Oversized galactosides as a probe for conformationaldynamics in LacYIrina Smirnovaa, Vladimir Kashoa, Xiaoxu Jianga, Hong-Ming Chenb, Stephen G. Withersb, and H. Ronald Kabacka,c,d,1

aDepartment of Physiology, University of California, Los Angeles, CA 90095; bDepartment of Chemistry, University of British Columbia, Vancouver, BC V6T1Z1, Canada; cDepartment of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095; and dMolecular BiologyInstitute, University of California, Los Angeles, CA 90095

Contributed by H. Ronald Kaback, February 23, 2018 (sent for review January 18, 2018; reviewed by Phillip E. Klebba and Christopher Miller)

Binding kinetics of α-galactopyranoside homologs with fluores-cent aglycones of different sizes and shapes were determinedwith the lactose permease (LacY) of Escherichia coli by FRET fromTrp151 in the binding site of LacY to the fluorophores. Fast bindingwas observed with LacY stabilized in an outward-open conforma-tion (kon = 4–20 μM−1·s−1), indicating unobstructed access to thebinding site even for ligands that are much larger than lactose.Dissociation rate constants (koff) increase with the size of the agly-cone so that Kd values also increase but remain in the micromolarrange for each homolog. Phe27 (helix I) forms an apparent con-striction in the pathway for sugar by protruding into the periplas-mic cavity. However, replacement of Phe27 with a bulkier Trp doesnot create an obstacle in the pathway even for large ligands, sincebinding kinetics remain unchanged. High accessibility of the bind-ing site is also observed in a LacY/nanobody complex with par-tially blocked periplasmic opening. Remarkably, E. coli expressingWT LacY catalyzes transport of α- or β-galactopyranosides withoversized aglycones such as bodipy or Aldol518, which may re-quire an extra space within the occluded intermediate. The re-sults confirm that LacY specificity is strictly directed toward thegalactopyranoside ring and also clearly indicate that the openingon the periplasmic side is sufficiently wide to accommodate thelarge galactoside derivatives tested here. We conclude that the ac-tual pathway for the substrate entering from the periplasmic side iswider than the pore diameter calculated in the periplasmic-openX-ray structures.

membrane transport proteins | lactose permease | fluorescence |nanobodies | stopped-flow

The mechanism of lactose/H+ symport catalyzed by the lactosepermease (LacY) of Escherichia coli is based on the alter-

nating access of single sugar- and H+-binding sites in the middleof the molecule to either side of the membrane (1, 2). Openingand closing of hydrophilic cavities in the LacY molecule havebeen demonstrated by numerous biochemical/spectroscopic ap-proaches (3–10) and also are depicted in X-ray crystal structures(11–17). Nevertheless, the dynamic aspects of the mechanismneed further clarification.The highly dynamic nature of LacY is well documented. Thus,

the entire backbone is accessible to water, since hydrogen/deu-terium exchange of backbone amide protons occurs at a veryrapid rate with LacY either solubilized in detergent (18, 19) orembedded in a phospholipid membrane (20). The turnovernumber of the overall lactose transport cycle is relatively low(∼20 s−1) (21) and is virtually identical to the rate of opening ofthe periplasmic cavity (7, 8). The kinetic scheme of transportincludes multiple conformations (1), and at least some confor-mational transitions appear to be fast (200–250 s−1) (22–24).Kinetic parameters for substrate binding have been de-

termined with high-affinity sugars by FRET from Trp151,which is in the sugar-binding site, to bound 4-nitrophenyl α-D-galactopyranoside (α-NPG) by direct measurement or by dis-placement of bound α-NPG by β-D-galactopyranosyl-1-thio-β-D-galactopyranoside (TDG), which is not a FRET acceptor (22).The studies show clearly that the rate of cavity opening limits

sugar binding on either side of LacY and that sugar has freeaccess to the binding site when the outward- or inward-facingcavity is open (8, 24).The extent of opening of the periplasmic cavity that provides

access to the binding site and the rate of opening are importantelements for understanding the transport mechanism, and theyhave been subjected to a number of studies (4–10, 25, 26). Theconformation of WT LacY with an open periplasmic cavity isstabilized by bound sugar (3–5) or by interaction with nano-bodies (Nbs) developed against a periplasmic-open mutantG46W/G262W (LacYWW) (27). Both outward- and inward-openconformations are characterized kinetically by linear concen-tration dependence of sugar-binding rates with high association-rate constants (kon = 5–10 μM−1·s−1) (10, 27).All X-ray structures of LacYWW in an outward-open confor-

