Transient conformers of LacY are trapped by

Transient conformers of LacY are trapped by nanobodies Irina Smirnovaa, Vladimir Kashoa, Xiaoxu Jianga, Els Pardonb,c, Jan Steyaertb,c,1, and H. Ronald Kabacka,d,e,1 a Department of Physiology, University of California, Los Angeles, CA 90095-7327; bStructural Biology Research Center, Vrije Universiteit Brussel, 1050 Brussel, Belgium; cStructural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussel, Belgium; dMicrobiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095-7327; and eMolecular Biology Institute, University of California, Los Angeles, CA 90095-7327

The lactose permease of Escherichia coli (LacY), a highly dynamic membrane protein, catalyzes symport of a galactopyranoside and an H+ by using an alternating access mechanism, and the transport cycle involves multiple conformational states. Single-domain camelid nanobodies (Nbs) developed against a LacY mutant immobilized in an outward (periplasmic)-open conformation bind to the flexible WT protein and stabilize the open-outward conformation(s). Here, we use site-directed, distance-dependent Trp quenching/ unquenching of fluorescent probes inserted on opposite surfaces of LacY to assess the conformational states of the protein complexed with each of eight unique Nbs that bind exclusively to the periplasmic side and block transport, but increase the accessibility of the sugar-binding site. Nb binding involves conformational selection of LacY molecules with exposed binding epitopes. Each of eight Nbs induces quenching with three pairs of cytoplasmic Trp/ fluorophore probes, indicating closing of cytoplasmic cavity. In reciprocal fashion, the same Nbs induce unquenching of fluorescence in three pairs of periplasmic probes due to opening of the periplasmic cavity. Because the extent of fluorescence change with various Nbs differs and the differences correlate with changes in the rate of sugar binding, it is also concluded that the Nbs stabilize several different outward-open conformations of LacY.

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membrane transport proteins lactose permease conformers alternating access

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ymport of lactose and an H+ across the membrane is catalyzed by lactose permease of Escherichia coli (LacY) by an alternating access mechanism using reciprocal opening and closing of deep water-filled cavities on periplasmic and cytoplasmic sides of the protein. By this means, sugar- and H+-binding sites are exposed to either side of the membrane without providing a continuous pathway. The overall transport cycle involves multiple states that include conformers with outward- or inward-open cavities and occluded intermediates (1). LacY is highly dynamic with a distribution of conformational intermediates. Several X-ray structures of LacY in an inward-facing conformation with a tightly closed periplasmic side have been solved (2–5). More recently, a narrowly outward-open conformation with occluded galactosides and a tightly closed cytoplasmic side was reported (6, 7). Distance measurements using site-directed, double nitroxide-labeled mutants of LacY demonstrate the presence of several intermediates, the distribution of which is altered by sugar binding that shifts the equilibrium toward periplasmic-open forms (8, 9). However, the structure of each intermediate and the details of the transitions during the transport cycle are unclear. Therefore, stabilization of intermediate states is critically important for understanding the partial reactions in the overall transport cycle. Utilization of camelid single-domain nanobodies (Nbs) for trapping specific conformational states of G protein-coupled receptors has led to significant progress in understanding their mechanism(s) (10–14). The small size of the Nbs and a unique ability to stabilize intermediate states of flexible proteins by insertion of antigen-specific binding loops into clefts and cavities are extremely valuable for trapping different conformers of proteins.

