Lactose Permease Research Articles

Kinetic Effects of Inside‐Positive Membrane Potential on the Lactose Efflux of LacY

LacY, the lactose permease of E. coli., utilizes the free energy released from the downhill movement of H+ in response of an electrochemical H+ gradient (Δμ̃H+, interior negative and/or alkaline) to catalyze the accumulation of lactose against a concentration gradient. In the absence of Δμ̃H+, the downhill transport of lactose in response of a concentration gradient drives uphill translocation of H+ to generate a H+ gradient, and the polarity of the H+ gradient depends on the direction of lactose gradient. In other words, LacY is a lactose/H+ symporter, in which the lactose transport and H+ translocation are obligatorily coupled. LacY functions as a monomer with a total of 12 transmembrane helices arranged into two 6‐helix bundles tethered by a long loop between helix VI and VII. The sugar‐ and H+‐binding sites are located at approximate the center of the permease and can be accessed from either side of the membrane via an alternating access conformational change driven by the binding and dissociation of sugar. In LacY, Glu325 plays an essential role in H+ translocation since replacing Glu325 with neutral amino residues abolishes transport activities involving H+ translocation, but the mutants still bind sugar with normal affinity and catalyze equilibrium exchange. Previous studies have shown that the presence of Δμ̃H+ (interior negative) significantly accelerates lactose influx by decreasing the Km 50‐100‐fold with no change in Kd. The goal of this study is to investigate kinetic effects of Δμ̃H+ on lactose efflux in right‐side‐out membrane vesicles. We measured the initial rate of efflux at different pHs in the absence or presence of an imposed membrane potential (ΔΨ, generated by K+ influx in response of a concentration gradient in the presence of valinomycin, interior positive). The results revealed that the lactose efflux of WT LacY exhibits an apparent pK value near 7.2, which is not affected by the imposition of ΔΨ (interior positive). The initial rate of lactose efflux increases in the presence of imposed ΔΨ (interior positive) at all tested pHs, although enhancement is about two orders of magnitude less than that observed in lactose influx. Moreover, the effect of ΔΨ (interior positive) on the Km of lactose efflux is almost negligible compared to the dramatic change of the Km of lactose influx caused by ΔΨ (interior negative). In contrast to WT LacY, the E325A mutant does not carry out lactose efflux in the absence or presence of imposed ΔΨ (interior positive) at all tested pHs.Support or Funding InformationNSF Eager Grant MCB‐1547801 to H.R.K., NIH Grant 1R01 GM120043 to H.R.K., and a grant from Ruth and Bucky Stein to H.R.K..

Production of Ethanol from Lactose in Engineered S. cerevisiae

Wisconsin is the dairy capitol of the United States. However, all this cheese production leaves behind a lot of unused by product in the form of cheese whey. If cheese whey could be fermented into consumable ethanol there would be less discarded into the trash and could also be a new revenue stream for Wisconsin dairies. Our plan is to modify yeast that can already produce ethanol from sucrose to also be able to make the ethanol from cheese whey that contains lactose.By engineering two strains of Saccharomyces cerevisiae to metabolize lactose we predict they will be able to digest and ferment cheese whey. This is done by allowing the yeast to express lactose permease and beta galactosidase, LAC12 and LAC4 respectively. These genes can be found in Kluyveromyces marxianus and Kluyveromyces lactis.With this production of ethanol from cheese whey we plan to patent this newly found technique which in return will lead to lots of vodka and money.Support or Funding InformationThis project is funded by UW ‐ Stout's Office of Research and Sponsored Programs.

Origin of Lactose Fermentation inKluyveromyces lactisby Interspecies Transferof a Neo-functionalized Gene Cluster during Domestication.

SummaryHumans have used yeasts to make cheese and kefir for millennia, but the ability to ferment the milk sugar lactose is found in only a few yeast species, of which the foremost is Kluyveromyces lactis [1]. Two genes, LAC12 (lactose permease) and LAC4 (lactase), are sufficient for lactose uptake and hydrolysis to glucose and galactose [2]. Here, we show that these genes have a complex evolutionary history in the genus Kluyveromyces that is likely the result of human activity during domestication. We show that the ancestral Lac12 was bifunctional, able to import both lactose and cellobiose into the cell. These disaccharides were then hydrolyzed by Lac4 in the case of lactose or Cel2 in the case of cellobiose. A second cellobiose transporter, Cel1, was also present ancestrally. In the K. lactis lineage, the ancestral LAC12 and LAC4 were lost and a separate upheaval in the sister species K. marxianus resulted in loss of CEL1 and quadruplication of LAC12. One of these LAC12 genes became neofunctionalized to encode an efficient lactose transporter capable of supporting fermentation, specifically in dairy strains of K. marxianus, where it formed a LAC4-LAC12-CEL2 gene cluster, although another remained a cellobiose transporter. Then, the ability to ferment lactose was acquired very recently by K. lactis var. lactis by introgression of LAC12 and LAC4 on a 15-kb subtelomeric region from a dairy strain of K. marxianus. The genomic history of the LAC genes shows that strong selective pressures were imposed on yeasts by early dairy farmers.