mation (15–17) exhibit a tightly sealed cytoplasmic side and aperiplasmic cavity with a relatively narrow opening in sugar-bound conformers with either TDG [Protein Data Bank (PDB)ID code 4OAA] or α-NPG (PDB ID code 4ZYR) and in anNb9039-bound structure without sugar (PDB ID code 5GXB).All three structures show that Phe27, at a kink in helix I, bulgesinto the periplasmic cavity and forms an apparent constriction inthe pathway for sugar (Fig. 1). Trp151 and other residues thatcomprise the sugar-binding site are positioned further inside theperiplasmic opening. In addition, the X-ray structure of the LacY/Nb9039 complex (17) shows that complementarity-determiningregion 3 (CDR3) of the Nb extends into the periplasmic opening

Significance

LacY catalyzes stoichiometric symport of lactose and an H+

across the membrane by an alternating access mechanism and isa paradigm for the major facilitator superfamily, the largestfamily of membrane transport proteins. The established mech-anism is expanded here by kinetic studies involving binding andtransport of greatly oversized substrates. Data demonstrate thehigh-affinity galactoside-specific interaction of LacY with largefluorescent substrates and, importantly, unrestricted accessi-bility of the sugar-binding site in the periplasmic-open confor-mation of the transporter. Moreover, oversized aglycones ofgalactosidic substrates do not preclude active transport; to ac-commodate a large-size ligand within occluded intermediate, itis likely that in the highly flexible LacY molecule additionalmovements in helical bends and kinks occur during the forma-tion of extra space.

Author contributions: I.S., V.K., and H.R.K. designed research; I.S., V.K., and X.J. per-formed research; I.S., V.K., H.-M.C., and S.G.W. contributed new reagents/analytic tools;I.S., V.K., and H.R.K. analyzed data; and I.S., V.K., S.G.W., and H.R.K. wrote the paper.

Reviewers: P.E.K., Kansas State University; and C.M., Howard Hughes Medical Institute,Brandeis University.

The authors declare no conflict of interest.

Published under the PNAS license.1To whom correspondence should be addressed. Email: rkaback@mednet.ucla.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1800706115/-/DCSupplemental.

Published online March 30, 2018.

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between N- and C-terminal six-helix bundles and could be an ob-stacle with respect to accessibility of the binding site (Fig. 1).The calculated diameter of the narrowest part of the peri-

plasmic cavity at Phe27 in the outward-open conformer of LacYis 3–5 Å (15–17), as determined by the HOLE (28) or PoreWalker(29) programs. This pore size appears to be too narrow for di-saccharide sugars (like lactose) to enter from the periplasmic sideand conflicts with direct demonstrations of rapid sugar binding.For example, α-NPG binds to LacYWW reconstituted into pro-teoliposomes with a kon of 10 μM−1·s−1 in the absence or presenceof bound Nb9039 (17), indicating free access to the binding site.Moreover, it was shown that substrates larger than lactose arerecognized and translocated by WT LacY. Thus, binding (30) andtransport (31) of dansyl-galactosides have been described earlier.Also, accumulation of bodipy-labeled N-acetyl-β-D-galactosamineby E. coli was observed only if the lacY gene was expressed (32).

The smallest transport substrate for LacY is D-galactose, sincerecognition is directed toward the galactopyranosyl ring with ahydroxyl at carbon atom C-4 as the major determinant for speci-ficity, as well as hydroxyls at C-2, C-3, and C-6 (33–35). Bindingaffinity for galactose is low (Kd = 30 mM) but increases dra-matically with hydrophobic aglycones at the anomeric oxygen,particularly in α-substituted D-galactopyranosides. Thus, theaffinity for α-NPG is three orders of magnitude better (Kd =14 μM) than for galactose. Sugar binding is the first step and isthe driving force for the alternating access mechanism, whichinvolves conformational change to an occluded intermediate,followed by the opening of the inward-facing cavity. Theseconformational transitions are expected to be altered by the li-gand, in which the structural fragment attached to the anomericposition of galactose is larger and far more irregularly shapedthan the glucose moiety in lactose. Here galactopyranosidescontaining different fluorescent aglycones were used to study thebinding kinetics and transport activity of LacY, with special at-tention to the role of Phe27 and the effect of the CDR3 loop ofNb9039 on accessibility of the sugar-binding site.