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Recently, we reported development of Nbs that specifically bind to the periplasmic side of LacY and trap outward-open conformation(s) (15). The Nbs were prepared against the double-Trp mutant of LacY G46W/G262W reconstituted into proteoliposomes, where the protein is oriented with periplasmic side out, as in the bacterial membrane, so that the periplasmic epitopes are exposed. Purified Nbs bind with nanomolar or subnanomolar affinity to the periplasmic face of WT LacY and inactivate lactose transport. However, the sugar-binding site in the LacY/Nb complexes becomes much more accessible to galactosides (with up to 50 times increased kon values for sugar binding), indicating stabilization of the outward-open conformation(s). In addition, opening of the periplasmic cavity in an Nb-stabilized conformer was demonstrated by unquenching of a bimane-labeled Cys/Trp double-mutant F29W/G262C on the periplasmic side of LacY. Quenching of bimane fluorescence by Trp has been described in T4 lysozyme when distances between Cα atoms of bimane-labeled Cys and Trp residues are within 7–11 Å (16). Unquenching of bimane-labeled Cys262 in the LacY/Nb9036 complex is due to separation of bimane from the quencher (Trp29), which clearly indicates opening of the periplasmic cavity. Moreover, rates of bimane unquenching and binding of Nb9036 measured directly are identical (the kon value is 0.4 μM−1·s−1 in both cases). Thus, the Nb interacts with an existing outward-open conformer of LacY and stabilizes the structure rather than inducing a conformational change, suggesting binding by conformational selection. In this paper, we report the effects of eight unique Nbs on the overall conformation of LacY. Each of the Nbs binds to the periplasmic side of WT LacY and completely blocks active Significance To obtain stable conformers of multiple intermediates in the transport cycle of lactose permease of Escherichia coli (LacY), we use camelid single-chain nanobodies (Nbs) that are ∼15 kDa in size and have a unique structure with flexible antigen-binding loops that can insert into clefts and cavities. Site-directed, distance-dependent quenching/unquenching of fluorescent probes on opposite surfaces of LacY are used in a unique fashion to assess LacY conformers trapped by Nbs that bind exclusively to the periplasmic side and block transport with increased accessibility of the sugar-binding site. The studies conclusively document an alternating access mechanism for transport and provide evidence that the Nbs stabilize several different periplasmicopen conformations of LacY, thereby providing a novel general approach for structure-function studies of membrane proteins. Author contributions: I.S., V.K., and H.R.K. designed research; I.S. and V.K. performed research; I.S., V.K., X.J., E.P., and J.S. contributed new reagents/analytic tools; I.S., V.K., and H.R.K. analyzed data; and I.S., V.K., 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. 1

To whom correspondence may be addressed. Email: [email protected] or jan. [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1519485112/-/DCSupplemental.

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Contributed by H. Ronald Kaback, October 2, 2015 (sent for review September 8, 2015; reviewed by Phillip E. Klebba and Christopher Miller)

transport because of stabilization of specific conformer(s) with increased accessibility of the sugar-binding site (15). Conformational states of the LacY/Nb complexes were examined by site-directed, distance-dependent Trp-induced quenching of fluorophores (16) in LacY mutants with paired fluorophore-labeled Cys and Trp residues located on the cytoplasmic or periplasmic surface of LacY. Quenching with three cytoplasmic pairs and unquenching with three periplasmic pairs are observed in the LacY/Nb complexes, thereby extending the conclusion that the Nbs stabilize conformers in which the periplasmic cavity is open and the cytoplasmic cavity is closed. Moreover, the magnitude of the fluorescence changes differs with different Nbs, and is correlated with changes in the rate of sugar binding, thereby indicating that the Nbs stabilize several distinct outward-open conformers of LacY. Results Bimane-labeled Cys-Trp pairs introduced on opposite sides of LacY are sensitive to closing/opening of cytoplasmic and periplasmic cavities caused by binding or dissociation of sugar, as demonstrated by quenching/unquenching of fluorescent probes (17). Moreover, the rates of conformational changes were measured by stopped-flow using Trp-induced fluorescence quenching/unquenching of bimane located on either side of LacY (17). Testing of bimane- or ATTO655-labeled mutants for an effect of Nb binding on fluorescence led to the selection of three cytoplasmic (R135C/ V343W, N137C/F334W, and F140W/V343C) and three periplasmic (I32C/N245W, K42C/F378W, and F29W/G262C) pairs for detailed analyses. In each mutant, the inserted Trp is in the N-terminal sixhelix bundle and the labeled Cys is in the C-terminal 6-helix bundle, or vice versa (i.e., fluorophore and quencher are placed on opposite sides of cytoplasmic or periplasmic cavities). In the X-ray structures of LacY, Cα-Cα distances between cytoplasmic residues 135/343 (helix IV/loop 10–11), 137/334 (loop 4–5/helix X), and 140/ 334 (helix V/loop 10–11) are 25.1, 25, and 22.5 Å in the inwardopen structure, and 12.4, 15.7, and 14.8 Å in the outward-open structure, respectively. Cα-Cα distances between periplasmic residues 32/245 (helix I/helix VII), 42/378 (helix II/loop 11–12), and 29/262 (helix I/helix VIII) are 6.2, 9.1, and 8.1 Å in the inward-open structure, and 12.4, 18.1, and 10.4 Å in the outward-open structure, respectively. Thus, significant distance changes occur during transition from the inward- to outward-open conformation that are in the range suitable for measurements by Trp-induced fluorophore quenching/unquenching (17). Closing the Cytoplasmic Cavity. LacY is highly dynamic and exhibits multiple conformations. Therefore, Nb-binding epitopes in WT LacY are always accessible in a fraction of conformers with an open periplasmic cavity. However, interaction of Nbs with mutants may be altered by insertion of bulky Trp residues and labeling with fluorophores that may change the conformational distribution. Therefore, the effect of Nb9063 was tested in the absence and presence of sugar, which shifts the equilibrium toward conformers with open periplasmic and closed cytoplasmic cavities (18). In the absence of sugar, Trp-induced quenching of bimane fluorescence after addition of Nb9063 is apparent only in mutant 135C/343W, and little or no effect is observed with the 137C/334W or 140W/343C pair (Fig. 1). However, subsequent addition of p-nitrophenyl-α-D-galactopyranoside (NPG) results in a rapid drop in bimane fluorescence followed by a slower decrease. Furthermore, β-D-galactopyranosyl-1-thio-β-D-galactopyranoside (TDG) binding elicits the same effect (Fig. S1). Nbs 9055 and 9036 exhibit similar results (Fig. S2)–quenching of bimane with the 135C/343W pair, but no effect on the 137C/334W pair in the absence of sugar. NPG added before Nb results in different extents of fluorescence quenching for each mutant (15% for 135C/343W and 10% for 137C/334W), but the total magnitude of quenching does not depend on the order of addition of sugar and Nb (compare green and gray traces in Fig. S2). 2 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1519485112