Importance of phosphorylation/dephosphorylation cycles on lipid-dependent modulation of membrane protein topology by posttranslational phosphorylation

Posttranslational modifications of proteins, such as phosphorylation and dephosphorylation, play critical roles in cellular functions through diverse cell signaling pathways. Protein kinases and phosphatases have been described early on as key regulatory elements of the phosphorylated state of proteins. Tight spatial and temporal regulation of protein kinase and phosphatase activities has to be achieved in the cell to ensure accurate signal transduction. We demonstrated previously that phosphorylation of a membrane protein can lead to its topological rearrangement. Additionally, we found that both the rate and extent of topological rearrangement upon phosphorylation are lipid charge- and lipid environment-dependent. Here, using a model membrane protein (the bacterial lactose permease LacY reconstituted in proteoliposomes) and a combination of real-time measurements and steady-state assessments of protein topology, we established a set of experimental conditions to dissect the effects of phosphorylation and dephosphorylation of a membrane protein on its topological orientation. We also demonstrate that the phosphorylation-induced topological switch of a membrane protein can be reversed upon protein dephosphorylation, revealing a new regulatory role for phosphorylation/dephosphorylation cycles. Furthermore, we determined that the rate of topological rearrangement reversal is correlated with phosphatase activity and is influenced by the membrane's lipid composition, presenting new insights into the spatiotemporal control of the protein phosphorylation state. Together, our results highlight the importance of the compartmentalization of phosphorylation/dephosphorylation cycles in controlling membrane protein topology and, therefore, function, which are influenced by the local lipid environment of the membrane protein.

Engineering the Substrate Transport and Cofactor Regeneration Systems for Enhancing 2'-Fucosyllactose Synthesis in Bacillus subtilis.

Human milk oligosaccharides (HMOs) have been proven to be beneficial to infants' intestinal health and immune systems. 2'-Fucosyllactose (2'-FL) is the most abundant and thoroughly studied HMO and has been approved to be an additive of infant formula. How to construct efficient and safe microbial cell factories for the production of 2'-FL attracts increasing attention. In this work, we engineered the Bacillus subtilis as an efficient 2'-FL producer by engineering the substrate transport and cofactor guanosine 5'-triphosphate (GTP) regeneration systems. First, we constructed a synthesis pathway for the 2'-FL precursor guanosine 5'-diphosphate-l-fucose (GDP-l-fucose) by introducing the salvage pathway gene fkp from Bacteriodes fragilis and improved the fucose importation by overexpressing the transporters. Then, the complete synthesis pathway of 2'-FL was constructed by introducing the heterologous fucosyltransferases from different sources, and it was found that the gene from Helicobacter pylori was the best one for 2'-FL synthesis. We also improved the substrate lactose importation by introducing heterologous lactose permeases and eliminated endogenous β-galactosidase (yesZ) to block the lactose degradation. Next, the production of 2'-FL and GDP-l-fucose was improved by fine-tuning the expression of cofactor guanosine 5'-triphosphate regeneration module genes gmd, ndk, guaA, guaC, ykfN, deoD, and xpt. Finally, a 3 L fed-batch fermentation was performed, and the highest 2'-FL titer reached 5.01 g/L with a yield up to 0.85 mol/mol fucose. We optimized the synthesis modules of 2'-FL in B. subtilis, and this provides a good starting point for metabolic engineering to further improve 2'-FL production in the future.