ResultsKinetic Parameters of Galactosides Binding.Spectral properties of galactoside derivatives. Fluorescent deriva-tives of substituted α-galactopyranosides were selected for theirability to act as acceptors of FRET from Trp151 located in thesugar-binding site of LacY (Fig. 1) as demonstrated for α-NPG(22). Purified LacY exhibits a Trp emission spectrum with amaximum at 340 nm, which overlaps with the absorptionspectra of 6-bromo-2-naphthyl α-D-galactopyranoside (α-BNG),4-methylumbelliferyl α-D-galactopyranoside (α-MUG), bodipyα-D-galactopyranoside (α-bodipy-Gal), and Aldol518 α-D-gal-actopyranoside (α-Aldol-Gal) (Fig. S1). As shown, the spectralproperties are favorable for energy transfer from Trp151 to theaglycones if the galactosides bind to LacY.Binding of galactosides to LacY with a periplasmic-open cavity. The ac-cessibility of the sugar-binding site to selected α-galactosides wasexamined with LacY stabilized in an outward-open conformationby using two constructs: (i) WT LacY with bound Nb9048 (WTLacY/Nb9048 complex) (27) and (ii) WT LacY with two Trpresidues in the region of contact between the N- and C-terminalhalves (G46W/G262W; LacYWW) (10). Binding of ligands wastested by measuring stopped-flow rates of the decrease in Trp(donor) fluorescence after mixing LacY with a given α-galacto-side as shown for α-NPG (22). As described previously, both theWT LacY/Nb9048 complex and mutant LacYWW bind α-NPGwith a high kon (∼5–6 μM−1·s−1), indicating unrestricted access tothe binding site in contrast toWT LacY (kon = 0.2 μM−1·s−1) (10, 27).Binding rates (kobs) were measured at increasing concentra-

tions of the substituted α-galactosides (Fig. S2A). All four (BNG,MUG, bodipy-Gal, and Aldol-Gal) are characterized by high konvalues that vary from 4 to 20 μM−1·s−1 as estimated from linearconcentration dependencies of the kobs (Fig. 2, black symbolsand Table S1). Clearly, each α-galactoside, even those with a sizemore than three times that of galactose, binds readily to LacYwith a periplasmic-open cavity, and there is no indication of con-formational change preceding binding. No correlation is observedbetween accessibility and the overall size/shape of the ligand.Dissociation rates of bound α-galactosides (true koff values) were

measured by displacement with excess TDG (Fig. S3A). Trp fluo-rescence increases due to the release of bound galactoside replacedby TDG, which is not a FRET acceptor. Displacement rates areindependent of concentration and are specific for each galactosidederivative. Measured koff values are in good agreement with thoseestimated from binding experiments, as the intersection of linear fitswith the y axis (Fig. 2, open black symbols and black lines; TableS1). There is a clear correlation between the size of the ligand andkoff : the larger the galactoside, the higher the off rate (Table S1).The increase in koff results in an increase in Kd (calculated as theratio of dissociation and association rate constants: Kd = koff/kon),and binding affinity decreases with the size of the ligand, but in

Fig. 1. Periplasmic-open conformer LacYWW. The structural model viewedfrom the side (PDB ID code 5GXB) is shown with N- and C-terminal six-helicalbundles colored gray and cyan, respectively, and Nb9039 attached to theperiplasmic side shown in red. Residues participating in sugar binding arepresented as yellow sticks. Residue Phe27 located in the middle of the per-iplasmic cavity was replaced with Trp and is shown as pink spheres. Inter-acting residues in the area of contact between the CDR3 loop of the Nb(Tyr104) and the periplasmic end of helix I of LacY (Lys42) are shown as redand gray spheres, respectively. Structural models of the α-galactosides withdifferent aglycones are also shown.