Fig. 1. Effect of sugar on interaction of Nb with LacY mutants. Structural models of inward- and outward-facing conformers viewed from cytoplasmic side (PDB ID codes 2CFQ and 4OAA on top left and right, respectively) are shown with the backbone rainbow colored (blue to red for helices I to XII, respectively). Residues replaced by site-directed mutagenesis are presented as spheres. Time courses of fluorescence change with three bimane-labeled cytoplasmic mutants 137C/334W, 140W/343C, and 135C/343W were recorded after addition of Nb9063 (red arrow) followed by addition of NPG (black arrow). Single-exponential fits are shown as red lines.

Therefore, in mutants 137C/334W and 140W/343C, the periplasmic epitopes for Nb are not accessible in the absence of substrate, but after galactoside binding, a conformational change occurs and the epitopes became accessible. Possibly, insertion of bulky Trp residues (140W or 334W) in the region of close contact between the N- and C-terminal six-helix bundles on the cytoplasmic surface of LacY (Fig. S3) causes a conformational shift toward the cytoplasmic-open/periplasmic-closed form with inaccessible periplasmic epitopes. Rates of closing of the cytoplasmic cavity triggered by NPG binding (4–200 s−1 for different bimane-labeled Cys-Trp pairs and independent of sugar concentration; ref. 17) are much faster than the rate of quenching induced by Nb9063 (∼0.02 s−1; see red lines in Fig. 1), suggesting that the Nb does not cause a conformational change, but binds to a conformer that already exists. In any case, the dynamic conformational equilibrium in the presence of galactoside defines the starting level of fluorescence in experiments with the cytoplasmic Cys-Trp pairs, and fluorescence quenching after addition of Nb represents slow accumulation of stabilized LacY/Nb complexes. Closing of the cytoplasmic cavity is observed in complexes of all three fluorophore-labeled Cys-Trp mutants with each of eight Nbs (Fig. 2). Time courses of fluorescence change are colored specifically for each Nb and presented on the same time scale in Fig. 2, demonstrating a similar effect of each Nb on all three labeled pairs. Thus, bimane-labeled pairs 135C/343W, 137C/334W, 140W/343C, and ATTO655-labeled 140W/343C exhibit relatively slow decreases in fluorescence upon binding of Nb; rates of fluorescence quenching are specific for each Nb and increase from Nb9055 to Nb9065 (ranging from 0.01 to 0.13 s−1) with nearly identical results for the bimane- or ATTO655-labeled 140W/343C pair. In addition, no significant shifts in emission spectra maximum are observed with each Nb (Fig. S4), and control experiments with bimanelabeled Cys mutants lacking the appropriate Trp quencher exhibit no fluorescence decrease after Nbs addition (Fig. S5). Therefore, Smirnova et al.

