Substrate-induced changes in the structural properties of LacY

The lactose permease (LacY) of Escherichia coli, a paradigm for the major facilitator superfamily, catalyzes the coupled stoichiometric translocation of a galactopyranoside and an H⁺ across the cytoplasmic membrane. To catalyze transport, LacY undergoes large conformational changes that allow alternating access of sugar- and H⁺-binding sites to either side of the membrane. Despite strong evidence for an alternating access mechanism, it remains unclear how H⁺- and sugar-binding trigger the cascade of interactions leading to alternating conformational states. Here we used dynamic single-molecule force spectroscopy to investigate how substrate binding induces this phenomenon. Galactoside binding strongly modifies kinetic, energetic, and mechanical properties of the N-terminal 6-helix bundle of LacY, whereas the C-terminal 6-helix bundle remains largely unaffected. Within the N-terminal 6-helix bundle, the properties of helix V, which contains residues critical for sugar binding, change most radically. Particularly, secondary structures forming the N-terminal domain exhibit mechanically brittle properties in the unbound state, but highly flexible conformations in the substrate-bound state with significantly increased lifetimes and energetic stability. Thus, sugar binding tunes the properties of the N-terminal domain to initiate galactoside/H⁺ symport. In contrast to wild-type LacY, the properties of the conformationally restricted mutant Cys154➝Gly do not change upon sugar binding. It is also observed that the single mutation of Cys154➝Gly alters intramolecular interactions so that individual transmembrane helices manifest different properties. The results support a working model of LacY in which substrate binding induces alternating conformational states and provides insight into their specific kinetic, energetic, and mechanical properties.The lactose permease of Escherichia coli (LacY) of the major facilitator superfamily (MFS) (1, 2) catalyzes the coupled stoichiometric translocation of a galactopyranoside and an H⁺ (sugar/H⁺ symport) (3–6). Uphill (i.e., active) symport of galactoside against a concentration gradient is achieved by transduction of free energy released from the downhill movement of H⁺ with the electrochemical H⁺ gradient (Δ$\tilde{\mu }$_H⁺; interior negative and/or alkaline). Conversely, because coupling between sugar and H⁺ is obligatory, downhill galactoside transport from a high to a low sugar concentration is coupled to uphill H⁺ transport with the generation of Δ$\tilde{\mu }$_H+, the polarity of which depends upon the direction of the sugar concentration gradient (7–10).LacY monomers reconstituted into proteoliposomes are functional (11, 12), and X-ray crystal structures reveal 12, mostly irregular, transmembrane α-helices organized into two pseudosymmetrical 6-helix bundles surrounding a large interior hydrophilic cavity open to the cytoplasm (13–16). At the apex of the hydrophilic cavity, which is at the approximate middle of the molecule, the galactoside- and H⁺-binding sites are located. Side chains important for sugar recognition are located in both the N- and the C-terminal 6-helix bundles, whereas those involved in H⁺ binding are largely in the C-terminal 6-helix bundle. Most X-ray structures obtained thus far exhibit a tightly sealed periplasmic side with the sugar-binding site at the apex of the cavity and inaccessible from the periplasm and an open cytoplasmic side (an inward-facing conformation). LacY is structurally highly dynamic, and binding of a galactoside closes the deep inward-facing cavity with opening of a complementary outward-facing cavity (reviewed in refs. 17, 18). Therefore, transport involves a large conformational change that allows alternating access of sugar- and H⁺-binding sites to either side of the cellular membrane, and a recent structure indicates that an occluded intermediate is involved (19). Although structural models of LacY provide insight into the conformational states involved in transport, a crystal structure represents a static structural snapshot, and therefore an understanding of how sugar binding triggers the cascade of events that results in dynamic alternating access remains unclear. Furthermore, because these interactions alter the physical properties of LacY (reviewed in ref. 9), the energetic, kinetic, and mechanical properties of LacY that fulfill different functional roles during transport remain to be characterized.Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) has been applied to localize and quantify interactions that stabilize structural elements of an increasing number of native membrane proteins (20–25). Because SMFS can be used with membrane proteins embedded in native or synthetic lipid membranes under physiological conditions, the method has been used to assess interactions that change upon substrate binding, insertion of mutations, and assembly or lipid composition of the membrane (26–35). Moreover, when operated in the dynamic mode, dynamic single-molecule force spectroscopy (DFS) localizes and quantifies the kinetic, energetic, and mechanical properties of the structural elements in a membrane protein in a physiologically relevant environment (20, 21).LacY binds galactopyranosides, and 4-nitrophenyl-α-d-galactopyranoside (αNPG) is among the lactose analogs with highest affinity (∼30 µM) (36). In the absence of substrate, LacY preferentially occupies an inward-facing open conformation, and substrate binding causes closing of the inward-facing cavity with opening of a reciprocal outward-facing cavity (reviewed in refs. 17, 18) with an occluded intermediate conformation (19). To understand the structural perturbations and properties associated with these conformations, we describe here the conformational, kinetic, energetic, and mechanical properties of LacY in the apo state and how these properties change upon substrate binding. SMFS and DFS are used to characterize the properties of individual structural segments of LacY and to describe how these regions change properties upon galactoside binding. To understand further how a single point mutation alters LacY, the conformationally restricted LacY mutant C154G (37), which crystallized originally (13), was also investigated. All measurements were conducted with wild-type (WT) or mutant C154G LacY embedded in a phospholipid membrane under physiological conditions. The findings quantify the structural properties of WT LacY, which change drastically upon sugar binding. In contrast, the structural properties of mutant C154G LacY remain largely unaffected by ligand binding.     

Outward-facing conformers of LacY stabilized by nanobodies

The lactose permease of Escherichia coli (LacY), a highly dynamic polytopic membrane protein, catalyzes stoichiometric galactoside/H⁺ symport by an alternating access mechanism and exhibits multiple conformations, the distribution of which is altered by sugar binding. We have developed single-domain camelid nanobodies (Nbs) against a LacY mutant in an outward (periplasmic)-open conformation to stabilize this state of the WT protein. Twelve purified Nbs inhibit lactose transport in right-side–out membrane vesicles, indicating that the Nbs recognize epitopes on the periplasmic side of LacY. Stopped-flow kinetics of sugar binding by WT LacY in detergent micelles or reconstituted into proteoliposomes reveals dramatic increases in galactoside-binding rates induced by interaction with the Nbs. Thus, WT LacY in complex with the great majority of the Nbs exhibits varied increases in access of sugar to the binding site with an increase in association rate constants (k_on) of up to ∼50-fold (reaching 10⁷ M⁻¹⋅s⁻¹). In contrast, with the double-Trp mutant, which is already open on the periplasmic side, the Nbs have little effect. The findings are clearly consistent with stabilization of WT conformers with an open periplasmic cavity. Remarkably, some Nbs drastically decrease the rate of dissociation of bound sugar leading to increased affinity (greater than 200-fold for lactose).Typical of many transport proteins, from organisms as widely separated evolutionarily as Archaea and Homo sapiens, the lactose permease of Escherichia coli (LacY), a paradigm for the Major Facilitator Superfamily (1), catalyzes the coupled, stoichiometric translocation of a galactopyranoside and an H⁺ (galactoside/H⁺ symport) across the cytoplasmic membrane (reviewed in refs. 2 and 3). Although it is now generally accepted that membrane transport proteins operate by an alternating access mechanism, this has been documented almost exclusively for LacY (reviewed in refs. 4 and 5). By this means, galactoside- and H⁺-binding sites become alternatively accessible to either side of the membrane as the result of reciprocal opening/closing of cavities on the periplasmic and cytoplasmic sides of the molecule. LacY is highly dynamic, and alternates between different conformations (6, 7).Until recently, six X-ray structures of LacY have exhibited the same inward-facing conformation with an aqueous cavity open to the cytoplasmic side, a tightly sealed periplasmic side, and sugar- and H⁺-binding sites in the middle of the molecule (8–11). Numerous studies confirm that this conformation prevails in the absence of sugar (12–16). Recently, however, the X-ray structure of double-Trp mutant G46W/G262W with bound sugar reveals a conformation with a narrowly open periplasmic pathway and a tightly sealed cytoplasmic side (PDB ID code 4OAA) (17), thereby providing structural evidence that an intermediate occluded conformation occurs between the outward- and inward-facing conformations in the transport cycle.Rates of opening/closing of periplasmic and cytoplasmic cavities have been determined in real time from changes in fluorescence of Trp or attached fluorophores with LacY either in detergent micelles or in reconstituted proteoliposomes (PLs) (15, 18, 19). Sugar-binding rates with WT LacY in PLs measured by Trp151→4-nitrophenyl-α-d-galactopyranoside (NPG) FRET are independent of sugar concentration, whereas the mutant with an open periplasmic cavity is characterized by a linear concentration dependence of sugar binding rates with k_on of ∼10 μM⁻¹⋅s⁻¹ (18, 20), which approximates diffusion controlled access to the binding site (21). Therefore, with WT LacY embedded in PLs, the periplasmic side is sealed, and substrate binding is limited by opening of the periplasmic cavity at a rate of 20–30 s⁻¹ (19). This rate is very similar to the turnover number of WT LacY in right-side–out (RSO) membrane vesicles or reconstituted PLs (22) and is consistent with the notion that opening of the periplasmic cavity may be a limiting step in the overall transport mechanism.To define and characterize partial reactions in the LacY transport cycle, stable conformers would be particularly useful. In this regard, remarkable progress has been made with G protein-coupled receptors through the use of camelid single-domain nanobodies (Nbs), which stabilize specific conformers (23–27). Advantages of Nbs include small size and a unique structure that allows flexible antigen-binding loops to insert into clefts and cavities. Here we report that Nbs prepared against the outward (periplasmic)-open LacY mutant G46W/G262W effectively bind to WT LacY and inactivate transport activity. However, the sugar-binding site becomes much more accessible to galactosides as a result of Nb binding, indicating stabilization of the open-outward conformations of LacY, and providing the means for detailed studies of galactoside binding to these conformers. Remarkably, several Nbs significantly increase affinity for galactosides by slowing the dissociation rate of the sugar while maintaining a high association rate. It is also apparent that the Nbs have the potential for crystallizing LacY trapped as otherwise unstable transient intermediates.     