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general, the affinity of LacY for all the α-galactosides tested isrelatively high (Kd = 2–18 μM).Effect of the F27W mutation. The F27W mutation places a bulkierTrp residue in the middle of the periplasmic cavity, which shouldcreate a greater obstacle to sugar binding than the native Phe.However, the binding of α-galactoside derivatives to the complexF27W LacY/Nb9048 is similar to that observed for the WTLacY/Nb9048 complex (Fig. 2A, red and black symbols; Figs. S2and S3). With each ligand, kon is high and similar for WT LacYand mutant F27W, koff increases with the size of the ligand, andKds are in the micromolar range (Table S1). The replacementF27W in the LacYWW background does not significantly changethe binding rates of the ligands or the high-affinity Kds (Fig. 2B,red and black symbols and Table S1). Therefore, despite anapparently obstructed periplasmic pathway due to the insertionof Trp in place of Phe27, the sugar-binding site is freely acces-sible even to the galactoside with the largest aglycone.The LacY/Nb9039 complex. The X-ray structure of the LacYWW/Nb9039 reveals that the Nb binds primarily to the periplasmicface of the C-terminal half of LacY. However, Tyr104 in theCDR3 forms an H-bond with Lys42 in the N-terminal six-helixbundle and appears to hamper periplasmic access to the bindingsite (Fig. 1). Accessibility of the sugar-binding site in LacYWW/Nb9039 was tested with α-galactosides NPG, BNG, MUG, andbodipy-Gal. The dissociation rate constant (koff) for each ligandis about 1.5–2 times higher with the LacYWW/Nb9039 complexthan with LacYWW alone, which hardly increases Kd significantly

(Table S1). However, all ligands exhibit linear concentration de-pendencies with fast binding rates (kon = 5–8 μM−1·s−1) and high-affinity binding (Kd = 10–25 μM), which is very similar to thatobserved with LacYWW in the absence of Nb (Fig. 3, red and blacksymbols; Figs. S4 and S5 and Table S1). Therefore, the sugar-binding site is easily accessible to each galactoside despite theapparent obstacle that created by CDR3 in the structural model.

Transport of Oversized Galactosides by WT LacY.Fluorescent substrates. Accumulation of α- and β-galactopyrano-sides containing fluorescent aglycones was measured byemploying the distinctive spectral properties of each galactosidehomolog. The fluorescence spectra of MUG and Aldol-Galmanifest dramatic changes after enzymatic cleavage by galacto-sidase. In both cases, release of the free fluorophore is accom-panied by an increase in fluorescence intensity with a spectralshift to a longer wavelength, from 379 to 445 nm for MUG andfrom 490 to 595 nm for Aldol-Gal (Fig. S6 A and B). Uptake ofβ-MUG (4-methylumbelliferyl β-D-galactopyranoside) or β-Al-dol-Gal by E. coli T184 cells expressing LacY was measuredafter the disruption of cells preincubated with galactosides andtreatment of accumulated galactoside homologs with addedβ-galactosidase, since cells lack β-galactosidase activity (lacZ−).Galactoside derivatives with attached bodipy exhibit a high levelof fluorescence that does not change after cleavage with galac-tosidase (Fig. S6C), and enzymatic treatment was omitted(Methods).Accumulation of MUG. β-MUG transport was measured with E. coliT184 cells (ΔlacZY) expressing WT LacY or mutants that do notcatalyze active lactose transport, E325A with blocked H+ trans-fer (36) or LacYWW with blocked conformational change (10).Cells with WT LacY exhibit an increase in fluorescence withtime (Fig. 4A) due to β-MUG uptake, while control cells showpractically no fluorescence change for 30 min (Fig. 4B). Asteady-state level of accumulation is reached after ∼20 min in-cubation with 50-μM β-MUG and corresponds to ∼3 nmol/mg oftotal cell protein (Fig. 4C) as calculated from a calibration curve(Fig. S6D).With α-MUG, a different methodology was used. Accumula-

tion of galactoside after the addition of α-MUG to a suspensionof intact E. coli T184 cells expressing WT LacY was continuouslyrecorded as the fluorescence increase due to intracellular

Fig. 2. Accessibility of the sugar-binding site in LacY with an outward-opencavity and the effect of the Phe27→Trp mutation. Binding of five α-galac-tosides was measured directly as FRET from Trp151 of LacY to bound ligandas described in Methods. Two outward-facing constructs were used: the WTLacY/Nb9048 complex (A) and mutant LacYWW (B). Binding rates (kobs, filledsymbols) were estimated from single-exponential fits of stopped-flow tracesat each galactoside concentration (Fig. S2), and koffs (open symbols) weremeasured as the rate of displacement of bound ligand with an excess of TDG(Fig. S3). The slopes of the linear fits of the concentration dependencies ofbinding rates gave estimates of kon for each galactoside derivative. Blackand red symbols represent data obtained before and after replacement ofPhe27 with Trp, respectively, for each galactoside (circles: NPG; squares: BNG;triangles: MUG; stars: bodipy-Gal; diamonds: Aldol-Gal) (Table S1). NPGbinding rates measured with WT LacY without Nb9048 are shown forcomparison (green circles).

