and “open” states for substrate access, respectively. Moreover, because of the high-resolution analysis, many water molecules were observed in the cryo-EM structure of PKT. Structural features of the water molecules in the cryo-EM structure are discussed and compared with water molecules observed in the crystal structure.     

Structure of the Human Cholesterol Transporter ABCG1

ABCG1 is an ATP binding cassette (ABC) transporter that removes excess cholesterol from peripheral tissues. Despite its role in preventing lipid accumulation and the development of cardiovascular and metabolic disease, the mechanism underpinning ABCG1-mediated cholesterol transport is unknown. Here we report a cryo-EM structure of human ABCG1 at 4 Å resolution in an inward-open state, featuring sterol-like density in the binding cavity. Structural comparison with the multidrug transporter ABCG2 and the sterol transporter ABCG5/G8 reveals the basis of mechanistic differences and distinct substrate specificity. Benzamil and taurocholate inhibited the ATPase activity of liposome-reconstituted ABCG1, whereas the ABCG2 inhibitor Ko143 did not. Based on the structural insights into ABCG1, we propose a mechanism for ABCG1-mediated cholesterol transport.     

Structural Basis of Drug Recognition by the Multidrug Transporter ABCG2

ABCG2 is an ATP-binding cassette (ABC) transporter whose function affects the pharmacokinetics of drugs and contributes to multidrug resistance of cancer cells. While its interaction with the endogenous substrate estrone-3-sulfate (E₁S) has been elucidated at a structural level, the recognition and recruitment of exogenous compounds is not understood at sufficiently high resolution. Here we present three cryo-EM structures of nanodisc-reconstituted, human ABCG2 bound to anticancer drugs tariquidar, topotecan and mitoxantrone. To enable structural insight at high resolution, we used Fab fragments of the ABCG2-specific monoclonal antibody 5D3, which binds to the external side of the transporter but does not interfere with drug-induced stimulation of ATPase activity. We observed that the binding pocket of ABCG2 can accommodate a single tariquidar molecule in a C-shaped conformation, similar to one of the two tariquidar molecules bound to ABCB1, where tariquidar acts as an inhibitor. We also found single copies of topotecan and mitoxantrone bound between key phenylalanine residues. Mutagenesis experiments confirmed the functional importance of two residues in the binding pocket, F439 and N436. Using 3D variability analyses, we found a correlation between substrate binding and reduced dynamics of the nucleotide binding domains (NBDs), suggesting a structural explanation for drug-induced ATPase stimulation. Our findings provide additional insight into how ABCG2 differentiates between inhibitors and substrates and may guide a rational design of new modulators and substrates.     

Principles of Alternating Access in Multidrug and Toxin Extrusion (MATE) Transporters

The multidrug and toxin extrusion (MATE) transporters catalyze active efflux of a broad range of chemically- and structurally-diverse compounds including antimicrobials and chemotherapeutics, thus contributing to multidrug resistance in pathogenic bacteria and cancers. Multiple methodological approaches have been taken to investigate the structural basis of energy transduction and substrate translocation in MATE transporters. Crystal structures representing members from all three MATE subfamilies have been interpreted within the context of an alternating access mechanism that postulates occupation of distinct structural intermediates in a conformational cycle powered by electrochemical ion gradients. Here we review the structural biology of MATE transporters, integrating the crystallographic models with biophysical and computational studies to define the molecular determinants that shape the transport energy landscape. This holistic analysis highlights both shared and disparate structural and functional features within the MATE family, which underpin an emerging theme of mechanistic diversity within the framework of a conserved structural scaffold.     

Formation of Kiss1R/GPER Heterocomplexes Negatively Regulates Kiss1R-mediated Signalling through Limiting Receptor Cell Surface Expression

Kisspeptin receptor (Kiss1R) is an important receptor that plays central regulatory roles in reproduction by regulating hormone release in the hypothalamus. We hypothesize that the formation of heterocomplexes between Kiss1R and other hypothalamus G protein-coupled receptors (GPCRs) affects their cellular signaling. Through screening of potential interactions between Kiss1R and hypothalamus GPCRs, we identified G protein-coupled estrogen receptor (GPER) as one interaction partner of Kiss1R. Based on the recognised function of kisspeptin and estrogen in regulating the reproductive system, we investigated the Kiss1R/GPER heterocomplex in more detail and revealed that complex formation significantly reduced Kiss1R-mediated signaling. GPER did not directly antagonize Kiss1R conformational changes upon ligand binding, but it rather reduced the cell surface expression of Kiss1R. These results therefore demonstrate a regulatory mechanism of hypothalamic hormone receptors via receptor cooperation in the reproductive system and modulation of receptor sensitivity.     