Structure of sugar-bound LacY

Here we describe the X-ray crystal structure of a double-Trp mutant (Gly46→Trp/Gly262→Trp) of the lactose permease of Escherichia coli (LacY) with a bound, high-affinity lactose analog. Although thought to be arrested in an open-outward conformation, the structure is almost occluded and is partially open to the periplasmic side; the cytoplasmic side is tightly sealed. Surprisingly, the opening on the periplasmic side is sufficiently narrow that sugar cannot get in or out of the binding site. Clearly defined density for a bound sugar is observed at the apex of the almost occluded cavity in the middle of the protein, and the side chains shown to ligate the galactopyranoside strongly confirm more than two decades of biochemical and spectroscopic findings. Comparison of the current structure with a previous structure of LacY with a covalently bound inactivator suggests that the galactopyranoside must be fully ligated to induce an occluded conformation. We conclude that protonated LacY binds d-galactopyranosides specifically, inducing an occluded state that can open to either side of the membrane.The lactose permease of Escherichia coli (LacY), a paradigm for the major facilitator superfamily (MFS), binds and catalyzes transport of d-galactose and d-galactopyranosides specifically with an H⁺ (1, 2). In contrast, LacY does not recognize d-glucose or d-glucopyranosides, which differ only in the orientation of the C4-OH of the pyranosyl ring. By using the free energy released from the energetically downhill movement of H⁺ in response to the electrochemical H⁺ gradient (), LacY catalyzes the uphill (active) transport of galactosides against a concentration gradient. Because coupling between sugar and H⁺ translocation is obligatory, in the absence of , LacY also can transduce the energy released from the downhill transport of sugar to drive uphill H⁺ transport with the generation of , the polarity of which depends upon the direction of the sugar gradient.It also has been shown that LacY binds sugar with a pK_a of ∼10.5 and that sugar binding does not induce a change in ambient pH; both findings indicate that the protein is protonated over the physiological range of pH (3–5). These observations and many others (1, 2) provide evidence for an ordered kinetic mechanism in which protonation precedes galactoside binding on one side of the membrane and follows sugar dissociation on the other side. Recent considerations (6) suggest that a similar ordered mechanism may be common to other members of the MFS.Because equilibrium exchange and counterflow are unaffected by imposition of , it is apparent that the alternating accessibility of sugar- and H⁺-binding sites to either side of the membrane is the result of sugar binding and dissociation and not of (reviewed in refs. 1 and 2). Moreover, downhill lactose/H⁺ symport from a high to a low lactose concentration exhibits a primary deuterium isotope effect that is not observed for -driven lactose/H⁺ symport, equilibrium exchange, or counterflow (7, 8). Thus, it is likely that the rate-limiting step for downhill symport is deprotonation (9, 10), whereas in the presence of either dissociation of sugar or a conformational change leading to deprotonation is rate-limiting.X-ray crystal structures of WT LacY (11), the conformationally restricted mutant C154G (12, 13), and a single-Cys mutant with covalently bound methanethiosulfonyl-galactopyranoside (MTS-Gal) (14) have been determined in an inward-facing conformation. The structures consist of two six-helix bundles that are related by a quasi twofold symmetry axis in the membrane plane, linked by a long cytoplasmic loop between helices VI and VII. Furthermore, in each six-helix bundle, there are two three-helix bundles with inverted symmetry (6, 15). The two six-helix bundles surround a deep hydrophilic cavity tightly sealed on the periplasmic face and open only to the cytoplasmic side (an inward-open conformation). Although crystal structures reflect only a single lowest-energy conformation under conditions of crystallization, the entire backbone appears to be accessible to water (16–18), and an abundance of biochemical and spectroscopic data (19–27) demonstrate that sugar binding causes the molecule to open alternatively to either side of the membrane, thereby providing strong evidence for an alternating-access model (reviewed in refs. 28 and 29).The initial X-ray structure of conformationally restricted C154G LacY was obtained with density at the apex of the central cavity, but because of limited resolution, the identity of the bound sugar at this site and/or side-chain interactions are difficult to specify with certainty. However, biochemical and spectroscopic studies show that LacY contains a single galactoside-binding site and that the residues involved in sugar binding are located at or near the apex of the central cavity. Although the specificity of LacY is strongly directed toward the C4-OH of the galactopyranosyl ring, other OH groups also are important in the following order: C4-OH >> C6-OH > C3-OH > C2-OH (30, 31). Cys-scanning mutagenesis, site-directed alkylation, and direct binding assays show that Glu126 (helix IV) and Arg144 (helix V) are irreplaceable for substrate binding and probably are charge-paired (4, 32–35). Trp151 (helix V), two turns removed from Arg144, stacks aromatically with the galactopyranosyl ring (36, 37). Glu269 (helix VIII), another irreplaceable residue (38–40), also is essential for sugar recognition and binding and cannot be replaced even with Asp without markedly decreasing affinity (4, 41). It has been shown recently (42) that Asn272 (helix VIII) also is essential for binding and transport. In contrast, Cys148 (helix V), which is protected from alkylation by substrate, and Ala122 (helix IV), where bulky replacements make LacY specific for galactose, are close to the binding site but probably do not contact the sugar directly.Among the conserved residues in LacY and other MFS members are two Gly–Gly pairs between the N- and C-terminal six-helix bundles on the periplasmic side of LacY at the ends of helices II and XI (Gly46 and Gly370, respectively) and helices V and VIII (Gly159 and Gly262, respectively) (43). When Gly46 (helix II) and Gly262 (helix VIII) are replaced with bulky Trp residues (Fig. 1), transport activity is abrogated with little or no effect on galactoside binding (44). Moreover, site-directed alkylation and stopped-flow binding kinetics indicate that the G46W/G262W mutant is open on the periplasmic side (open outward). In addition, the detergent-solubilized mutant exhibits much greater thermal stability than WT LacY (44).Open in a separate windowFig. 1.Side view of LacY G46W/G262W molecules A (Center) and B (Left) in an almost occluded, outward-facing conformation shown in green and gray ribbons, respectively. The two molecules in the asymmetric unit are shown adjacent to one another from the perspective of the membrane plane. The two molecules have a similar conformation with bound TDG, and the Trp replacements at positions 46 and 262 are shown. TDG and the Trp replacements are represented as spheres, with carbon atoms in magenta (for Trp) or orange (for TDG), oxygen atoms in red, nitrogen atoms in blue, and sulfur in yellow. Dashed lines depict the quasi twofold axes relating the N- and C-terminal helix bundles. (Right) The change in structure between LacY G46W/G262W (green; helices numbered) versus the apo WT structure (PDB ID code 2V8N, blue). The orientation matches chain A, and the alignment of the two structures is based on alignment of the N-terminal six-helix bundle of the apo structure onto the G46W/G262W.The G46W/G262W mutant now has been crystallized in the presence of a high-affinity lactose analog. Remarkably, the structure presented here depicts an almost occluded, outward-open conformation with a reliably defined model of the bound sugar molecule.     