Modular pathway engineering of key precursor supply pathways for lacto-N-neotetraose production in Bacillus subtilis

BackgroundLacto-N-neotetraose (LNnT) is one of the important ingredients of human milk oligosaccharides, which can enhance immunity, regulate intestinal bacteria and promote cell maturation.ResultsIn this study, the synthetic pathway of LNnT was constructed by co-expressing the lactose permease (LacY) β-1,3-N-acetylglucosaminyltransferase (LgtA) and β-1,4-galactostltransferase (LgtB) in Bacillus subtilis, resulting in an LNnT titer of 0.61 g/L. Then, by fine-tuning the expression level of LgtB, the growth inhibition was reduced and the LNnT titer was increased to 1.31 g/L. In addition, by modular pathway engineering, the positive-acting enzymes of the UDP-GlcNAc and UDP-Gal pathways were strengthened to balance the two key precursors supply, and the LNnT titer was improved to 1.95 g/L. Finally, the LNnT titer reached 4.52 g/L in a 3-L bioreactor with an optimal glucose and lactose feeding strategy.ConclusionsIn general, this study showed that the LNnT biosynthesis could be significantly increased by optimizing enzymes expression levels and modular pathway engineering for balancing the precursors supply in B. subtilis.

The lipid-dependent structure and function of LacY can be recapitulated and analyzed in phospholipid-containing detergent micelles

Membrane proteins play key roles in cellular functions, their activity mainly depending on their topological arrangement in membranes. Structural studies of membrane proteins have long adopted a protein-centric view regarding the determinants of membrane protein topology and function. Several studies have shown that the orientation of transmembrane domains of polytopic membrane proteins with respect to the plane of the lipid bilayer can be largely determined by membrane lipid composition. However, the mechanism by which membrane proteins exhibit structural and functional duality in the same membrane or different membranes is still unknown. Here we show that lipid-dependent structural and functional assessment of a membrane protein can be conducted in detergent micelles, opening the possibility for the determination of lipid-dependent high-resolution crystal structures. We found that the lactose permease purified from Escherichia coli cells exhibiting varied phospholipid compositions exhibits the same topology and similar function as in its membrane of origin. Furthermore, we found several conditions, including protein mutations and micelle lipid composition, that lead to increased protein stability, correlating with a higher yield of two-dimensional crystal formation. Altogether, our results demonstrate how the membrane lipid environment influences membrane protein topology and arrangement, both in native membranes and in mixed detergent micelles.

Genomic analysis and lactose transporter expression in Kluyveromyces marxianus CCT 7735

Kluyveromyces marxianus CCT 7735 has been used to produce ethanol, aromatic compounds, enzymes and heterologous proteins besides assimilates lactose as carbon source. Its genome has 10.7 Mb and encodes 4787 genes distributed in 8 nuclear chromosomes and one mitochondrial. Contrary to Kluyveromyces lactis, which has a unique LAC12 gene (encodes lactose permease), K. marxianus possesses four. The presence of degenerated copies and Solo-LTRs related to retrotransposon TKM close to the LAC12 genes in K. marxianus indicates ectopic recombinations. The Lac12 permeases of K. marxianus and K. lactis are conserved, however the conservation is higher between the copy of the left side of the chromosome three and the unique copy of K. lactis, indicating that this copy is the ancestor. The expression of the four LAC12 genes occurred in aerobiosis and hypoxia. Notably, the high lactose consumption in hypoxia seems to be related to the high expression of the LAC12 genes.

It takes two to tango: The dance of the permease.

The lactose permease (LacY) of Escherichia coli is the prototype of the major facilitator superfamily, one of the largest families of membrane transport proteins. Structurally, two pseudo-symmetrical six-helix bundles surround a large internal aqueous cavity. Single binding sites for galactoside and H+ are positioned at the approximate center of LacY halfway through the membrane at the apex of the internal cavity. These features enable LacY to function by an alternating-access mechanism that can catalyze galactoside/H+ symport in either direction across the cytoplasmic membrane. The H+-binding site is fully protonated under physiological conditions, and subsequent sugar binding causes transition of the ternary complex to an occluded intermediate that can open to either side of the membrane. We review the structural and functional evidence that has provided new insight into the mechanism by which LacY achieves active transport against a concentration gradient.

Transport cycle of Escherichia coli lactose permease in a nonhomogeneous random walk model.

