Design of Nanohydrogels for Targeted Intracellular Drug Transport to the Trans-Golgi Network.

Nanohydrogels combine advantages of hydrogels and nanoparticles. In particular, they represent promising drug delivery systems. Nanogel synthesis by oxidative condensation of polyglycidol prepolymers, that are modified with thiol groups, results in crosslinking by disulfide bonds. Hereby, biomolecules like the antidiabetic peptide RS1-reg, derived from the regulatory protein RS1 of the Na+ -D-glucose cotransporter SGLT1, can be covalently bound by cysteine residues to the nanogel in a hydrophilic, stabilizing environment. After oral uptake, the acid-stable nanogels protect their loading during gastric passage from proteolytic degradation. Under alkaline conditions in small intestine the nanohydrogels become mucoadhesive, pass the intestinal mucosa and are taken up into small intestinal enterocytes by endocytosis. Using Caco-2 cells as a model for small intestinal enterocytes, by confocal laser scanning microscopy and structured illumination microscopy, the colocalization of fluorescent-labeled RS1-reg with markers of endosomes, lysosomes, and trans-Golgi-network after uptake with polyglycidol-based nanogels formed by precipitation polymerization is demonstrated. This indicates that RS1-reg follows the endosomal pathway. In the following, the design of bespoken nanohydrogels for specific targeting of RS1-reg to its site of action at the trans-Golgi network is described that might also represent a way of targeted transport for other drugs to their targets at the Golgi apparatus.

An in silico quest for next-generation antimalarial drugs by targeting Plasmodium falciparum hexose transporter protein: a multi-pronged approach.

The emergence of artemisinin resistance by malaria parasites is a major challenge in the fight against malaria, thus posing serious threat to the public health across the world. To tackle this, antimalarial drugs with unconventional mechanisms are therefore urgently needed. It has been reported that selective starvation of Plasmodium falciparum by blocking the function of hexose transporter 1 (PfHT1) protein, the only known transporter for glucose uptake in P. falciparum, could provide an alternative approach to fight the drug resistant malaria parasites. In this study, three high affinity molecules (BBB_25784317, BBB_26580136 and BBB_26580144) that have shown the best docked conformation and least binding energy with PfHT1 were shortlisted. The docking energy of BBB_25784317, BBB_26580136 and BBB_26580144 with PfHT1 were -12.5, -12.1 and -12.0 kcal/mol, respectively. In the follow up simulation studies, the protein 3D structure maintains considerable stability in the presence of the compounds. It was also observed that the compounds produced a number of hydrophilic and hydrophobic interactions with the protein allosteric site residues. This demonstrates strong intermolecular interaction guided by close distance hydrogen bonds of compounds with Ser45, Asn48, Thr49, Asn52, Ser317, Asn318, Ile330 and Ser334. Revalidation of compounds binding affinity was conducted by more appropriate simulation based binding free energy techniques (MM-GB/PBSA and WaterSwap). Additionally, entropy assay was performed that further strengthen the predictions. In silico pharmacokinetics confirmed that the compounds would be suitable candidates for oral delivery due to their high gastrointestinal absorption and less toxic reaction. Overall, the predicted compounds are promising and could be further sought as antimalarial leads and subjected to thorough experimental investigations.Communicated by Ramaswamy H. Sarma.

CLEC16A interacts with retromer and TRIM27, and its loss impairs endosomal trafficking and neurodevelopment.

CLEC16A is a membrane-associated C-type lectin protein that functions as a E3-ubiquitin ligase. CLEC16A regulates autophagy and mitophagy, and reportedly localizes to late endosomes. GWAS studies have associated CLEC16A SNPs to various auto-immune and neurological disorders, including multiple sclerosis and Parkinson disease. Studies in mouse models imply a role for CLEC16A in neurodegeneration. We identified bi-allelic CLEC16A truncating variants in siblings from unrelated families presenting with a severe neurodevelopmental disorder including microcephaly, brain atrophy, corpus callosum dysgenesis, and growth retardation. To understand the function of CLEC16A in neurodevelopment we used in vitro models and zebrafish embryos. We observed CLEC16A localization to early endosomes in HEK293T cells. Mass spectrometry of human CLEC16A showed interaction with endosomal retromer complex subunits and the endosomal ubiquitin ligase TRIM27. Expression of the human variant leading to C-terminal truncated CLEC16A, abolishes both its endosomal localization and interaction with TRIM27, suggesting a loss-of-function effect. CLEC16A knockdown increased TRIM27 adhesion to early endosomes and abnormal accumulation of endosomal F-actin, a sign of disrupted vesicle sorting. Mutagenesis of clec16a by CRISPR-Cas9 in zebrafish embryos resulted in accumulated acidic/phagolysosome compartments, in neurons and microglia, and dysregulated mitophagy. The autophagocytic phenotype was rescued by wild-type human CLEC16A but not the C-terminal truncated CLEC16A. Our results demonstrate that CLEC16A closely interacts with retromer components and regulates endosomal fate by fine-tuning levels of TRIM27 and polymerized F-actin on the endosome surface. Dysregulation of CLEC16A-mediated endosomal sorting is associated with neurodegeneration, but it also causes accumulation of autophagosomes and unhealthy mitochondria during brain development.