Structure of LacY with an α-substituted galactoside: Connecting the binding site to the protonation site

The X-ray crystal structure of a conformationally constrained mutant of the Escherichia coli lactose permease (the LacY double-Trp mutant Gly-46→Trp/Gly-262→Trp) with bound p-nitrophenyl-α-d-galactopyranoside (α-NPG), a high-affinity lactose analog, is described. With the exception of Glu-126 (helix IV), side chains Trp-151 (helix V), Glu-269 (helix VIII), Arg-144 (helix V), His-322 (helix X), and Asn-272 (helix VIII) interact directly with the galactopyranosyl ring of α-NPG to provide specificity, as indicated by biochemical studies and shown directly by X-ray crystallography. In contrast, Phe-20, Met-23, and Phe-27 (helix I) are within van der Waals distance of the benzyl moiety of the analog and thereby increase binding affinity nonspecifically. Thus, the specificity of LacY for sugar is determined solely by side-chain interactions with the galactopyranosyl ring, whereas affinity is increased by nonspecific hydrophobic interactions with the anomeric substituent.The lactose permease of Escherichia coli (LacY) binds and catalyzes the coupled stoichiometric transport of d-galactose or β-d-galactopyranosides and H⁺ (galactoside/H⁺ symport) but does not interact with glucopyranosides. Biochemical studies (1–7) indicate that affinity and specificity are distinct properties determined by different interactions with LacY. Specificity is determined entirely by interactions with the galactopyranosyl ring, whereas affinity is better with α- than β-galactopyranosides (anomeric at C1) and can be increased dramatically by hydrophobic anomeric substituents with no effect on specificity.By using the free energy released from the energetically downhill movement of H⁺ in response to the electrochemical H⁺ gradient (∆µ̃_H+), LacY catalyzes uphill (active) transport of galactosides against a concentration gradient. Because coupling between sugar and H⁺ translocation is obligatory, in the absence of ∆µ̃_H+, LacY can also transduce the free energy released from the downhill transport of sugar to drive uphill H⁺ transport with the generation of ∆µ̃_H+, the polarity of which depends upon the direction of the sugar gradient (reviewed in refs. 8–10).Rates of equilibrium exchange and counterflow (exchange of one substrate molecule for another labeled molecule from the other side of the membrane) are unaffected by imposition of ∆µ̃_H+. Therefore, it is apparent that alternating accessibility of sugar- and H⁺-binding sites to either side of the membrane is the result of galactoside binding and dissociation and not ∆µ̃_H+ (reviewed in refs. 8–10). Moreover, downhill lactose/H⁺ symport from a high to a low lactose concentration in the absence of ∆µ̃_H+ exhibits a primary deuterium isotope effect that is not observed for ∆µ̃_H+-driven lactose/H⁺ symport, equilibrium exchange, or counterflow (11, 12). Thus, it is likely that the rate-limiting step for downhill symport is deprotonation (13, 14), whereas in the presence of ∆µ̃_H+, opening of a cavity on the other side of the membrane after dissociation of sugar and H⁺ is limiting (15). Based on these and other findings, a detailed mechanism for symport by LacY has been proposed (10).Initial X-ray structures of LacY without bound sugar exhibit two pseudosymmetrical bundles of mostly irregular transmembrane helices surrounding a large aqueous cavity in the middle of the molecule; these initial structures were open on the cytoplasmic side and sealed on the periplasmic side (an inward-open conformation) (16–19). However, our recent X-ray crystallography studies (20) of the conformationally trapped double-Trp mutant G46W/G262W cocrystallized with β-d-galactopyranosyl-1-thio-β-d-galactopyranoside (TDG) reveal an almost occluded conformation with a narrowly outward (periplasmic)-open conformation and a tightly sealed cytoplasmic side [Protein Data Bank (PDB) ID code 4OAA]. In addition, a molecule of TDG is bound in a central cavity. The evidence shows that specific galactoside binding is consistent with prior findings from mutagenesis (21–23) and uses induced fit to interact with the surrounding protein (20). The findings also provide a strong indication that the transport mechanism of LacY involves a substrate-bound, occluded, intermediate conformation.Lactose has only one galactopyranosyl ring. Similarly, one galactopyranosyl ring of TDG lies against Trp-151 (helix V), confirming hydrophobic stacking between the bottom of the galactopyranosyl ring and the aromatic indole ring as suggested (24, 25). Glu-269 (helix VIII) is the acceptor of hydrogen bonds from the C4-OH group of the galactopyranosyl ring (21, 26). The η1 NH₂ group of Arg-144 (helix V) donates a hydrogen bond to O5 in the ring and is within hydrogen-bond distance of the C6-OH, whereas the η2 NH₂ group of Arg-144 donates hydrogen bonds to the C2′-OH (the other galactopyranosyl ring) of TDG and to Glu-126 Oε2 (27–29). Glu-126 (helix IV) acts as hydrogen-bond acceptor from the C2′-OH of TDG (27–29). His-322 (helix X) acts as a hydrogen-bond donor/acceptor between the εNH of the imidazole ring and the C3-OH of TDG (29–33) and is stabilized by a hydrogen-bond donor/acceptor interaction with the δNH of the imidazole and the OH of Tyr-236 (29, 34, 35). Finally, Asn-272 (helix VIII) donates a hydrogen bond to the C4-OH of TDG (23). These interactions define the specificity of LacY (summarized in ref. 20).Cys-148 (helix V), well known with respect to substrate protection against alkylation (reviewed in ref. 22), is also close to bound TDG but not sufficiently close to interact directly. Similarly, replacement of Ala-122 (helix IV) with a bulky side chain or alkylation of A122C with bulky thiol reagents causes LacY to become specific for the monosaccharide galactose. Disaccharide binding and transport are blocked sterically (36). Although Ala-122 does not make direct contact with TDG, bulky substituents at position 122 would clearly impact disaccharide binding.p-Nitrophenyl-α-d-galactopyranoside (α-NPG) is a FRET acceptor from Trp-151 (37) and binds to LacY with ∼eight times higher affinity than the twofold symmetric TDG, ∼two orders of magnitude better than β-NPG, and ∼three orders of magnitude better than the physiological substrate lactose or the monosaccharide galactose (6, 7, 38, 39). As we show here, the side-chain interactions with the galactopyranosyl moiety of α-NPG that provide specificity are almost identical to those described for TDG. In contrast, the increased affinity of α-NPG versus TDG is probably attributable primarily to hydrophobic interactions between the nitrophenyl group of NPG and Phe-20, Met-23, and Phe-27 from helix I. In addition, the nitro group is in close contact with polar groups that can sustain polar interactions of the type that also pertain to the glucose moiety of lactose.     

Aggregation propensities of superoxide dismutase G93 hotspot mutants mirror ALS clinical phenotypes