Opening the Periplasmic Cavity. Galactoside binding to LacY with fluorophore-labeled Cys-Trp pairs on the periplasmic side (32C/ 245W, 42C/378W, and 29W/262C) exhibits unquenching of fluorescence due to opening of periplasmic cavity (17). However, the Nbs contain 2–4 Trp residues (Fig. S6), they bind to periplasmic epitopes on LacY, and fluorophores introduced on periplasmic side may be sufficiently close to the Trp residues in the Nbs for quenching to occur (15). Therefore, as controls, the effects of Nb binding were tested first in the absence of sugar with fluorophore-labeled mutants devoid of an introduced Trp residue (Figs. S6 and S7). The size and location of the fluorophore appear to be important factors with respect to the ability of the Nbs to alter fluorescence. Thus, 32C LacY labeled with the small bimane probe exhibits quenching upon binding of Nb9063 only (Fig. S6 A and C), whereas six of the eight Nbs tested quench the ATTO655labeled mutant (Fig. S6 B and D). Various degrees of quenching are observed with bimane-labeled 42C LacY upon binding of eight Nbs, whereas with bimane-labeled 262C LacY, only Nbs 9063 and 9048 are quenchers (Fig. S7). When the Nbs are added to bimane-labeled double mutants 32C/245W, 42C/378W, or 29W/262C, unquenching of fluorescence is observed in most cases (Fig. 4). Fluorescence changes induced by Nbs were recorded in the absence of sugar and depict relatively slow increases in fluorescence to different levels (Fig. 4 A–C), indicating that the Nb-binding epitopes are accessible. The total magnitude of fluorescence change does not depend on the order of addition of sugar and Nb in each case (Fig. S8). Rates of unquenching are similar to the rates of quenching measured with the cytoplasmic pairs (compare time scales on Figs. 2 and 4). The fluorescence changes here represent slow binding of Nbs to accessible epitopes (rates vary from 0.02 to 0.15 s−1) and do not represent the rate of conformational change on periplasmic side (27–32 s−1 as determined with the bimane-labeled 32C/245W pair; Fig. 2. Effect of eight Nbs on distances between fluorophore-labeled Cys and Trp residues introduced on the cytoplasmic side of LacY. Cartoons on the right show the location of Cα atoms of introduced Trp (red spheres) and bimane-labeled Cys replacements (blue spheres) in each mutant in the cytoplasmicopen conformer viewed from the side with N- and C-terminal six-helix bundles indicated. Structures of fluorophores after reaction with Cys are presented at the bottom right for comparison. Time courses of fluorescence quenching resulting from addition of Nbs to bimane-labeled mutants 135C/343W (A), 137C/334W (B), and 140W/343C (C) or ATTO655-labeled 140W/343C (D) are shown as colored lines with a given corresponding Nb. Traces were recorded in the presence of 0.1 mM NPG and displayed on the same time scale for comparison. Black arrows indicate addition of Nb. Single-exponential fits are shown as black lines.

the bound Nbs cause practically no change in the local environment around the fluorophore. Clearly, fluorescence quenching in these LacY/Nb complexes results from a decrease in distance between Trp and the fluorophore in all labeled pairs and, therefore, reflects closing of the cytoplasmic cavity. The magnitude of fluorescence quenching estimated from single-exponential fits is different for each Nb tested. Remarkably, however, comparison of the normalized extent of fluorescence change in four sets of data (Fig. 2) reveals a similar pattern of quenching for at least six Nbs (Fig. 3). The largest change in each labeled pair is observed with bound Nbs 9063 (3) or 9048 (4), Smirnova et al.

Fig. 3. Comparison of the extent of fluorescence quenching in complexes of three labeled cytoplasmic LacY mutants with eight Nbs. The magnitude of quenching was calculated (as ΔF/F0) from the time courses shown on Fig. 2 and normalized as percent of maximum fluorescence change in each dataset. The extent of Trp/fluorophore quenching induced by each individual Nb in the four datasets was averaged and presented as a bar graph with SDs. The numbers on x axis correspond to Nbs: 9055 (1), 9036 (2), 9063 (3), 9048 (4), 9047 (5), 9033 (6), 9043 (7), and 9065 (8) and are color coded as in Fig. 2.

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whereas other Nbs display smaller effects. Nbs 9047 (5), 9033 (6), and 9043 (7) exhibit similar extents of quenching for each pair, which exceeds that of 9065 (8). Because the degree of quenching directly depends on the distance between fluorophore and quencher, the data suggest the presence of various conformers with respect to the cytoplasmic-closed LacY molecule in complex with different Nbs.