Functional characterization of Plasmodium vivax hexose transporter 1.

Plasmodium vivax is the most widely distributed human malaria parasite. The eradication of vivax malaria remains challenging due to transmission of drug-resistant parasite and dormant liver form. Consequently, anti-malarial drugs with novel mechanisms of action are urgently demanded. Glucose uptake blocking strategy is suggested as a novel mode of action that leads to selective starvation in various species of malaria parasites. The role of hexose transporter 1 in Plasmodium species is glucose uptake, and its blocking strategies proved to successfully induce selective starvation. However, there is limited information on the glucose uptake properties via P. vivax hexose transporter 1 (PvHT1). Thus, we focused on the PvHT1 to precisely identify its properties of glucose uptake. The PvHT1 North Korean strain (PvHT1NK) expressed Xenopus laevis oocytes mediating the transport of [3H] deoxy-D-glucose (ddGlu) in an expression and incubation time-dependent manner without sodium dependency. Moreover, the PvHT1NK showed no exchange mode of glucose in efflux experiments and concentration-dependent results showed saturable kinetics following the Michaelis-Menten equation. Non-linear regression analysis revealed a Km value of 294.1 µM and a Vmax value of 1,060 pmol/oocyte/hr, and inhibition experiments showed a strong inhibitory effect by glucose, mannose, and ddGlu. Additionally, weak inhibition was observed with fructose and galactose. Comparison of amino acid sequence and tertiary structure between P. falciparum and P. vivax HT1 revealed a completely conserved residue in glucose binding pocket. This result supported that the glucose uptake properties are similar to P. falciparum, and PfHT1 inhibitor (compound 3361) works in P. vivax. These findings provide properties of glucose uptake via PvHT1NK for carbohydrate metabolism and support the approaches to vivax malaria drug development strategy targeting the PvHT1 for starving of the parasite.

Structural Insights into the Substrate Transport Mechanisms in GTR Transporters through Ensemble Docking.

Glucosinolate transporters (GTRs) are part of the nitrate/peptide transporter (NPF) family, members of which also transport specialized secondary metabolites as substrates. Glucosinolates are defense compounds derived from amino acids. We selected 4-methylthiobutyl (4MTB) and indol-3-ylmethyl (I3M) glucosinolates to study how GTR1 from Arabidopsis thaliana transports these substrates in computational simulation approaches. The designed pipeline reported here includes massive docking of 4MTB and I3M in an ensemble of GTR1 conformations (in both inward and outward conformations) extracted from molecular dynamics simulations, followed by clustered and substrate-protein interactions profiling. The identified key residues were mutated, and their role in substrate transport was tested. We were able to identify key residues that integrate a major binding site of these substrates, which is critical for transport activity. In silico approaches employed here represent a breakthrough in the plant transportomics field, as the identification of key residues usually takes a long time if performed from a purely wet-lab experimental perspective. The inclusion of structural bioinformatics in the analyses of plant transporters significantly speeds up the knowledge-gaining process and optimizes valuable time and resources.

Unravelling the mechanism of glucose binding in a protein-based fluorescence probe: molecular dynamics simulation with a tailor-made charge model.

Fluorophores linked to the glucose/galactose-binding protein (GGBP) are a promising class of glucose sensors with potential application in medical devices for diabetes patients. Several different fluorophores at different positions in the protein were tested experimentally so far, but a deeper molecular understanding of their function is still missing. In this work, we use molecular dynamics simulations to investigate the mechanism of glucose binding in the GGBP-Badan triple mutant and make a comparison to the GGBP wild-type protein. The aim is to achieve a detailed molecular understanding of changes in the glucose binding site due to the mutations and their effect on glucose binding. Free simulations give an insight into the changes of the hydrogen-bonding network in the active site and into the mechanisms of glucose binding. Additionally, metadynamics simulations for wild type and mutant unravel the energetics of binding/unbinding in these proteins. Computed free energies for the opening of the binding pocket for the wild-type and the mutant agree well with the experimental data. Further, the simulations also give an insight into the changes of the chromophore conformations upon glucose binding, which can help to understand fluorescence changes. Therefore, the molecular details unravelled in this work may support effective optimisation strategies for the construction of more efficient glucose sensors.