Protein framework alterations in heritable Cu, Zn superoxide dismutase (SOD) mutants cause misassembly and aggregation in cells affected by the motor neuron disease ALS. However, the mechanistic relationship between superoxide dismutase 1 (SOD1) mutations and human disease is controversial, with many hypotheses postulated for the propensity of specific SOD mutants to cause ALS. Here, we experimentally identify distinguishing attributes of ALS mutant SOD proteins that correlate with clinical severity by applying solution biophysical techniques to six ALS mutants at human SOD hotspot glycine 93. A small-angle X-ray scattering (SAXS) assay and other structural methods assessed aggregation propensity by defining the size and shape of fibrillar SOD aggregates after mild biochemical perturbations. Inductively coupled plasma MS quantified metal ion binding stoichiometry, and pulsed dipolar ESR spectroscopy evaluated the Cu²⁺ binding site and defined cross-dimer copper–copper distance distributions. Importantly, we find that copper deficiency in these mutants promotes aggregation in a manner strikingly consistent with their clinical severities. G93 mutants seem to properly incorporate metal ions under physiological conditions when assisted by the copper chaperone but release copper under destabilizing conditions more readily than the WT enzyme. Altered intradimer flexibility in ALS mutants may cause differential metal retention and promote distinct aggregation trends observed for mutant proteins in vitro and in ALS patients. Combined biophysical and structural results test and link copper retention to the framework destabilization hypothesis as a unifying general mechanism for both SOD aggregation and ALS disease progression, with implications for disease severity and therapeutic intervention strategies.ALS is a lethal degenerative disease of the human motor system (1). Opportunities for improved understanding and clinical intervention arose from the discovery that up to 23.5% of familial ALS cases and 7% of spontaneous cases are caused by mutations in the superoxide dismutase 1 (SOD1) gene encoding human Cu, Zn SOD (2–4). SOD is a highly conserved (5), dimeric, antioxidant metalloenzyme that detoxifies superoxide radicals (6, 7), but overexpression of SOD1 ALS mutants is sufficient to cause disease in mice (8). Misfolded and/or aggregated SOD species are deposited within mouse neuronal and glial inclusions (9, 10), even before symptoms appear (11, 12). Although human familial ALS has a symptomatic phenotype indistinguishable from sporadic cases (13), individual SOD1 mutations can result in highly variable disease progression and penetrance (14, 15).Many nongeneral mechanisms, including loss of activity or gain of function, were postulated to explain the roles of SOD mutants in ALS (3, 16–19). Recently, however, an initial hypothesis proposing that SOD manifests disease symptoms by framework destabilization (protein instability caused by structural defects) and consequent protein misassembly and aggregation has gained renewed support (2, 10, 14, 20–23). Ironically, WT SOD is an unusually stable protein (7, 24–26), and precisely how SOD mutations cause disease remains unclear. For instance, human SOD free cysteine residues C6 and C111 have been implicated in protein aggregation by promoting cross-linking (27, 28) and/or stability changes associated with oxidative modifications (29–33). Mutation of the chemically reactive thiols significantly decreases the irreversible denaturation rate for human and bovine SOD (24, 34). However, ALS mutants in a C6A/C111S SOD (AS-SOD) background (35, 36) maintain the native C57–C146 disulfide bond but can still undergo aggregation, and mutations of the free cysteines can cause ALS (37, 38). These results imply that free cysteines are not strictly required but rather, may alter aggregation kinetics (20). SOD also contains two metal ion cofactors in each subunit: a catalytic copper ion (6) and a structurally stabilizing zinc ion (34, 39, 40) (Fig. 1A). In higher eukaryotes, a copper chaperone for SOD (CCS) plays an important role in catalyzing both the copper incorporation and native disulfide bond formation (41). Structural analyses of apo WT SOD point to greater flexibility or increased solvent accessibility of C6 otherwise buried in the stable dimer interface (42, 43), and molecular dynamics simulations also suggest a critical role for metal ions in protein structure, because SOD’s β-sheet propensity decreases in the absence of metals (44). As a result, apo SOD readily forms protein aggregates (45, 46), but the molecular structures of SOD aggregates are likely polymorphic and represent a controversial topic (23, 47–51). The intertwined effects of the aggregation-enhancing free cysteines, dimer-stabilizing metal ions, and CCS maturation of SOD complicate the study of the ALS-causing SOD mutations themselves, and therefore, a clear cause-and-effect relationship remains obscure and requires deconvolution.Open in a separate windowFig. 1.Comparison of crystallographic and solution structures of WT and G93A SOD. (A) Overall architecture of the WT SOD dimer is displayed in 90° rotated views. G93 (small red spheres) resides on a surface-exposed interstrand loop between the fifth and sixth sequential β-strands of SOD and is expected to be innocuous in facilitating protein stability; however, this site harbors the most substitutions observed to result in ALS. G93 is also distant from both (Upper) the dimer interface and (Lower Left) the SOD active site (gold and silver spheres), which are generally implicated as the major determinants for SOD stability. Small blue spheres denote free cysteines. (Lower Right) The close-up view of the mutation site (boxed region in Lower Left tilted forward) shows high similarity between WT (purple) and G93A (red) SOD crystal structures [Protein Data Bank ID codes 1PU0 (WT) and 2ZKY (G93A)]. Hydrogen bonds characteristic of a β-bulge motif are indicated, whereby G93 (or A93) represents position 1. The main chain carbonyl group of β-barrel cork residue L38 is adjacent to the G93 site. (B) SAXS-derived electron pair P(r) distributions from WT (purple) and G93A (red) SOD samples in solution are compared with the theoretical curve for 1PU0. P(r) plots are normalized to peak height. Ab initio models of WT SOD derived from P(r) data are depicted in purple, with crystal structure docked into mesh envelope. Contributions to major and minor peaks from subunit and dimer dimensions are indicated.To better understand the structural effects of ALS mutations on SOD architecture, we coupled the wealth of crystallographic knowledge on SOD structure (7, 52, 53) with small-angle X-ray scattering (SAXS) experiments to characterize misassembly aggregates of ALS mutant SODs in solution. Over 20 y ago, we solved the first atomic structure of the human WT SOD protein (Fig. 1A) (20, 34) and proposed the framework destabilization hypothesis to explain how diverse mutations located throughout the 153-residue β-barrel enzyme might produce a similar disease phenotype (2), albeit with distinctions in the progression trajectory. Since that time, a staggering number of ALS mutations has been documented in patients [178 (mostly missense) (54)], with a similar phenotype in dogs (55, 56). Solution-based techniques are increasingly being applied to connect structure to biological outcome, for instance, through examination of intermolecular interactions within stress-activated pathways, for instance (57, 58). SAXS, which can probe structures for a wide size range of species, also provides higher resolution insights (59), for instance, over visible light-scattering techniques, readily distinguishing unfolded from folded proteins (60).Here, we monitor the initial events of protein aggregation in a subset of ALS mutants localized to a mutational hotspot site at glycine 93. Specifically, we wished to test a possible structural basis for how G93 mutations (to A, C, D, R, S, or V) modulate age of onset and clinical severity in ALS patients (14, 15). The G93 substitution occurs in a β-bulge region (61) between sequential β-strands of the protein (Fig. 1A) on a protruding loop roughly ∼20 Å from T54, the nearest residue of the opposing subunit, and the metal-containing active site (Fig. S1). A priori, mutation of this outer loop position would not be expected to interfere with active site chemistry or buried molecular interfaces. However, we discovered correlations of aggregation nucleation kinetics of SOD proteins with ALS mutations at this site, the stabilizing effects of metal ion retention, and available data for clinical phenotypes in patients with the same mutation. Furthermore, by measuring and exploiting the dimer geometry to observe intrinsic SOD conformers, we show that G93 mutant proteins natively reveal increased intradimer conformational flexibility in the absence of aggregation, which may reflect an increased tendency for ALS mutants to become metal-deficient and misfolding-prone and further explain the correlation to disease severity. Collective results on G93 mutants, thus, support and extend the framework destabilization hypothesis.     

Functional architecture of MFS d-glucose transporters

The Major Facilitator Superfamily (MFS) is a diverse group of secondary transporters with over 10,000 members, found in all kingdoms of life, including Homo sapiens. One objective of determining crystallographic models of the bacterial representatives is identification and physical localization of residues important for catalysis in transporters with medical relevance. The recently solved crystallographic models of the d-xylose permease XylE from Escherichia coli and GlcP from Staphylococcus epidermidus, homologs of the human d-glucose transporters, the GLUTs (SLC2), provide information about the structure of these transporters. The goal of this work is to examine general concepts derived from the bacterial XylE, GlcP, and other MFS transporters for their relevance to the GLUTs by comparing conservation of functionally critical residues. An energy landscape for symport and uniport is presented. Furthermore, the substrate selectivity of XylE is compared with GLUT1 and GLUT5, as well as a XylE mutant that transports d-glucose.The exceptionally diverse Major Facilitator Superfamily (MFS), with over 10,000 sequenced members, includes 74 families with members from Archaea to Homo sapiens (1–3). One goal of determining crystallographic structures of bacterial representatives in this family is identification and physical localization of residues important for catalysis in transporters with medical relevance. The d-xylose permease XylE from Escherichia coli is recognized as a homolog of human d-glucose transporters, the GLUTs (SLC2) (4), although XylE catalyzes d-xylose/H⁺ symport, and uphill (i.e., active) transport is driven by the H⁺ electrochemical gradient (∆_H⁺; interior negative or alkaline). Furthermore, d-glucose competitively inhibits d-xylose translocation (5). The recent high-resolution crystal structure of XylE with bound d-xylose or d-glucose (5) provides important detailed information about the organization of the bacterial transporter. Here we compare these transport proteins and describe functionally relevant similarities and differences.One or more of the 14 GLUT proteins are expressed in virtually every cell type in the human body (6), and all have well-established roles as transporters in various tissues and cell types (7). The GLUTs are grouped into three different classes based on sequence similarities (8), and XylE displays a bias to class III. Unlike XylE, most GLUTs are uniporters that catalyze equilibration of d-glucose across the cell membrane without a coupling ion (9). The sole known exception is GLUT13, the myo-inositol/H⁺ symporter (HMIT) (10). All GLUTs appear to transport hexoses or polyols, but the primary physiological substrate for many of the GLUTs remains uncertain (9). On the basis of mutational analyses, specific residues have been proposed to participate in substrate recognition by GLUT1 as well as other isoforms [e.g., Gln282, Tyr293 (11, 12)]. The QLS sequence motif in helix 7 is thought to be involved in d-glucose specificity based on its presence in GLUT1, -3, and -4, which transport d-glucose but not fructose, and its absence in GLUT2, which transports d-fructose as well as d-glucose (13). Other positions that have been implicated in the specificity of GLUT1–4 for d-glucose include the STSIF-motif in loop 7 (14), as well as Trp388, and Gln161 in helix 5 (15). However, a crystal structure with or without bound sugar has not been obtained yet for any GLUT.XylE contains a remarkably conserved sugar-binding site. All residues in contact with d-glucose in the crystal structure are also suggested to be involved in d-glucose binding in GLUT1 (12). Based on the d-glucose–bound XylE structure, a more precise structural model of GLUT1 was generated that allows mapping of disease-related positions and localization of side-chains ligating d-glucose in GLUT1 (5). Although the side-chains postulated to be involved in binding in GLUT1 are conserved in XylE, d-glucose is not transported (16).The paradigm of the MFS, lactose permease from E. coli (LacY), a galactoside/H⁺ symporter, is arguably the most intensively studied secondary transporter at present (17–19). Each of the 417 aminoacyl side-chains in LacY has been mutated (20). Remarkably, fewer than 10 residues are irreplaceable for active lactose transport: Glu126 (helix IV) and Arg144 (helix V), which are critical for substrate binding, as well as Trp151 (helix V), where an aromatic side-chain is essential; Glu269 (helix VIII), His322 (helix X), and Tyr236 (helix VII), which may be involved in coupling between protonation and sugar binding; and Arg302 (helix IX), and Glu325 (helix X), which are exclusively involved in H⁺ translocation (17, 19). It is striking that neutral replacement mutants for Glu325 are specifically defective in all steps involving net H⁺ translocation, but affinity is unaffected, and the mutants catalyze equilibrium exchange and counterflow as well or better than WT LacY (reviewed in ref. 21). Thus, Glu325 is clearly required for deprotonation of LacY. Although only a few residues are absolutely irreplaceable, Cys replacement of 82 additional residues has a significant effect on activity, inhibiting the steady-state level of accumulation by 50–80% (20, 22).Converging lines of evidence demonstrate that LacY functions by an alternating access mechanism (18). The galactoside-binding site in LacY is located in a deep cavity in the approximate middle of the molecule, and LacY contains 12 mostly irregular transmembrane helices organized in two pseudosymmetrical six-helix bundles (23–26), a common structural feature in the MFS (5, 23, 27–35). Binding of galactosides to LacY induces a widespread conformational transition, increasing the open probability of a hydrophilic cleft on the periplasmic side of the molecule with closure of the cytoplasmic cavity in reciprocal fashion (18, 36). These coordinated conformational transitions are fundamental to secondary transport and represent the basis for the alternating access mechanism. Accordingly, the catalytic cycle of a transporter does not involve significant movement of sugar- and H⁺-binding sites relative to the membrane. Rather, the protein essentially moves around the substrate, reciprocally exposing the binding sites to either side of the membrane (i.e., alternating access in ref. 37). Recent X-ray structures of the bacterial l-fucose/H⁺ symporter FucP (29), d-xylose/H⁺ symporter XylE (5, 38), and d-glucose/H⁺ symporter GlcP_Se (39) indicate that other MFS sugar transporters probably function in similar fashion.Interspin distances obtained from double electron–electron resonance measurements suggest the presence of intermediates in the transport cycle of LacY, in addition to the inward- and outward-open conformations (40, 41). Further analysis of interspin distances combined with homology modeling (42) provide support for an occluded unprotonated, sugar-free apo intermediate, as well as a protonated, sugar-bound occluded intermediate. Moreover, de-convolution of the conformational distributions of the LacY molecule has allowed an assessment of relative energy levels of the respective conformations. A hypothetical energy landscape for the transport cycle was proposed in which the occluded intermediates accommodate a higher energy level relative to a moderately facile energy profile between the open conformers (42). Although all MFS symporters likely operate by an alternating access mechanism (43), almost incontrovertible evidence is available for LacY only (reviewed in refs. 18 and 36). Recently, crystallographic support for this hypothesis was obtained for LacY, showing the molecule in an outward-open conformation with an almost entirely occluded sugar derivative in the binding-pocket (44).A principal difficulty in comparing MFS proteins is low sequence conservation. Despite a conserved fold and in some cases overlapping function, sequence identity ranges around 12–18% (22). Recently, we described an example of flexibility in design between LacY and the l-fucose permease (FucP), two MFS symporters (22). The order of the helix-triplets in FucP was permuted from their natural order relative to LacY to obtain better sequence conservation. The alignment was tested for conservation by comparing the 92 LacY mutants that impair function with 34 analogous functional mutations in FucP. In contrast to a conventional alignment, homology of the sugar- and H⁺-binding sites in the two proteins are observed. It was suggested that LacY and FucP (22), and many other MFS members (43), might have evolved from primordial noncovalently fused helix-triplets that formed functional transporters, and the functional segments assembled in a different consecutive order. Using this notion of evolution, we can begin to identify functionally related residues that do not correlate when primary sequences are compared, but do correlate in the tertiary structures of MFS transport proteins.     