Fig. 3. Accessibility of the sugar-binding site in the LacYWW/Nb9039 complex.Binding rates of different α-galactosides were measured as described in Fig. 2using the outward-open LacY conformer with or without Nb9039 (red andblack symbols, respectively). The values of kobs (filled symbols) and koff (opensymbols) were estimated from single-exponential fits of stopped-flow traces(Figs. S4 and S5). The slopes of the linear fits of the concentration dependenciesof binding rates gave estimates of the kon for each galactoside derivative(circles: NPG; squares: BNG; triangles: MUG; stars: bodipy-Gal) (Table S1).

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α-galactosidase activity and the release of free dye (Fig. 5). Incontrast to WT LacY, the E325A or LacYWW mutants exhibit noα-MUG uptake, nor do cells with no LacY or with WT LacYpreincubated with N-ethylmaleimide (NEM), an inactivator oflactose transport. In addition, no fluorescence change was ob-served when β-MUG was added to the cells expressing WT LacY(Fig. 5, red dots), confirming the absence of intracellular β-ga-lactosidase (lacZ−), although uptake of β-MUG by the same cellsis documented in Fig. 4.Accumulation of bodipy- or Aldol-galactosides. Remarkably, galacto-sides containing aglycones larger than MUG (α- or β-bodipy-Gal,β-bodipy-Lact, and β-Aldol-Gal) are transported by WT LacY(Fig. 6 and Fig. S7). Essentially no fluorescence change is detectedin cells without LacY, and transport of each homolog to a steady-state level of accumulation with cells containing WT LacY isobserved (Fig. 6B, open and filled symbols, respectively). Trans-port was measured at a galactoside concentration of 50 μM, anddifferent steady-state levels of accumulation were achieved (0.7–6.2 nmol/mg protein) (Fig. 6B), as estimated from calibrationcurves for each fluorophore (Fig. S6). This level is about 3% orless relative to the transport of lactose (the steady-state level is210 nmol/mg) measured at 0.4 mM, the approximate Km for lac-tose (Fig. 6A, black circles). A similar low level of accumulationwas also observed with the high-affinity substrate TDG (1.2 nmol/mgprotein at 10-μM TDG) under similar conditions (37). Surpris-ingly, transport of lactose is unaffected by the F27W mutation(Fig. 6A, stars), which is consistent with results of the bindingassays described above (Fig. 2).The size of substrate clearly affects the rate of transport:

Slower accumulation of galactosides with larger aglycones isobserved. Thus, a steady-state level is achieved after 10-min in-cubation with lactose (Fig. 6A) and after 20–40 min with β-MUGor β-bodipy-Lact but only after about 2 h with α- or β-bodipy-Galor β-Aldol-Gal (Fig. 6B). α-Aldol-Gal accumulation by cells

expressing WT LacY is clearly detected, but at a much lower ratethan observed with β-Aldol-Gal (Fig. S8), and may reflect thelow intracellular α-galactosidase activity. Transport of β-bodipy-Lact is slower than the transport of lactose (steady-state levels ofaccumulation are reached in 40 and 10 min, respectively), which isconsistent with significantly lower koff for β-bodipy-Lact, as shownby displacement kinetics with LacYWW. Thus, koff values mea-sured for β-bodipy-Lact (Fig. S9 A and B) and lactose (Fig. S9C)are 70 ± 12 s−1 and 425 ± 24 s−1, respectively. Apparently, bodipyincreases binding affinity by decreasing koff and also decreases therate of β-bodipy-Lact translocation across the membrane.