unquenching indicating opening of the periplasmic cavity occurs together with quenching by the Trp in the Nb (red and open blue bars on Fig. 4 D–F). Therefore, unquenching is partially or completely masked by the quenching effect of the Nb. For example, Nb9055 (1) is a strong quencher of bimane attached to 42C, but not 32C or 262C (Fig. 4 D–F, open blue bar 1). However, the unquenching effect of Nb9055 on bimane-labeled pairs 32C/245W and 29W/262C is dramatically different (Fig. 4 D and F, red bars 1). The quenching effect of Nbs 9036 (2), 9063 (3), and 9048 (4) on bimane-labeled 42C gradually increases from 3 to 24% (Fig. 4E, open blue bars 2–4). Therefore, the unquenching effect of the same Nbs on the corresponding double mutant 42C/378W gradually decreases from 18 to 5% (Fig. 4E, red bars 2–4). The degree of unquenching observed with pair 32C/245W located in the middle of the periplasmic side (helices I and VII) is generally bigger than that with two other pairs (compare y axes on Fig. 4 D–F). Notably, addition of sugar alone also results in bigger unquenching in the middle of the periplasmic side than in the periphery of the molecule (Fig. S8 A, B, and D; TDG effect is 75%, versus 22% and 9% for the 32C/245W, 42C/378W, and 29W/ 262C pairs, respectively). This data likely reflect larger movements of helices I and VII relative to helices on the periphery of LacY upon opening of the periplasmic cavity. Strikingly, sugar binding to complexes of LacY with different Nbs results in additional unquenching on the periplasmic side, indicating further conformational change. For instance, five Nbs that do not cause fluorescence quenching in the control 32C mutant devoid of an inserted Trp (Fig. 4D, open blue bars) form complexes with 32C/245W LacY characterized by different degrees of bimane unquenching in the absence of sugar (Fig. 5 A and B, red bars). Subsequent addition of TDG increases unquenching to the maximum level observed with TDG alone (Fig. 5B, blue bars). All three periplasmic pairs exhibit rapid fluorescence increases when sugar is added to the preformed LacY/Nb complexes (Fig. 5A and Figs. S8 and S9), indicating further opening of the periplasmic cavity. Thus, Nb binding in the absence of sugar stabilizes outward-facing conformers that are different structurally from those with bound sugar.

Fig. 4. Effect of eight Nbs on distances between bimane-labeled Cys and Trp residues on the periplasmic side of LacY. Structural models of inwardand outward-facing conformers (PDB ID codes 2CFQ and 4OAA on top left and right, respectively) viewed from periplasmic side are shown with the helices rainbow colored (from blue to red for helices I to XII, respectively). Introduced Cys-Trp pairs are presented as spheres with indicated replacements (red for Trp, blue for Cys). Time courses of fluorescence unquenching resulted from addition of Nbs to bimane-labeled mutants 32C/245W (A), 42C/378W (B), and 29W/262C (C) are shown as colored lines with corresponding Nb specified. Traces were recorded without sugar present and displayed on the same time scale as shown in Fig. 2 for comparison. Black arrows indicate addition of Nb. Single-exponential fits are shown as black lines. Bar graphs D–F illustrate the magnitude of the fluorescence changes resulting from interaction of each of the eight Nbs with the three given LacY mutants. Data are presented for bimane-labeled Cys-Trp pairs (red bars, see traces in A–C) and control mutants devoid of the introduced Trp residue (open blue bars, Fig. S7): 32C/245W and 32C (D), 42C/378W and 42C (E), 29W/ 262C and 262C (F). The numbers on x axis correspond to Nbs: 9055 (1), 9036 (2), 9063 (3), 9048 (4), 9047 (5), 9033 (6), 9043 (7), and 9065 (8) and color coded as in Figs. 2 and 3.

ref. 17). Therefore, fluorescence unquenching after addition of Nb is due to slow binding of Nb and accumulation of stabilized LacY/Nb complexes with periplasmic-open cavity. Comparison of the magnitude of fluorescence change resulting from interaction of eight Nbs with LacY mutants demonstrates that each Nb has a specific effect. In several cases, fluorophore 4 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1519485112

Dissociation of Nbs from LacY/Nb Complexes. Quantitative analysis of the extent of fluorescence changes (Figs. 2–5) is accurate only if formation of LacY/Nb complexes is close to 100%. Because the LacY concentrations used were low (80–100 nM with a threefold excess of Nbs), the affinity of the Nbs for LacY becomes critical.

Fig. 5. Further opening of the periplasmic cavity after sugar addition to LacY/Nb complexes. (A) Time courses of fluorescence changes in bimanelabeled 32C/245W LacY after addition of each given Nb followed by TDG are shown as colored lines. The arrows indicate addition of Nb or TDG. Singleexponential fits are presented as black lines. (B) Bar graph illustrates the extent of bimane unquenching in LacY/Nb complexes (red bars) and total fluorescence increase after TDG binding (blue bars) calculated from individual traces in A. The time course of the fluorescence change with bimanelabeled 32C/245W after addition of TDG alone is shown on Fig. S8A.