Dynamic membrane protein topological switching upon changes in phospholipid environment

A fundamental objective in membrane biology is to understand and predict how a protein sequence folds and orients in a lipid bilayer. Establishing the principles governing membrane protein folding is central to understanding the molecular basis for membrane proteins that display multiple topologies, the intrinsic dynamic organization of membrane proteins, and membrane protein conformational disorders resulting in disease. We previously established that lactose permease of Escherichia coli displays a mixture of topological conformations and undergoes postassembly bidirectional changes in orientation within the lipid bilayer triggered by a change in membrane phosphatidylethanolamine content, both in vivo and in vitro. However, the physiological implications and mechanism of dynamic structural reorganization of membrane proteins due to changes in lipid environment are limited by the lack of approaches addressing the kinetic parameters of transmembrane protein flipping. In this study, real-time fluorescence spectroscopy was used to determine the rates of protein flipping in the lipid bilayer in both directions and transbilayer flipping of lipids triggered by a change in proteoliposome lipid composition. Our results provide, for the first time to our knowledge, a dynamic picture of these events and demonstrate that membrane protein topological rearrangements in response to lipid modulations occur rapidly following a threshold change in proteoliposome lipid composition. Protein flipping was not accompanied by extensive lipid-dependent unfolding of transmembrane domains. Establishment of lipid bilayer asymmetry was not required but may accelerate the rate of protein flipping. Membrane protein flipping was found to accelerate the rate of transbilayer flipping of lipids.There is limited understanding of how lipid environment affects membrane protein folding during exit from the translocon and insertion into the lipid bilayer or how dynamic changes in lipid environment affect the structure and folding of membrane proteins. Membrane protein lipid environment can change rapidly through lateral movement within a membrane, during trafficking along the secretion pathway, and locally in response to stimuli. Lipid environment is a determinant of transmembrane domain (TMD) orientation for several secondary transporters of Escherichia coli at the time of initial protein folding, as well as after final folding in the cell membrane (1). We used lactose permease (LacY), the paradigm of secondary transporters throughout nature (2), from E. coli as a model membrane protein to study how lipid–protein interactions determine inherently dynamic protein organization and function. When assembled in cells lacking phosphatidylethanolamine (PE), the net neutral and major phospholipid of E. coli (3) (Fig. 1), LacY exhibits inversion of the N-terminal (NT) six-TMD α-helical bundle with respect to the plane of the membrane bilayer and the C-terminal (CT) five-TMD bundle (4); TMD VII becomes an extramembrane domain (EMD) exposed to the periplasm and acts as a molecular hinge allowing the two halves of LacY to respond independently to the lipid environment (4). By use of genetically modified strains of E. coli in which the levels of PE in the bilayer can be controlled and titrated, the reversibility in both directions of LacY topological arrangement after membrane insertion and initial folding was demonstrated (5, 6). Multiple topological isoforms of LacY stably coexist at steady state in the membrane governed by the level of PE in a dose-dependent manner (5). Therefore, structural and topological organization of LacY is highly dynamic in a lipid-dependent, reversible, and bidirectional manner.Open in a separate windowFig. 1.Effect of changes in PE content on the orientation of domains C6, P7, or NT of LacY in lipid bilayers subjected to lipid exchange. TMD (I–XII) orientation is summarized for LacY in cell membranes or proteoliposomes containing 70% (Left), intermediate (Center), or 0% (Right) PE; the remaining lipid is PG plus CL. Stars indicate positions of single Cys or Trp replacements in EMDs used to determine topological orientation. The bold P7 EMD indicates proper folding of the P7 epitope only in the presence of PE.The contribution of additional cellular factors, such as the membrane protein insertion machinery, molecular chaperones, or membrane potential, cannot be ruled out by in vivo experiments alone. To determine the necessary and sufficient determinants for initial and postassembly lipid-dependent membrane protein dynamic topological organization, purified LacY was reconstituted into liposomes containing only lipids and LacY (7, 8). The in vitro results mirrored the in vivo results in that the ratio of native to inverted topological LacY isoforms increased in proportion to the ratio of PE to the anionic lipids phosphatidylglycerol (PG) and cardiolipin (CL) in the proteoliposomes. Changes in the PE content after protein integration into proteoliposomes [fliposomes (7, 8)] resulted in postassembly TMD flipping (Fig. 1). Therefore, membrane protein folding and dynamic postinsertional rearrangements are thermodynamically driven processes dependent on intrinsic lipid–protein interactions that may not require other cellular factors.We proposed the charge balance rule (1), which incorporates the influence of lipid environment into the positive inside rule (9–12) that governs the orientation of TMDs in the membrane. We found a synergistic relationship between the effective positive charge of EMDs as cytoplasmic retention signals and the negative charge density of the membrane surface determined by the ratio of net neutral [e.g., PE, phosphatidylcholine (PC)] to anionic (e.g., PG, CL) lipids. Therefore, postassembly topological rearrangements in response to alterations in the lipid environment and/or posttranslational modifications, such as phosphorylation, may dynamically alter organization of membrane proteins (1). The charge balance rule also provides a molecular basis for TMD orientation of proteins, such as the multi-drug transporter EmrE (13), and low-molecular-weight transporters (14) in E. coli, which coexist as stable dual topological conformers in the membrane. Although several recent reports (15–17) independently demonstrate that initial lipid composition of proteoliposomes determines steady-state TMD orientation, evidence for dynamic reorientation postassembly in vitro is limited (7).The existence of reversible multiple topologies of a protein dependent on the lipid environment raises new questions and challenges current dogma concerning the mechanism of membrane protein topogenesis (1). However, due to the length of time between lipid exchange and biochemical assessment of the change in topological organization in proteoliposomes (7), no insight was possible into the kinetics, mechanism, or structural intermediates occurring during a change in lipid composition. How fast are the events occurring? Are they happening concomitantly or sequentially? Do the proteoliposomes exhibit some form of lipid asymmetry that assists the flipping process? Is a membrane protein extensively unfolded during reorientation? Fast topological switching would allow a protein to escape degradation due to protein quality control or to adopt a new structural organization, and possibly a new function, quickly. Herein, we have used time-resolved fluorescence spectroscopy to show that the process involves a sequence of events that starts with a change in the lipid composition of the outer leaflet of proteoliposomes, quickly followed by protein topological reorientation, and ending with a slower equilibration of lipid composition between the inner and outer bilayer leaflets.     