DiscussionWe demonstrate here that galactoside derivatives containinglarge, variously shaped fluorescent aglycones bind to LacY andare transported across the E. coli cytoplasmic membrane. Bindingof different-sized galactosides was studied specifically with WTLacY stabilized in the outward-open conformation (WT LacY/Nb9048 complex) or with the outward-facing mutant LacYWW,which exhibits a rather narrow periplasmic pathway to the sugar-binding site in the crystal structure (15–17). Surprisingly, the re-sults clearly demonstrate that access to the binding site is un-obstructed even for the largest ligands. Binding rates are rapid,and concentration dependencies are linear (kon = 4–20 μM−1·s−1).In other words, if the cavity is already open, no additional openingoccurs. Rates (kobs) increase linearly up to 500–800 s−1, whichexcludes the involvement of a global conformational change with aslower rate, e.g., opening of the cavity, as a rate-limiting process.In LacY the fastest rates of conformational change, which involvemovements of transmembrane helices, are less than 300 s−1 (8, 22,24). Therefore, the opening of the cavity on the periplasmic side issufficiently wide to accommodate each of the galactoside deriva-tives tested here, and they vary considerably in both size andshape. Moreover, increasing the bulk of a side chain at the nar-rowest region of the periplasmic cavity in the F27W mutant orbinding of Nb9039, which forms a bridge between the two six-helixbundles of LacY across the periplasmic cavity, has no effect on theaccessibility of the binding site. So, the question remains: How dosuch large and variously shaped substrates gain ready access to thebinding site of LacY through a relatively narrow opening of theperiplasmic cavity?

Fig. 4. Transport of β-MUG. The accumulation of MUG was measured usingE. coli T184 cells (ΔlacZY) expressing WT LacY (red), the E325A (green) orLacYWW (blue) mutants, or no LacY (cyan) as described in the text and inMethods (see also Fig. S6). Cells incubated with β-MUG for a given time werewashed with acidic Na-acetate (pH 4.8), disrupted by sonication, and treatedwith β-galactosidase. (A) Fluorescence change in cells containing WT LacYduring a 30-min incubation with 50-μM β-MUG. (B) Fluorescence change incells containing LacY mutants or no LacY after a 30-min incubation underthe same conditions. Dotted lines show fluorescence at time 0. (C) Timecourse of β-MUG uptake. Transport activity was calculated from fluorescenceintensities at 445 nm in A and B using the calibration curve (Fig. S6D).

Fig. 5. Transport of α-MUG. The accumulation of MUG was recorded as thefluorescence increase with time in intact E. coli T184 cells (ΔlacZY) expressingWT LacY (red), the E325A (green) or LacYWW (blue) mutants, or no LacY(cyan) as described in the text and in Methods. The arrow indicates theaddition of 10-μM α-MUG to cells at 0.6 mg/mL total protein. Uptake ofα-MUG was detected as an increase in fluorescence due to enzymaticcleavage of accumulated α-galactoside by intracellular α-galactosidase. Ascontrols, cells containing WT LacY were either pretreated with NEM, aninactivator of lactose transport (10 mM for 20 min) (black dots) or weremixed with 10-μM β-MUG (red dots).

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In the X-ray structures of outward-open LacYWW (15–17) thewidth of the periplasmic cavity is defined by the side chains ofthe residues lining the cavity, such as Phe27 or Gln241, and bythe configuration of the helices, particularly I and VII. Therelative positions of the periplasmic ends of the transmembranehelices are in good agreement with distances obtained previouslyby double electron–electron resonance (DEER) measurementsusing sugar-bound LacY (5) or by thiol cross-linking experimentswith homobifunctional reagents of different lengths (9). Cross-linking data reveal that flexible reagents greater than 15 Å inlength do not block lactose transport, which is totally consistentwith the distances measured in the periplasmic-open X-raystructure between residues in Cys pairs used for cross-links (Fig.S10A). In addition, structural alignment of the same periplasmic-open mutant LacYWW with the model of the outward-facingconformer based on DEER data (5) demonstrates remarkablesimilarity between the crystal structure and the model (Fig. S10B).The structural similarity suggests that the actual cleft betweentransmembrane helices on the periplasmic side observed in X-raystructures is large enough to provide free access to the binding siteand is not hindered by amino acid residues facing the cavity.Structural fluctuations of the side chains protruding into the cavityare expected to be extremely fast and apparently do not precludefree access to the binding site, even for bulky ligands. In that re-spect, the observations provide a strong indication that the open-periplasmic pathway in LacY is unlikely to be a smooth, cylindricaltube with a diameter of 3–5 Å, as estimated in outward-open X-raystructures using pore-measuring programs designed for channels(28, 29).