We present Monte Carlo simulations for the transport cycle of Escherichia coli lactose permease (LacY), using as a starting point the model proposed by Kaback etal. [Nat. Rev. Mol. Cell Biol. 2, 610 (2001)NRMCBP1471-007210.1038/35085077], which is based on functional properties of mutants and x-ray structures. Kaback's model suggests the existence of six states for the whole cycle of lactose-H^{+} symport. However, the free-energy differences between these states have not yet been reported in the literature. Here, we analyzed the biochemical structure of each state and determined a range of possible values for each one of the five free-energy variations. Then, using the Metropolis algorithm in a nonhomogeneous random walk model, we tested all the possible combinations with these values to find the free-energy curve that best reproduces the dynamics of LacY. The agreement between our model predictions and the experimental data suggests that our free-energy curve is appropriate for describing the lactose-H^{+} symport. We found not only this curve, but also the time of occupancy of the permease at each conformation. In addition, we paved the way in this work to solve an open question related to this transport mechanism, which is the importance of protonation for lactose binding.

Arg302 governs the pKa of Glu325 in LacY

Lactose permease is a paradigm for the major facilitator superfamily, the largest family of ion-coupled membrane transport proteins known at present. LacY carries out the coupled stoichiometric symport of a galactoside with an H+, using the free energy released from downhill translocation of H+ to drive accumulation of galactosides against a concentration gradient. In neutrophilic Escherichia coli, internal pH is kept at ∼7.6 over the physiological range, but the apparent pK (pKapp) for galactoside binding is 10.5. Surface-enhanced infrared absorption spectroscopy (SEIRAS) demonstrates that the high pKa is due to Glu325 (helix X), which must be protonated for LacY to bind galactoside effectively. Deprotonation is also obligatory for turnover, however. Here, we utilize SEIRAS to study the effect of mutating residues in the immediate vicinity of Glu325 on its pKa The results are consistent with the idea that Arg302 (helix IX) is important for deprotonation of Glu325.

Uptake Dynamics in the Lactose Permease (LacY) Membrane Protein Transporter

The sugar transporter Lactose permease (LacY) of Escherichia coli has become a prototype to understand the underlying molecular details of membrane transport. Crystal structures have trapped the protein in sugar-bound states facing the periplasm, but with narrow openings unable to accommodate sugar. Therefore, the molecular details of sugar uptake remain elusive. In this work, we have used extended simulations and metadynamics sampling to explore a putative sugar-uptake pathway and associated free energy landscape. We found an entrance at helix-pair 2 and 11, which involved lipid head groups and residues Gln 241 and Gln 359. Furthermore, the protein displayed high flexibility on the periplasmic side of Phe 27, which is located at the narrowest section of the pathway. Interactions to Phe 27 enabled passage into the binding site, which was associated with a 24 ± 4 kJ/mol binding free energy in excellent agreement with an independent binding free energy calculation and experimental data. Two free energy minima corresponding to the two possible binding poses of the lactose analog β-D-galactopyranosyl-1-thio-β-D-galactopyranoside (TDG) were aligned with the crystal structure-binding pocket. This work outlines the chemical environment of a putative periplasmic sugar pathway and paves way for understanding substrate affinity and specificity in LacY.

Insertion and folding pathways of single membrane proteins guided by translocases and insertases.

Biogenesis in prokaryotes and eukaryotes requires the insertion of α-helical proteins into cellular membranes for which they use universally conserved cellular machineries. In bacterial inner membranes, insertion is facilitated by YidC insertase and SecYEG translocon working individually or cooperatively. How insertase and translocon fold a polypeptide into the native protein in the membrane is largely unknown. We apply single-molecule force spectroscopy assays to investigate the insertion and folding process of single lactose permease (LacY) precursors assisted by YidC and SecYEG. Both YidC and SecYEG initiate folding of the completely unfolded polypeptide by inserting a single structural segment. YidC then inserts the remaining segments in random order, whereas SecYEG inserts them sequentially. Each type of insertion process proceeds until LacY folding is complete. When YidC and SecYEG cooperate, the folding pathway of the membrane protein is dominated by the translocase. We propose that both of the fundamentally different pathways along which YidC and SecYEG insert and fold a polypeptide are essential components of membrane protein biogenesis.