Cyclic nucleotide-gated channel 18 is an essential Ca2+ channel in pollen tube tips for pollen tube guidance to ovules in Arabidopsis

In flowering plants, pollen tubes are guided into ovules by multiple attractants from female gametophytes to release paired sperm cells for double fertilization. It has been well-established that Ca²⁺ gradients in the pollen tube tips are essential for pollen tube guidance and that plasma membrane Ca²⁺ channels in pollen tube tips are core components that regulate Ca²⁺ gradients by mediating and regulating external Ca²⁺ influx. Therefore, Ca²⁺ channels are the core components for pollen tube guidance. However, there is still no genetic evidence for the identification of the putative Ca²⁺ channels essential for pollen tube guidance. Here, we report that the point mutations R491Q or R578K in cyclic nucleotide-gated channel 18 (CNGC18) resulted in abnormal Ca²⁺ gradients and strong pollen tube guidance defects by impairing the activation of CNGC18 in Arabidopsis. The pollen tube guidance defects of cngc18-17 (R491Q) and of the transfer DNA (T-DNA) insertion mutant cngc18-1 (+/−) were completely rescued by CNGC18. Furthermore, domain-swapping experiments showed that CNGC18’s transmembrane domains are indispensable for pollen tube guidance. Additionally, we found that, among eight Ca²⁺ channels (including six CNGCs and two glutamate receptor-like channels), CNGC18 was the only one essential for pollen tube guidance. Thus, CNGC18 is the long-sought essential Ca²⁺ channel for pollen tube guidance in Arabidopsis.Pollen tubes deliver paired sperm cells into ovules for double fertilization, and signaling communication between pollen tubes and female reproductive tissues is required to ensure the delivery of sperm cells into the ovules (1). Pollen tube guidance is governed by both female sporophytic and gametophytic tissues (2, 3) and can be separated into two categories: preovular guidance and ovular guidance (1). For preovular guidance, diverse signaling molecules from female sporophytic tissues have been identified, including the transmitting tissue-specific (TTS) glycoprotein in tobacco (4), γ-amino butyric acid (GABA) in Arabidopsis (5), and chemocyanin and the lipid transfer protein SCA in Lilium longiflorum (6, 7). For ovular pollen tube guidance, female gametophytes secrete small peptides as attractants, including LUREs in Torenia fournieri (8) and Arabidopsis (9) and ZmEA1 in maize (10, 11). Synergid cells, central cells, egg cells, and egg apparatus are all involved in pollen tube guidance, probably by secreting different attractants (9–15). Additionally, nitric oxide (NO) and phytosulfokine peptides have also been implicated in both preovular and ovular pollen tube guidance (16–18). Thus, pollen tubes could be guided by diverse attractants in a single plant species.Ca²⁺ gradients at pollen tube tips are essential for both tip growth and pollen tube guidance (19–27). Spatial modification of the Ca²⁺ gradients leads to the reorientation of pollen tube growth in vitro (28, 29). The Ca²⁺ gradients were significantly increased in pollen tubes attracted to the micropyles by synergid cells in vivo, compared with those not attracted by ovules (30). Therefore, the Ca²⁺ gradients in pollen tube tips are essential for pollen tube guidance. The Ca²⁺ gradients result from external Ca²⁺ influx, which is mainly mediated by plasma membrane Ca²⁺ channels in pollen tube tips. Thus, the Ca²⁺ channels are the key components for regulating the Ca²⁺ gradients and are consequently essential for pollen tube guidance. Using electrophysiological techniques, inward Ca²⁺ currents were observed in both pollen grain and pollen tube protoplasts (31–36), supporting the presence of plasma membrane Ca²⁺ channels in pollen tube tips. Recently, a number of candidate Ca²⁺ channels were identified in pollen tubes, including six cyclic nucleotide-gated channels (CNGCs) and two glutamate receptor-like channels (GLRs) in Arabidopsis (37–40). Three of these eight channels, namely CNGC18, GLR1.2, and GLR3.7, were characterized as Ca²⁺-permeable channels (40, 41) whereas the ion selectivity of the other five CNGCs has not been characterized. We hypothesized that the Ca²⁺ channel essential for pollen tube guidance could be among these eight channels.In this research, we first characterized the remaining five CNGCs as Ca²⁺ channels. We further found that CNGC18, out of the eight Ca²⁺ channels, was the only one essential for pollen tube guidance in Arabidopsis and that its transmembrane domains were indispensable for pollen tube guidance.     

Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle

To dissect the kinetics of structural transitions underlying the stepping cycle of kinesin-1 at physiological ATP, we used interferometric scattering microscopy to track the position of gold nanoparticles attached to individual motor domains in processively stepping dimers. Labeled heads resided stably at positions 16.4 nm apart, corresponding to a microtubule-bound state, and at a previously unseen intermediate position, corresponding to a tethered state. The chemical transitions underlying these structural transitions were identified by varying nucleotide conditions and carrying out parallel stopped-flow kinetics assays. At saturating ATP, kinesin-1 spends half of each stepping cycle with one head bound, specifying a structural state for each of two rate-limiting transitions. Analysis of stepping kinetics in varying nucleotides shows that ATP binding is required to properly enter the one-head–bound state, and hydrolysis is necessary to exit it at a physiological rate. These transitions differ from the standard model in which ATP binding drives full docking of the flexible neck linker domain of the motor. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily.Kinesin-1 is a motor protein that steps processively toward microtubule plus-ends, tracking single protofilaments and hydrolyzing one ATP molecule per step (1–6). Step sizes corresponding to the tubulin dimer spacing of 8.2 nm are observed when the molecule is labeled by its C-terminal tail (7–10) and to a two-dimer spacing of 16.4 nm when a single motor domain is labeled (4, 11, 12), consistent with the motor walking in a hand-over-hand fashion. Kinesin has served as an important model system for advancing single-molecule techniques (7–10) and is clinically relevant for its role in neurodegenerative diseases (13), making dissection of its step a popular ongoing target of study.Despite decades of work, many essential components of the mechanochemical cycle remain disputed, including (i) how much time kinesin-1 spends in a one-head–bound (1HB) state when stepping at physiological ATP concentrations, (ii) whether the motor waits for ATP in a 1HB or two-heads–bound (2HB) state, and (iii) whether ATP hydrolysis occurs before or after tethered head attachment (4, 11, 14–20). These questions are important because they are fundamental to the mechanism by which kinesins harness nucleotide-dependent structural changes to generate mechanical force in a manner optimized for their specific cellular tasks. Addressing these questions requires characterizing a transient 1HB state in the stepping cycle in which the unattached head is located between successive binding sites on the microtubule. This 1HB intermediate is associated with the force-generating powerstroke of the motor and underlies the detachment pathway that limits motor processivity. Optical trapping (7, 19, 21, 22) and single-molecule tracking studies (4, 8–11) have failed to detect this 1HB state during stepping. Single-molecule fluorescence approaches have detected a 1HB intermediate at limiting ATP concentrations (11, 12, 14, 15), but apart from one study that used autocorrelation analysis to detect a 3-ms intermediate (17), the 1HB state has been undetectable at physiological ATP concentrations.Single-molecule microscopy is a powerful tool for studying the kinetics of structural changes in macromolecules (23). Tracking steps and potential substeps for kinesin-1 at saturating ATP has until now been hampered by the high stepping rates of the motor (up to 100 s⁻¹), which necessitates high frame rates, and the small step size (8.2 nm), which necessitates high spatial precision (7). Here, we apply interferometric scattering microscopy (iSCAT), a recently established single-molecule tool with high spatiotemporal resolution (24–27) to directly visualize the structural changes underlying kinesin stepping. By labeling one motor domain in a dimeric motor, we detect a 1HB intermediate state in which the tethered head resides over the bound head for half the duration of the stepping cycle at saturating ATP. We further show that at physiological stepping rates, ATP binding is required to enter this 1HB state and that ATP hydrolysis is required to exit it. This work leads to a significant revision of the sequence and kinetics of mechanochemical transitions that make up the kinesin-1 stepping cycle and provides a framework for understanding functional diversity across the kinesin superfamily.     