Smirnova et al.

Discussion Nbs against the outward-facing G46W/G262W mutant of LacY interact with periplasmic epitopes of WT LacY to stabilize conformers with an open periplasmic cavity as judged by increased accessibility of the sugar-binding site to galactosides (15). Thus, NPG binding rates increase dramatically in complexes of WT LacY with different Nbs (kon values rise 5- to 50-fold), demonstrating varied increase in accessibility of the binding site to sugar. The data likely reflect stabilization of different outward-open conformers of LacY that represent physiological intermediates of the transport cycle. A site-directed, distance-dependent method of Trp-induced fluorescence quenching (16) is now used to ascertain the global conformational states of these stabilized LacY/Nb complexes. Three mutants with Cys-Trp pairs on either the cytoplasmic or periplasmic side of LacY were constructed, labeled with a thiolreactive fluorophore, and tested for changes in fluorescence after interaction with each of eight Nbs. Unquenching of fluorescence of periplasmic and quenching of cytoplasmic fluorophores-labeled

Fig. 6. Determination of koff values by displacement of Nbs bound to the ATTO655-labeled 32C periplasmic LacY mutant without an inserted Trp residue. The arrow indicates addition of 6- to 12-fold excess of Nb9063 to LacY itself (lowest line) or to LacY/Nb complexes (seven lines with indicated Nbs). The rate of fluorescence quenching resulted from binding of Nb9063 (110 nM) to ATTO655-labeled LacY (20 nM) was estimated as 0.13 s−1 (lowest line). Addition of 110–240 nM Nb9063 to 20 nM LacY complexed with indicated Nbs resulted in binding of Nb9063 due to release of bound Nb from LacY/Nb complex. Quenching rates estimated from single-exponential fits (black lines) report dissociation rates of individual Nbs (koff) and varied from 7 × 10−4 to 1.7 × 10−2 sec−1 (Table 1).

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Table 1. Dissociation rate constants (koff) measured as displacement rates of Nbs bound to ATTO655-labeled 32C LacY Nbs

koff s−1

Kd*, nM

kon*, μM−1·s−1

koff = Kdkon, s−1

9055 9036 9063 9048 9047 9033 9043 9065

1.2 x 10−3 0.7 x 10−3 1.1 x 10−3 2.3 x 10−3 6.7 x 10−3 1.2 x 10−3 9.2 x 10−3 17 x 10−3

2.8 0.6 0.9

0.16 0.43 1.3

0.4 x 10−3 0.3 x 10−3 1.2 x 10−3 — — — — —

† † †

†

2.7 †

†

†

†

†

Data are obtained as shown on Fig. 6 except koff value for Nb9063 that was determined from displacement of Nb9063 bound to LacY with excess of Nb9036. Data in third, fourth, and fifth columns are presented for comparison of measured koff with the values calculated from measured Kd and kon (15). *Directly measured by fluorescence quenching (15). † Very low or no fluorescence change precluded direct measurements of Nb binding.

pairs is observed, indicating opening of the periplasmic cavity with concomitant closing of the cytoplasmic cavity. Single-exponential fitting of time courses (Figs. 2 and 4) indicates that observed fluorescence change caused by each Nb is a single-step process. Moreover, the estimated rates of fluorescence change represent Nb binding rates (see koff and kon values in Table 1) and are much slower than the rates of conformational change triggered by substrate binding (17). Therefore, the Nbs do not induce conformational change in LacY. Rather, they stabilize natural intermediates by conformational selection of LacY molecules with accessible Nb epitopes. Closing of the cytoplasmic cavity is observed as quenching of fluorophores in LacY/Nb complexes. Thus, each of eight Nbs, which bind to the periplasmic side of LacY (15), induces changes on the cytoplasmic side of the molecule that are on the opposite face of the molecule 50–60 Å away (Fig. 2). This quenching clearly represents decreased distance between Trp and fluorophore because no changes in the environment around fluorophores are detected upon Nbs binding (Figs. S4 and S5). Virtually identical results are obtained with either bimane or ATTO655, which are dissimilar structurally in agreement with a notion that Trp-induced fluorescence quenching depends directly on distance as shown with lysozyme labeled with different fluorophores (16). Furthermore, the extent of closure is specific for each Nb with a distinct pattern when four sets of data with three Cys-Trp pairs and different size fluorophores are compared (Fig. 3). Nbs 9063 and 9048 stabilize LacY conformers with the greatest quenching, whereas Nbs 9047, 9033, 9043, and 9065 exert a lesser effect. Therefore, it appears that different Nbs trap structurally different cytoplasmicclosed conformers. Although direct quenching of fluorophores on the cytoplasmic side by Trp in Nb bound to the periplasmic side cannot occur because of excessively long distances (17), periplasmic location of the fluorophore probe results in combined effects in many instances–simultaneous quenching of fluorophore by Trp in the Nb (Fig. 4 D–F, open blue bars, and Fig. S7) and increased fluorescence (unquenching) due to opening of cavity (Fig. 4 D–F, red bars). For example, all of the Nbs quench bimane attached to Cys42 so that data obtained with the 42C/378W pair are a combination of two effects (Fig. 4E). Nevertheless, net unquenching of fluorophores in all three periplasmic pairs is consistent with increases in distance and indicates cavity opening to different extents depending on the particular Nb (Fig. 4). On the periplasmic side, the clearest results are observed with the 32C/245W pair located in the approximate middle of the periplasmic surface, because all of the Nbs (except for Nb9063) PNAS Early Edition | 5 of 6