Origin of Lactose Fermentation in <i>Kluyveromyces lactis</i> by Interspecies Transfer of a Neofunctionalized Gene Cluster During Domestication

Humans have used yeasts to make cheese and kefir for millennia, but the ability to ferment the milk sugar lactose is found in only a few yeast species, of which the foremost is Kluyveromyces lactis. Two genes, LAC12 (lactose permease) and LAC4 (lactase), are sufficient for lactose uptake and hydrolysis to glucose and galactose. Here, we show that these genes have a complex evolutionary history in the genus Kluyveromyces that is likely the result of human activity during domestication. We show that their ancestral role was in a combined lactose and cellobiose assimilation system, which used Lac12 to import both of these sugars into the cell, and then hydrolyzed lactose using Lac4, and cellobiose using a cellobiase, Cel2. A second cellobiose transporter Cel1 was also present ancestrally. In the K. lactis lineage, however, the ancestral LAC12 and LAC4 were lost and a separate upheaval in the sister species K. marxianus resulted in loss of CEL1 and quadruplication of LAC12. Among its four LAC12 genes, one became neofunctionalized as an efficient lactose transporter capable of supporting fermentation, specifically in dairy strains of K. marxianus where it formed a LAC4-LAC12-CEL2 gene cluster, while another remained a cellobiose transporter. Then, the ability to ferment lactose was acquired by K. lactis var. lactis by introgression of a 15 kb subtelomeric region containing LAC12 and LAC4 from a dairy strain of K. marxianus. The genomic history of the LAC genes shows that strong selective pressures were imposed on yeasts by early dairy farmers.

Presence of galactose in precultures induces lacS and leads to short lag phase in lactose-grown Lactococcus lactis cultures

Lactose conversion by lactic acid bacteria is of high industrial relevance and consistent starter culture quality is of outmost importance. We observed that Lactococcus lactis using the high-affinity lactose-phosphotransferase system excreted galactose towards the end of the lactose consumption phase. The excreted galactose was re-consumed after lactose depletion. The lacS gene, known to encode a lactose permease with affinity for galactose, a putative galactose–lactose antiporter, was upregulated under the conditions studied. When transferring cells from anaerobic to respiration-permissive conditions, lactose-assimilating strains exhibited a long and non-reproducible lag phase. Through systematic preculture experiments, the presence of galactose in the precultures was correlated to short and reproducible lag phases in respiration-permissive main cultivations. For starter culture production, the presence of galactose during propagation of dairy strains can provide a physiological marker for short culture lag phase in lactose-grown cultures.

Engineered occluded apo-intermediate of LacY

The lactose permease of Escherichia coli (LacY) utilizes an alternating access symport mechanism with multiple conformational intermediates, but only inward (cytoplasmic)- or outward (periplasmic)-open structures have been characterized by X-ray crystallography. It is demonstrated here with sugar-binding studies that cross-linking paired-Cys replacements across the closed cytoplasmic cavity stabilize an occluded conformer with an inaccessible sugar-binding site. In addition, a nanobody (Nb) that stabilizes a periplasmic-open conformer with an easily accessible sugar-binding site in WT LacY fails to cause the cytoplasmic cross-linked mutants to become accessible to galactoside, showing that the periplasmic cavity is closed. These results are consistent with tight association of the periplasmic ends in two pairs of helices containing clusters of small residues in the packing interface between N- and C-terminal six-helix bundles of the symporter. However, after reduction of the disulfide bond, the Nb markedly increases the rate of galactoside binding, indicating unrestricted access to the Nb epitope and the galactoside-binding site from the periplasm. The findings indicate that the cross-linked cytoplasmic double-Cys mutants resemble an occluded apo-intermediate in the transport cycle.

The putative \u03b2-glucosidase BGL3I regulates cellulase induction in Trichoderma reesei

BackgroundThe filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina) displays increased cellulase expression while growing on inducers such as lactose or cellulose. However, the mechanism of cellulase induction in T. reesei is not yet completely characterized. Here, a protein annotated as β-glucosidase (BGL3I) was found to be involved in cellulase induction in T. reesei. The effects of BGL3I on cellulase production have not yet been fully understood.ResultsDeletion of the bgl3i gene had no influence on the growth of T. reesei, but significantly increased its cellulase activities. Deletion of bgl3i also resulted in decreased extracellular galactosidase activity, but significantly increased transcription of lactose permeases, which might be involved in lactose transport. Furthermore, deletion of bgl3i enhanced the transcription levels of intracellular β-glucosidases cel1a, cel1b and the regulator xyr1, which are all essential for lactose induction in T. reesei. BGL3I was found to have a relatively high ability to hydrolyze sophorose, which is proposed to be the strongest natural inducer of cellulase synthesis in T. reesei.ConclusionsBGL3I may take part in the complex regulating system of cellulase induction. The deletion of bgl3i offers a new strategy to improve T. reesei strain performance.