Hyperpolarization-activated,cyclic nucleotide-gated cation channels in Aplysia: Contribution to classical conditioning

Hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels are critical regulators of neuronal excitability, but less is known about their possible roles in synaptic plasticity and memory circuits. Here, we characterized the HCN gene organization, channel properties, distribution, and involvement in associative and nonassociative forms of learning in Aplysia californica. Aplysia has only one HCN gene, which codes for a channel that has many similarities to the mammalian HCN channel. The cloned acHCN gene was expressed in Xenopus oocytes, which displayed a hyperpolarization-induced inward current that was enhanced by cGMP as well as cAMP. Similarly to its homologs in other animals, acHCN is permeable to K⁺ and Na⁺ ions, and is selectively blocked by Cs⁺ and ZD7288. We found that acHCN is predominantly expressed in inter- and motor neurons, including LFS siphon motor neurons, and therefore tested whether HCN channels are involved in simple forms of learning of the siphon-withdrawal reflex in a semiintact preparation. ZD7288 (100 μM) significantly reduced an associative form of learning (classical conditioning) but had no effect on two nonassociative forms of learning (intermediate-term sensitization and unpaired training) or baseline responses. The HCN current is enhanced by nitric oxide (NO), which may explain the postsynaptic role of NO during conditioning. HCN current in turn enhances the NMDA-like current in the motor neurons, suggesting that HCN channels contribute to conditioning through this pathway.Hyperpolarization-activated, cyclic nucleotide-gated (HCN), cation nonselective ion channels generate hyperpolarization-activated inward currents (I_h) and thus tend to stabilize membrane potential (1–3). In addition, binding of cyclic nucleotides (cAMP and cGMP) to the C-terminal cyclic nucleotide binding domain (CNBD) enhances I_h and thus couples membrane excitability with intracellular signaling pathways (2, 4). HCN channels are widely important for numerous systemic functions such as hormonal regulation, heart contractility, epilepsy, pain, central pattern generation, sensory perception (4–15), and learning and memory (16–24).However, in previous studies it has been difficult to relate the cellular effects of HCN channels directly to their behavioral effects, because of the immense complexity of the mammalian brain. We have therefore investigated the role of HCN channels in Aplysia, which has a numerically simpler nervous system (25). We first identified and characterized an HCN gene in Aplysia, and showed that it codes for a channel that has many similarities to the mammalian HCN channel. We found that the Aplysia HCN channel is predominantly expressed in motor neurons including LFS neurons in the siphon withdrawal reflex circuit (26, 27). We therefore investigated simple forms of learning of that reflex in a semiintact preparation (28–30) and found that HCN current is involved in classical conditioning and enhances the NMDA-like current in the motor neurons. These results provide a direct connection between HCN channels and behavioral learning and suggest a postsynaptic mechanism of that effect. HCN current in turn is enhanced by nitric oxide (NO), a transmitter of facilitatory interneurons, and thus may contribute to the postsynaptic role of NO during conditioning.     

Rewiring yeast sugar transporter preference through modifying a conserved protein motif

Utilization of exogenous sugars found in lignocellulosic biomass hydrolysates, such as xylose, must be improved before yeast can serve as an efficient biofuel and biochemical production platform. In particular, the first step in this process, the molecular transport of xylose into the cell, can serve as a significant flux bottleneck and is highly inhibited by other sugars. Here we demonstrate that sugar transport preference and kinetics can be rewired through the programming of a sequence motif of the general form G-G/F-XXX-G found in the first transmembrane span. By evaluating 46 different heterologously expressed transporters, we find that this motif is conserved among functional transporters and highly enriched in transporters that confer growth on xylose. Through saturation mutagenesis and subsequent rational mutagenesis, four transporter mutants unable to confer growth on glucose but able to sustain growth on xylose were engineered. Specifically, Candida intermedia gxs1 Phe³⁸Ile³⁹Met⁴⁰, Scheffersomyces stipitis rgt2 Phe³⁸ and Met⁴⁰, and Saccharomyces cerevisiae hxt7 Ile³⁹Met⁴⁰Met³⁴⁰ all exhibit this phenotype. In these cases, primary hexose transporters were rewired into xylose transporters. These xylose transporters nevertheless remained inhibited by glucose. Furthermore, in the course of identifying this motif, novel wild-type transporters with superior monosaccharide growth profiles were discovered, namely S. stipitis RGT2 and Debaryomyces hansenii 2D01474. These findings build toward the engineering of efficient pentose utilization in yeast and provide a blueprint for reprogramming transporter properties.Molecular transporter proteins facilitate monosaccharide uptake and serve as the first step in catabolic metabolism. In this capacity, the preferences, regulation, and kinetics of these transporters ultimately dictate total carbon flux (1–3); and optimization of intracellular catabolic pathways only increases the degree to which transport exerts control over metabolic flux (4, 5). Thus, monosaccharide transport profiles and rates are important design criteria and a driving force to enable metabolic engineering advances, ultimately resulting in a biorefinery concept whereby biomass is converted via microbes into a diverse set of molecules (6–10). Among possible host organisms, Saccharomyces cerevisiae is an emerging industrial organism with well-developed genetic tools and established industrial processes and track record (11–16). However, S. cerevisiae lacks an endogenous xylose catabolic pathway and thus is unable to natively use the second most abundant sugar in lignocellulosic biomass. Decades of research have been focused on improving xylose catabolic pathways in recombinant S. cerevisiae (17–22), but less work has been focused on the first committed step of the process—xylose transport, an outstanding limitation in the efficient conversion of lignocellulosic sugars (23, 24).In S. cerevisiae, monosaccharide uptake is mediated by transporters belonging to the major facilitator superfamily (MFS) (25, 26), a ubiquitous group of proteins found across species (27). The predominant transporters in yeast are members of the HXT family (28) and are marked by efficient hexose transport (29) with lower affinities to xylose thus contributing to diauxic growth and flux limitation when attempting pentose utilization in recombinant S. cerevisiae (30). Previous efforts have attempted to identify heterologous transporters with a higher affinity for xylose over glucose (31–36). However, the vast majority of these transporters are either nonfunctional, not efficient, or not xylose specific (24, 37). Furthermore, nearly all known wild-type transporters that enable growth on xylose in yeast confer higher growth rates on glucose than on xylose (24, 37). As an alternative to bioprospecting, we have previously reported that xylose affinity and exponential growth rates on xylose can be improved via directed evolution of Candida intermedia glucose-xylose symporter 1 (GXS1) and Scheffersomyces stipitis xylose uptake 3 (XUT3) (38). These results demonstrated that mutations at specific residues (e.g., Phe⁴⁰ in C. intermedia GXS1) can have a significant impact on the carbohydrate selectivity of these MFS transporters. The fact that single amino acid substitutions can have such a significant impact on transport phenotype (38–40) indicates how simple homology based searches can be ineffective at identifying efficient xylose transporters (35, 36). However, evidence of natural xylose exclusivity is seen in the Escherichia coli xylE transporter that has recently been crystallized (41). The sequence-function flexibility of MFS transporters potentiates the capability to rewire hexose transporters from being glucose favoring, xylose permissive into being xylose-exclusive transporters.In this work, we report on the discovery of a conserved Gly³⁶-Gly³⁷-Val³⁸-Leu³⁹-Phe⁴⁰-Gly⁴¹ motif surrounding the previously identified Phe⁴⁰ residue of C. intermedia GXS1 that controls transporter efficiency and selectivity. By evaluating 46 different heterologously expressed transporters, we find that this motif is conserved among functional transporters and highly enriched in transporters that confer growth on xylose, taking the general form G-G/F-XXX-G. We conduct saturation mutagenesis on Val³⁸, Leu³⁹, and Phe⁴⁰ within the variable region of this motif in C. intermedia GXS1 to demonstrate control of sugar selectivity. Next, we combine xylose-favoring mutations to create a unique mutant version of gxs1 that transports xylose, but not glucose. Finally, we demonstrate the importance of this motif in the capacity to rewire the sugar preference of other hexose transporters including S. cerevisiae hexose transporter 7 (HXT7) and S. stipitis glucose transporter/sensor (RGT2, similar to S. cerevisiae RGT2). This work serves to increase our understanding of the structure–function relationships for molecular transporter engineering and demonstrates complete rewiring of hexose transporters into transporters that prefer xylose as a substrate.     