Translocation of oversized galactosides across the membraneis measured with WT LacY in E. coli cells and therefore char-acterizes the mechanism of the entire transport cycle. It is clearthat Phe27 does not obstruct lactose transport, since no changein transport activity occurs after the replacement of this residuewith Trp (Fig. 6A) or with Cys (38). The observed translocation ofdifferent galactosides is completely abrogated in E. coli cells con-taining the E325A LacY mutant (Figs. 4 and 5) because this mu-tation blocks any reactions involving net H+ transfer, while bindingof the substrate and all conformational changes occur, as evidentfrom lactose equilibrium exchange (36). Therefore, transport ofoversized galactosides is H+-coupled. The restriction of the con-formational changes as shown for the outward-open LacYWW mu-tant (10) also eliminates the transport of galactosides (Figs. 4 and 5).The observed translocation of the large-sized galactoside de-

rivatives confirms that LacY specificity is strictly directed towardthe galactopyranoside ring, and aglycones do not preclude activetransport. However, large substrates are transported more slowlythan is lactose, and the rate is most likely limited by conforma-tional transitions of the LacY molecule. Formation of the oc-cluded intermediate with bound oversized ligand likely requiresextra space within the binding site, and additional movements inhelical bends and kinks may occur, which could slow the rate ofglobal conformational changes.

MethodsThe materials used in this study, construction of mutants, and purification ofLacY, are described in SI Methods.

Sugar Binding. Rates of galactoside binding (observed rates, kobs) were mea-sured directly by rapid (stopped-flow) mixing of purified protein (0.5 μM finalconcentration) with each ligand as a decrease in Trp fluorescence due to FRETfrom Trp151 in LacY to the aglycone of a given galactoside (22). Displacementrates (dissociation rate constants, koff) were measured as an increase in Trpfluorescence due to the release of the bound FRET acceptor in experimentswhere LacY was first premixed with a given concentration of galactosidederivative for 10 min and then rapidly mixed with a saturating concentrationof TDG (20 mM final concentration), which is not a FRET acceptor. Therefore,Trp151 fluorescence increases at a rate equal to the dissociation rate of boundgalactoside displaced by TDG. Where indicated, Nb9048 or Nb9039 was addedin a 1.2-fold molar excess to LacY (30–40 μM) and kept at room temperaturefor 20 min before mixing with galactoside. All binding experiments weredone in 50-mM NaPi/0.02% n-Dodecyl-β-D-maltoside (DDM; Affymetrix) (pH7.5). Linear fit of concentration dependence of observed rates (kobs = koff +kon[ligand]) was used to estimate the association rate constant, kon. Bindingaffinity was calculated as the ratio of rate constants: Kd = koff/kon. A filtereffect of the ligands on the fluorescence of Trp does not alter the ratesmeasured by stopped flow.

Transport Assays. Lactose transport in E. coli T184 cells (ΔlacZY) transformedwith pt7-5 plasmid containing WT LacY or no LacY was measured at roomtemperature with 0.4-mM [14C]lactose (5 mCi/mmol) in 50 μL of cell suspensions(35 μg total protein) in 100-mM potassium phosphate (KPi)/10 mM MgSO4

(pH 7.2) as described (10). Reactions were terminated at given times by di-lution in 3 mL 100-mM KPi/100 mM LiCl (pH 5.5) and rapid filtration to collectcells that were assayed for radioactivity by scintillation spectrometry (39).

Transport of fluorescent sugars was measured in E. coli T184 cells preparedin the same way as for lactose transport measurements. Two approacheswere used. (i) Cells at 2 mg/mL total protein in 100-mM KPi/10-mM MgSO4

(pH 7.2) were mixed with 50-μM galactoside derivative and incubated for thegiven times at room temperature. Reactions were terminated by 10-folddilution of the 0.2-mL cell suspension in ice-cold 50-mM sodium acetate(pH 4.8). Cells were collected by centrifugation at 20,000 × g for 3 min at4 °C, rinsed with the same buffer, suspended in 1 mL of 50-mM NaPi (pH 7.5),and disrupted by sonification for 30 s at power 4 using a Misonix 3000Sonifier equipped with a microtip (Misonix, Inc.). The sonicated samplescontaining β-MUG or β-Aldol-Gal were treated with β-galactosidase (50 units/mL) for 1 h at room temperature, since the free fluorophores exhibit dra-matically higher-intensity emission spectra shifted to a longer wavelengthrange. Fluorescence spectra were recorded in 2.5 mL of 50-mM NaPi (pH 7.5).The addition of detergent (0.1% DDM) to the samples containing β-Aldol-Gal during β-galactosidase treatment and fluorescence measurements wasessential for preventing Aldol518 precipitation. (ii) Continuous monitoring of