Uptake dynamics in the Lactose permease (LacY) membrane protein transporter

The sugar transporter Lactose permease (LacY) of Escherichia coli has become a prototype to understand the underlying molecular details of membrane transport. Crystal structures have trapped the protein in sugar-bound states facing the periplasm, but with narrow openings unable to accommodate sugar. Therefore, the molecular details of sugar uptake remain elusive. In this work, we have used extended simulations and metadynamics sampling to explore a putative sugar-uptake pathway and associated free energy landscape. We found an entrance at helix-pair 2 and 11, which involved lipid head groups and residues Gln 241 and Gln 359. Furthermore, the protein displayed high flexibility on the periplasmic side of Phe 27, which is located at the narrowest section of the pathway. Interactions to Phe 27 enabled passage into the binding site, which was associated with a 24 ± 4 kJ/mol binding free energy in excellent agreement with an independent binding free energy calculation and experimental data. Two free energy minima corresponding to the two possible binding poses of the lactose analog β-D-galactopyranosyl-1-thio-β-D-galactopyranoside (TDG) were aligned with the crystal structure-binding pocket. This work outlines the chemical environment of a putative periplasmic sugar pathway and paves way for understanding substrate affinity and specificity in LacY.

GENETIC CHARACTERISTICS OF THE ADAPTIVE POTENTIAL OF BIFIDOBACTERIA IN THE BIOTOPE OF DISTAL HUMAN INTESTINE

Aim. Determination of distinctive parameters of the B. bifidum and B. longum genomes, which characterizes their adaptive potential applied to distal intestine biotope of the human gut. Materials and methods. 5 strains of bifidobacteria have been used: B. bifidum ICIS-310, B. bifidum ICIS-643, B. bifidum ICIS-791, B. longum ICIS-505 (clinical isolates) и B. longum MC-42. Whole Genome Sequencing (WGS) has been performed by «MiSeq» DNA sequencer and «Nextera» DNA library preparation kit (Illumiina). Annotation and the primary analysis of known homologues gene content has been performed by RAST service (NMPDR). Results. B. bifidum ICIS-310 has not revealed lactose and galactose permease genes, that present in two other sequenced B. bifidum strains, but two exo-alph-sialidase genes has remained, as well as additional gene of DNA-methyltransferases family. Clinical isolates of B. longum has demonstrated a slightly more differences between each other: B. longum ICIS-505 strain contains more than 200 genes more than B. longum MC-42 reference strain, where are 29 genes - homologoues with known function. These genes are distributed uniformly by functional groups. Conclusion. Obtained data of genome analysis of the bifidobacteria reflect their specialization in occupied biotope and mutu-alistic reliability, determining dominance role of bifidoflora in human gut microsymbiocoenosis. Genome size, stability of signal census and predictability of reactions of the bifidobacteria allow to use them as a general model suitable for studying of symbiotic relations of human and his/her microbiota as well as construction of the experimental systems of intermicrobial interactions.

Crystal Structure of a ligand-bound LacY–Nanobody Complex

The lactose permease of Escherichia coli (LacY), a dynamic polytopic membrane transport protein, catalyzes galactoside/H+ symport and operates by an alternating access mechanism that exhibits multiple conformations, the distribution of which is altered by sugar-binding. Camelid nanobodies were made against a double-mutant Gly46 → Trp/Gly262 → Trp (LacYWW) that produces an outward-open conformation, as opposed to the cytoplasmic open-state crystal structure of WT LacY. Nanobody 9047 (Nb9047) stabilizes WT LacY in a periplasmic-open conformation. Here, we describe the X-ray crystal structure of a complex between LacYWW, the high-affinity substrate analog 4-nitrophenyl-α-d-galactoside (NPG), and Nb9047 at 3-Å resolution. The present crystal structure demonstrates that Nb9047 binds to the periplasmic face of LacY, primarily to the C-terminal six-helical bundle, while a flexible loop of the Nb forms a bridge between the N- and C-terminal halves of LacY across the periplasmic vestibule. The bound Nb partially covers the vestibule, yet does not affect the on-rates or off-rates for the substrate binding to LacYWW, which implicates dynamic flexibility of the Nb-LacYWW complex. Nb9047-binding neither changes the overall structure of LacYWW with bound NPG, nor the positions of side chains comprising the galactoside-binding site. The current NPG-bound structure exhibits a more occluded periplasmic vestibule than seen in a previous structure of a (different Nb) apo-LacYWW/Nb9039 complex that we argue is caused by sugar-binding, with major differences located at the periplasmic ends of transmembrane helices in the N-terminal half of LacY.