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Kd values for Nbs 9055, 9036, and 9063 as 2.8, 0.6, and 0.9 nM, respectively, were determined by fluorescence quenching of bimaneand ATTO655-labeled 32C LacY without an inserted Trp residue (15). Here, the affinity of the Nbs was estimated by competitive binding of Nb9063 to ATTO655-labeled 32C LacY preincubated individually with each Nb. The large quenching effect of Nb9063 (Fig. 6 and Fig. S6 B and D) allows determination of dissociation rates of other Nbs from LacY complexes by displacement with excess of Nb9063. The binding rate of Nb9063 to LacY alone is estimated under these experimental conditions as 0.13 s−1 (Fig. 6, lowest line). Nb9063 added to a LacY/Nb complex binds only after dissociation of bound Nb. Decrease of ATTO fluorescence is observed because other Nbs quench ATTO to a lesser extent (Fig. S6 B and D). Displacement rates correspond to dissociation rate constants (koff) of the released Nbs and vary from 7 × 10−4 to 1.7 × 10−2·s−1 (Table 1). These data indicate that the affinity of each Nb is quite high with Kd values less than 10 nM, because kon values are usually approximately 1 μM−1·s−1 (12), which is in a good agreement with the values measured previously for Nbs 9055, 9036, 9063, and 9047 (Table 1). Therefore, formation of LacY/Nb complexes is estimated as 95–99% of maximum in each experiment presented.

are not quenchers of bimane-labeled 32C (Fig. 4D, open blue bars). The degree of bimane unquenching gradually decreases in complexes of LacY with Nbs 9047, 9065, 9033, 9036, and 9055 (red bars on Fig. 5B), indicating that opening of periplasmic cavity is largest with Nb9047 and smallest with Nb9055. Remarkably, the different extents of periplasmic opening observed here are comparable to the accessibility of the sugar-binding site in WT LacY with bound Nbs derived from kinetic studies (15). Rates of NPG binding to WT LacY are faster in complexes with Nbs 9047, 9065, or 9033 (kon values of 7, 9, or 8 μM−1·s−1, respectively) and slower in complexes with Nbs 9036 or 9055 (kon values of 1 or 1.2 μM−1·s−1, respectively), indicating higher accessibility to sugar when first three Nbs are bound and lower accessibility when last two Nbs are bound. Binding of sugar to LacY/Nb complexes results in further opening of the periplasmic cavity as detected by additional unquenching with the periplasmic pairs (Fig. 5 and Figs. S8 and S9). Added sugar binds from periplasmic side, because increased accessibility of sugar from that side in the LacY/Nb complexes has been documented (15). Therefore, additional unquenching is explained by further opening of the periplasmic cavity after sugar binding. The following event, closing of periplasmic cavity, is prevented by the bound Nb. Therefore, each Nb stabilizes two intermediates, one with no sugar bound and another with bound sugar and a more open periplasmic cavity. Finally, it is concluded that kinetic determinations of the accessibility of the sugar-binding site and direct measurements of distance changes in LacY/Nb complexes report similar outcomes with respect to the interactions of the Nbs with LacY. Both types of measurements provide data that are consistent with the conclusion that binding of different Nbs stabilize outward-facing conformers with different degrees of cavity opening. These conformers likely represent physiological intermediates in the overall transport cycle. It has also been reported that in vivo-matured Nbs do not generate nonnative conformations of antigens (19). This notion is in total agreement with observations that the Nbs bind only to conformers in which the periplasmic cavity is open. Thus, Nbs do not actively change LacY conformation, but bind only to specific conformers and, thereby, capture transient states of the highly dynamic LacY molecule. 1. Kaback HR (2015) A chemiosmotic mechanism of symport. Proc Natl Acad Sci USA 112(5):1259–1264. 2. Abramson J, et al. (2003) Structure and mechanism of the lactose permease of Escherichia coli. Science 301(5633):610–615. 3. Mirza O, Guan L, Verner G, Iwata S, Kaback HR (2006) Structural evidence for induced fit and a mechanism for sugar/H+ symport in LacY. EMBO J 25(6):1177–1183. 4. Guan L, Mirza O, Verner G, Iwata S, Kaback HR (2007) Structural determination of wild-type lactose permease. Proc Natl Acad Sci USA 104(39):15294–15298. 5. Chaptal V, et al. (2011) Crystal structure of lactose permease in complex with an affinity inactivator yields unique insight into sugar recognition. Proc Natl Acad Sci USA 108(23):9361–9366. 6. Kumar H, et al. (2014) Structure of sugar-bound LacY. Proc Natl Acad Sci USA 111(5): 1784–1788. 7. Kumar H, Finer-Moore JS, Kaback HR, Stroud RM (2015) Structure of LacY with an α-substituted galactoside: Connecting the binding site to the protonation site. Proc Natl Acad Sci USA 112(29):9004–9009. 8. Smirnova I, et al. (2007) Sugar binding induces an outward facing conformation of LacY. Proc Natl Acad Sci USA 104(42):16504–16509. 9. Madej MG, Soro SN, Kaback HR (2012) Apo-intermediate in the transport cycle of lactose permease (LacY). Proc Natl Acad Sci USA 109(44):E2970–E2978. 10. Rasmussen SG, et al. (2011) Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature 477(7366):549–555.