Fig. 6. Transport of galactopyranosides with different-sized aglycones. (A)Time courses of the accumulation of [14C] lactose (0.4 mM) by E. coliT184 cells (ΔlacZY) expressing WT LacY (filled circles), mutant LacY F27W(stars), or no LacY (open circles) were measured by scintillation spectrometryas described in Methods. The steady-state level of 100% corresponds to210 nmol/mg of total protein. (B) Time courses of accumulation of differentgalactoside derivatives (50 μM) by cells expressing WT LacY (filled symbols) orno LacY (open symbols). Transport was measured with β-MUG (green circles),β-bodipy-Lact (purple squares), β-bodipy-Gal (blue triangles), α-bodipy-Gal(cyan triangles), and β-Aldol-Gal (red diamonds) as described in Fig. 4, butsamples containing bodipy-galactosides were not treated with β-galactosidase(Fig. S6). The fluorescence changes in cells during incubation with β-bodipy-Lact, β-bodipy-Gal, α-bodipy-Gal, and β-Aldol-Gal are shown in Fig. S7. Trans-port activity was calculated from fluorescence intensities using the calibrationcurves (Fig. S6 E and F for Aldol and bodipy, respectively). The steady-statelevel of 100% corresponds to 2.7, 0.72, 1.0, 0.86, and 6.2 nmol/mg of totalprotein for β-MUG, β-bodipy-Lact, β-bodipy-Gal, α-bodipy-Gal, and β-Aldol-Gal,respectively.

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α-MUG uptake by intact E. coli T184 cells: The accumulation of α-MUG wasdetected as the fluorescence increase due to enzymatic cleavage of trans-ported α-galactoside by intracellular α-galactosidase activity. The fluorescencechange indicating the release of free fluorophore with higher-intensity emis-sion was recorded directly in the fluorimeter cuvette. The reaction was startedby the addition of 10-μM α-MUG to the cell suspension (0.6 mg/mL of totalprotein) in 2.5 mL of 50-mM NaPi (pH 7.5).

Fluorescence Measurements. Steady-state fluorescence was measured at roomtemperature on a SPEX Fluorolog-3 spectrofluorometer (Horiba) in a 2.5-mLcuvette (1 × 1 cm) with constant stirring. Trp emission spectra of purified WTLacY (0.5 μM) were recorded at an excitation wavelength of 295 nm in50-mM NaPi/0.02% DDM (pH 7.5). Emission spectra of galactosides withfluorescent aglycones were recorded at excitation wavelengths of 320, 460,and 370 nm for methylumbelliferyl, bodipy, and Aldol518, respectively, in50-mM NaPi (pH 7.5) with the addition of 0.1% DDM to samples containingAldol518. Continuous recording of the uptake of α-MUG by E. coli T184 cellswas performed at excitation and emission wavelengths of 320 and 445 nm,respectively, in 2.5 mL of 50-mM NaPi (pH 7.5).

Stopped-flowmeasurements were performed at 25 °C in an SFM-300 rapidkinetic system equipped with a TC-50/10 cuvette (dead-time 1.2 ms) and aMOS-450 spectrofluorimeter (Bio-Logic USA, LLC). Binding of galactosideswas measured directly as FRET from Trp151 of LacY to bound ligand at anexcitation wavelength of 295 nm with collection of emitted light using anIF340 interference filter with bandwidth of 26 nm (Edmund Optics). Stopped-flow traces were recorded at final concentration 0.5–1 μMof LacY after mixingwith galactosides. In displacement experiments, LacY was preincubated withindicated galactoside for 10 min and then mixed with an excess of TDG(20 mM final concentration). Measurements with purified LacY were done in50-mM NaPi/0.02% DDM (pH 7.5). Typically, 10–30 traces were recorded foreach data point, averaged, and fitted with an exponential equation using thebuilt-in Bio-Kine32 software package or by using SigmaPlot 10 (Systat Software,Inc.). Calculated SDs were within 10% for each presented data point. All givenconcentrations were final after mixing.

ACKNOWLEDGMENTS. This work was supported by NIH Grant GM120043,National Science Foundation Eager Grant MCB1747705, and a gift from Ruthand Bucky Stein (to H.R.K.). S.G.W. received funding support from theCanadian Glycomics Network of Centres of Excellence.

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