Competitive Microtubule Binding of PEX14 Coordinates Peroxisomal Protein Import and Motility

Human PEX14 plays a dual role as docking protein in peroxisomal protein import and as peroxisomal anchor for microtubules (MT), which relates to peroxisome motility. For docking, the conserved N-terminal domain of PEX14 (PEX14-NTD) binds amphipathic alpha-helical ligands, typically comprising one or two aromatic residues, of which human PEX5 possesses eight. Here, we show that the PEX14-NTD also binds to microtubular filaments in vitro with a dissociation constant in nanomolar range. PEX14 interacts with two motifs in the C-terminal region of human ß-tubulin. At least one of the binding motifs is in spatial proximity to the binding site of microtubules (MT) for kinesin. Both PEX14 and kinesin can bind to MT simultaneously. Notably, binding of PEX14 to tubulin can be prevented by its association with PEX5. The data suggest that PEX5 competes peroxisome anchoring to MT by occupying the ß-tubulin-binding site of PEX14. The competitive correlation of matrix protein import and motility may facilitate the homogeneous dispersion of peroxisomes in mammalian cells.     

Conformational Change of the Hairpin-like-structured Robo2 Ectodomain Allows NELL1/2 Binding

Since neural epidermal growth factor-like-like (NELL) 2 was identified as a novel ligand for the roundabout (Robo) 3 receptor, research on NELL–Robo signaling has become increasingly important. We have previously reported that Robo2 can bind to NELL1/2 in acidic conditions but not at neutral pH. The NELL1/2-binding site that is occluded in neutral conditions is thought to be exposed by a conformational change of the Robo2 ectodomain upon exposure to acidic pH; however, the underlying structural mechanisms are not well understood. Here, we investigated the interaction between the immunoglobulin-like domains and fibronectin type III domains that form hairpin-like structure of the Robo2 ectodomain, and demonstrated that acidic pH attenuates the interaction between them. Alternative splicing isoforms of Robo2, which affect the conformation of the hairpin-like structure, were found to have distinct NELL1/2-binding affinities. We developed Förster resonance energy transfer-based indicators for monitoring conformational change of the Robo2 ectodomain by individually inserting donor and acceptor fluorescent proteins at its ends. These experiments revealed that the ends of the Robo2 ectodomain are close to each other in acidic conditions. By combining these findings with the results of size exclusion chromatography analysis, we suggest that, in acidic conditions, the Robo2 ectodomain has a compact conformation with a loose hairpin-like structure. These results may help elucidate the signaling mechanisms resulting from the interaction between Robo2 and NELL1/2 in acidic conditions.     

Activation of the PDGF β Receptor by a Persistent Artificial Signal Peptide

Most eukaryotic transmembrane and secreted proteins contain N-terminal signal peptides that mediate insertion of the nascent translation products into the membrane of the endoplasmic reticulum. After membrane insertion, signal peptides typically are cleaved from the mature protein and degraded. Here, we tested whether a small hydrophobic protein selected for growth promoting activity in mammalian cells retained transforming activity while also acting as a signal peptide. We replaced the signal peptide of the PDGF β receptor (PDGFβR) with a previously described 29-residue artificial transmembrane protein named 9C3 that can activate the PDGFβR in trans. We showed that a modified version of 9C3 at the N-terminus of the PDGFβR can function as a signal peptide, as assessed by its ability to support high level expression, glycosylation, and cell surface localization of the PDGFβR. The 9C3 signal peptide retains its ability to interact with the transmembrane domain of the PDGFβR and cause receptor activation and cell proliferation. Cleavage of the 9C3 signal peptide from the mature receptor is not required for these activities. However, signal peptide cleavage does occur in some molecules, and the cleaved signal peptide can persist in cells and activate a co-expressed PDGFβR in trans. Our finding that a hydrophobic sequence can display signal peptide and transforming activity suggest that some naturally occurring signal peptides may also display additional biological activities by interacting with the transmembrane domains of target proteins.     

Membrane transporter identification and modulation via adaptive laboratory evolution

Membrane transport proteins are potential targets for medical and biotechnological applications. However, more than 30% of reported membrane transporter families are either poorly characterized or lack adequate functional annotation. Here, adaptive laboratory evolution was leveraged to identify membrane transporters for a set of four amino acids as well as specific mutations that modulate the activities of these transporters. Specifically, Escherichia coli was adaptively evolved under increasing concentrations of L–histidine, L–phenylalanine, L–threonine, and L–methionine separately with multiple replicate evolutions. Evolved populations and isolated clones displayed growth rates comparable to the unstressed ancestral strain at elevated concentrations (four-to six-fold increases) of the targeted amino acids. Whole genome sequencing of the evolved strains revealed a diverse number of key mutations, including SNPs, small deletions, and copy number variants targeting the transporters leuE for histidine, yddG for phenylalanine, yedA for methionine, and brnQ and rhtC for threonine. Reverse engineering of the mutations in the ancestral strain established mutation causality of the specific mutations for the tolerant phenotypes. The functional roles of yedA and brnQ in the transport of methionine and threonine, respectively, are novel assignments and their functional roles were validated using a flow cytometry cellular accumulation assay. To demonstrate how the identified transporters can be leveraged for production, an L–phenylalanine overproduction strain was shown to be a superior producer when the identified yddG exporter was overexpressed. Overall, the results revealed the striking efficiency of laboratory evolution to identify transporters and specific mutational mechanisms to modulate their activities, thereby demonstrating promising applicability in transporter discovery efforts and strain engineering.