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Methods Production of Nbs, construction of mutants, purification of LacY, and materials used in this study are described in SI Methods. Labeling of LacY Mutants with Fluorophores. Purified LacY mutants with specified Cys replacements (50–100 μM protein in 50 mM NaPi/0.02% DDM, pH 7.5) were mixed with (2-pyridyl)dithiobimane (PDT-bimane) at 1:1 molar ratio, or with 1.2- to 1.5-fold molar excess of ATTO655-maleimide, and incubated at room temperature in dark (10 min with PDT-bimane or 30 min with ATTO655-maleimide). TDG (30 mM) was also included for labeling of Cys residues introduced on periplasmic side to increase the probability of opening the periplasmic cavity and also to protect native Cys148 from labeling. Cys replacements on cytoplasmic side are readily accessible without bound sugar; therefore, Cys148 was substituted with Met (C148M LacY has WT activity and sugar binding affinity), and labeling was carried out without TDG. Labeled samples were washed twice in the same buffer without TDG or fluorophores using an Amicon Ultra concentrator with 50-kDa cutoff (Millipore), frozen in liquid nitrogen, and stored at −80 °C before use. Fluorescence Measurements. Steady-state fluorescence emission spectra were collected at room temperature on a SPEX Fluorolog 3 spectrofluorometer (Edison) in a 2.5-mL cuvette (1 × 1 cm) in 50 mM NaPi/0.02% DDM (pH 7.5) as described (20) with excitation at 380 nm (for bimane) and 650 nm (for ATTO655). Time courses of fluorescence change were recorded at excitation/ emission wavelengths 380/465 nm and 660/677 nm for bimane- and ATTO655labeled protein, respectively. Fluorophore-labeled protein (80–100 nM) was mixed with a threefold excess of Nb and 0.1 mM NPG or 6 mM TDG as indicated. A single exponential equation was used for fitting to the data collected on a time scale that was sufficiently long to observe completion of the process. The magnitude of fluorescence change was expressed as the change in intensity relative to the initial level before a given addition (ΔF/F0, %). Typically, calculated SDs were within 10% of each presented value. In displacement experiments, ATTO655-labeled 32C LacY (20 nM) was preincubated with a small excess of Nb and 0.1 mM NPG, then a 6- to 12-fold excess of a second Nb was added, which binds upon dissociation of the first Nb. Dissociation rates were estimated from time courses of the change in fluorescence by fitting with a single-exponential equation. ACKNOWLEDGMENTS. We thank Junichi Sugihara for skillful technical assistance in preparation of LacY mutants. This work was supported by NIH Grant DK51131 and a National Science Foundation Eager Grant 1547801 (to H.R.K.).

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