Quantum mechanical calculations suggest that lytic polysaccharide monooxygenases use a copper-oxyl,oxygen-rebound mechanism

Lytic polysaccharide monooxygenases (LPMOs) exhibit a mononuclear copper-containing active site and use dioxygen and a reducing agent to oxidatively cleave glycosidic linkages in polysaccharides. LPMOs represent a unique paradigm in carbohydrate turnover and exhibit synergy with hydrolytic enzymes in biomass depolymerization. To date, several features of copper binding to LPMOs have been elucidated, but the identity of the reactive oxygen species and the key steps in the oxidative mechanism have not been elucidated. Here, density functional theory calculations are used with an enzyme active site model to identify the reactive oxygen species and compare two hypothesized reaction pathways in LPMOs for hydrogen abstraction and polysaccharide hydroxylation; namely, a mechanism that employs a η¹-superoxo intermediate, which abstracts a substrate hydrogen and a hydroperoxo species is responsible for substrate hydroxylation, and a mechanism wherein a copper-oxyl radical abstracts a hydrogen and subsequently hydroxylates the substrate via an oxygen-rebound mechanism. The results predict that oxygen binds end-on (η¹) to copper, and that a copper-oxyl–mediated, oxygen-rebound mechanism is energetically preferred. The N-terminal histidine methylation is also examined, which is thought to modify the structure and reactivity of the enzyme. Density functional theory calculations suggest that this posttranslational modification has only a minor effect on the LPMO active site structure or reactivity for the examined steps. Overall, this study suggests the steps in the LPMO mechanism for oxidative cleavage of glycosidic bonds.Carbohydrates are the most diverse set of biomolecules, and thus, many enzyme classes have evolved to assemble, modify, and depolymerize carbohydrates, including glycosyltransferases, glycoside hydrolases, carbohydrate esterases, and polysaccharide lyases (1). Recently, a new enzymatic paradigm was discovered that employs copper-dependent oxidation to cleave glycosidic bonds in polysaccharides (2–13). These newly classified enzymes, termed lytic polysaccharide monooxygenases (LPMOs), broadly resemble other copper monooxygenases and some hydroxylation catalysts (14–21).The discovery that LPMOs use an oxidative mechanism has attracted interest both because it is a unique paradigm for carbohydrate modification that employs a powerful C–H activation mechanism, and also because LPMOs synergize with hydrolytic enzymes in biomass conversion to sugars because they act directly on the crystalline polysaccharide surface without the requirement for depolymerization (4, 22, 23), making them of interest in biofuels production. LPMOs were originally characterized as Family 61 glycoside hydrolases (GH61s, reclassified as auxiliary activity 9, AA9) or Family 33 carbohydrate-binding modules (CBM33s, reclassified as AA10), which are structurally similar enzymes found in fungi and nonfungal organisms (22), respectively. In 2005, Vaaje-Kolstad et al. described the synergism (24) of a chitin-active CBM33 (chitin-binding protein, CBP21) with hydrolases, but the mechanism was not apparent. Harris et al. demonstrated that a GH61 boosts hydrolytic enzyme activity on lignocellulosic biomass (2). Vaaje-Kolstad et al. subsequently showed that CBP21 employs an oxidative mechanism to cleave glycosidic linkages in chitin (4).Following these initial discoveries, multiple features of LPMOs have been elucidated. LPMOs use copper (5–7) and produce either aldonic acids or 4-keto sugars at oxidized chain ends, believed to result from hydroxylation at the C1 or C4 carbon, respectively. Hydroxylation at the C1 carbon is proposed to spontaneously undergo elimination to a lactone followed by hydrolytic ring opening to an aldonic acid, whereas hydroxylation and elimination at C4 yields a 4-keto sugar at the nonreducing end (5–12). The active site is a mononuclear type(II) copper center ligated by a “histidine brace” (5, 12), comprising a bidentate N-terminal histidine ligand via the amino terminus and an imidazole ring nitrogen atom and another histidine residue also via a ring nitrogen atom. Hemsworth et al. reported a bacterial LPMO structure wherein the active site copper ion was photoreduced to Cu(I) (12), and Aachmann et al. demonstrated that Cu(I) binds with higher affinity than Cu(II) in CBP21 (13). A structural study of a fungal LPMO revealed an N-terminal methylation on a nitrogen atom in the imidazole ring of unknown function (5), but some LPMOs are active without this modification (6, 11). LPMOs require reducing agents for activity such as ascorbate (2–8, 10–12), and cellobiose dehydrogenase (CDH), a common fungal secretome component, can potentiate LPMO activity in lieu of a small-molecule reducing agent (7, 8).Overall, many structural and mechanistic insights have been reported since the discoveries that LPMOs are oxidative enzymes (4–10). However, many questions remain regarding LPMO function (22, 25). Here, we examine the LPMO catalytic mechanism with density functional theory (DFT) calculations on an active site model (ASM) of a fungal LPMO. We seek to (i) understand the identity of the reactive oxygen species (ROS), (ii) compare two hypothesized catalytic mechanisms, and (iii) examine the role of N-terminal methylation in catalysis.     

Effects of lymphocyte profile on development of EBV-induced lymphoma subtypes in humanized mice

Epstein-Barr virus (EBV) infection causes both Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphoma (NHL). The present study reveals that EBV-induced HL and NHL are intriguingly associated with a repopulated immune cell profile in humanized mice. Newborn immunodeficient NSG mice were engrafted with human cord blood CD34⁺ hematopoietic stem cells (HSCs) for a 8- or 15-wk reconstitution period (denoted ^8whN and ^15whN, respectively), resulting in human B-cell and T-cell predominance in peripheral blood cells, respectively. Further, novel humanized mice were established via engraftment of hCD34⁺ HSCs together with nonautologous fetal liver-derived mesenchymal stem cells (MSCs) or MSCs expressing an active notch ligand DLK1, resulting in mice skewed with human B or T cells, respectively. After EBV infection, whereas NHL developed more frequently in B-cell–predominant humanized mice, HL was seen in T-cell–predominant mice (P = 0.0013). Whereas human splenocytes from NHL-bearing mice were positive for EBV-associated NHL markers (hBCL2⁺, hCD20⁺, hKi67⁺, hCD20⁺/EBNA1⁺, and EBER⁺) but negative for HL markers (LMP1⁻, EBNA2⁻, and hCD30⁻), most HL-like tumors were characterized by the presence of malignant Hodgkin’s Reed–Sternberg (HRS)-like cells, lacunar RS (hCD30⁺, hCD15⁺, IgJ⁻, EBER⁺/hCD30⁺, EBNA1⁺/hCD30⁺, LMP⁺/EBNA2⁻, hCD68⁺, hBCL2⁻, hCD20^-/weak, Phospho STAT6⁺), and mummified RS cells. This study reveals that immune cell composition plays an important role in the development of EBV-induced B-cell lymphoma.Epstein Barr virus (EBV) infects human B lymphocytes and epithelial cells in >90% of the human population (1, 2). EBV infection is widely associated with the development of diverse human disorders that include Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphomas (NHL), including diffused large B-cell lymphoma (DLBCL), follicular B-cell lymphoma (FBCL), endemic Burkitt’s lymphoma (BL), and hemophagocytic lymphohistiocytosis (HLH) (3).HL is a malignant lymphoid neoplasm most prevalent in adolescents and young adults (4–6). Hodgkin/Reed–Sternberg (HRS) cells are the sole malignant cells of HL. HRS cells are characterized by CD30⁺/CD15⁺/BCL6⁻/CD20^+/− markers and appear large and multinucleated owing to multiple nuclear divisions without cytokinesis. Although HRS cells are malignant in the body, surrounding inflammatory cells greatly outnumber them. These reactive nonmalignant inflammatory cells, including lymphocytes, histiocytes, eosinophils, fibroblasts, neutrophils, and plasma cells, compose the vast majority of the tumor mass. The presence of HRS cells in the context of this inflammatory cellular background is a critical hallmark of the HL diagnosis (4). Approximately 50% of HL cases are EBV-associated (EBVaHL) (7–11). EBV-positive HRS cells express EBV latent membrane protein (LMP) 1 (LMP1), LMP2A, LMP2B, and EBV nuclear antigen (EBNA) 1 (EBNA1), but lack EBNA2 (latency II marker) (12). LMP1 is consistently expressed in all EBV-associated cases of classical HL (13, 14). LMP1 mimics activated CD40 receptors, induces NF-κB, and allows cells to become malignant while escaping apoptosis (15).The etiologic role of EBV in numerous disorders has been studied in humanized mouse models in diverse experimental conditions. Humanized mouse models recapitulate key characteristics of EBV infection-associated disease pathogenesis (16–24). Different settings have given rise to quite distinct phenotypes, including B-cell type NHL (DLBCL, FBCL, and unspecified B-cell lymphomas), natural killer/T cell lymphoma (NKTCL), nonmalignant lymphoproliferative disorder (LPD), extremely rare HL, HLH, and arthritis (16–24). Despite considerable efforts (16–24), EBVaHL has not been properly produced in the humanized mouse setting model, owing to inappropriate animal models and a lack of in-depth analyses. After an initial report of infected humanized mice, HRS-like cells appeared to be extremely rare in the spleens of infected humanized mice; however, the findings were inconclusive (18). Here we report direct evidence of EBVaHL or HL-like neoplasms in multiple humanized mice in which T cells were predominant over B cells. Our study demonstrates that EBV-infected humanized mice display additional EBV-associated pathogenesis, including DLBCL and hemophagocytic lymphohistiocytosis (16, 17).     

Transient conformers of LacY are trapped by nanobodies

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.Symport 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.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 k_on 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 k_on 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 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.