Isolation and functional characterization of a glucose-6-phosphate/phosphate translocator (IbG6PPT1) from sweet potato (Ipomoea batatas (L.) Lam.).

Sweet potato (Ipomoea batatas (L.) Lam.) is a good source of carbohydrates, an excellent raw material for starch-based industries, and a strong candidate for biofuel production due to its high starch content. However, the molecular basis of starch biosynthesis and accumulation in sweet potato is still insufficiently understood. Glucose-6-phosphate/phosphate translocators (GPTs) mediate the import of glucose-6-phosphate (Glc6P) into plastids for starch synthesis. Here, we report the isolation of a GPT-encoding gene, IbG6PPT1, from sweet potato and the identification of two additional IbG6PPT1 gene copies in the sweet potato genome. IbG6PPT1 encodes a chloroplast membrane-localized GPT belonging to the GPT1 group and highly expressed in storage root of sweet potato. Heterologous expression of IbG6PPT1 resulted in increased starch content in the leaves, root tips, and seeds and soluble sugar in seeds of Arabidopsis thaliana, but a reduction in soluble sugar in the leaves. These findings suggested that IbG6PPT1 might play a critical role in the distribution of carbon sources in source and sink and the accumulation of carbohydrates in storage tissues and would be a good candidate gene for controlling critical starch properties in sweet potato.

Photophysical Properties of BADAN Revealed in the Study of GGBP Structural Transitions.

The fluorescent dye BADAN (6-bromoacetyl-2-dimetylaminonaphtalene) is widely used in various fields of life sciences, however, the photophysical properties of BADAN are not fully understood. The study of the spectral properties of BADAN attached to a number of mutant forms of GGBP, as well as changes in its spectral characteristics during structural changes in proteins, allowed to shed light on the photophysical properties of BADAN. It was shown that spectral properties of BADAN are determined by at least one non-fluorescent and two fluorescent isomers with overlapping absorbing bands. It was found that BADAN fluorescence is determined by the unsolvated "PICT" (planar intramolecular charge transfer state) and solvated "TICT" (twisted intramolecular charge transfer state) excited states. While "TICT" state can be formed both as a result of the "PICT" state solvation and as a result of light absorption by the solvated ground state of the dye. BADAN fluorescence linked to GGBP/H152C apoform is quenched by Trp 183, but this effect is inhibited by glucose intercalation. New details of the changes in the spectral characteristics of BADAN during the unfolding of the protein apo and holoforms have been obtained.

Impact of Increased Membrane Realism on Conformational Sampling of Proteins.

The realism and accuracy of lipid bilayer simulations through molecular dynamics (MD) are heavily dependent on the lipid composition. While the field is pushing toward implementing more heterogeneous and realistic membrane compositions, a lack of high-resolution lipidomic data prevents some membrane protein systems from being modeled with the highest level of realism. Given the additional diversity of real-world cellular membranes and protein-lipid interactions, it is still not fully understood how altering membrane complexity affects modeled membrane protein functions or if it matters over long-timescale simulations. This is especially true for organisms whose membrane environments have little to no computational study, such as the plant plasma membrane. Tackling these issues in tandem, a generalized, realistic, and asymmetric plant plasma membrane with more than 10 different lipid species is constructed herein. Classical MD simulations of pure membrane constructs were performed to evaluate how altering the compositional complexity of the membrane impacted the plant membrane properties. The apo form of a plant sugar transporter, OsSWEET2b, was inserted into membrane models where lipid diversity was calculated in either a size-dependent or size-independent manner. An adaptive sampling simulation regime validated by Markov-state models was performed to capture the gating dynamics of OsSWEET2b in each of these membrane constructs. In comparison to previous OsSWEET2b simulations performed in a pure POPC bilayer, we confirm that simulations performed within a native-like membrane composition alter the stabilization of apo OsSWEET2b conformational states by ∼1 kcal/mol. The free-energy barriers of intermediate conformational states decrease when realistic membrane complexity is simplified, albeit roughly within sampling error, suggesting that protein-specific responses to membranes differ due to altered packing caused by compositional fluctuations. This work serves as a case study where a more realistic bilayer composition makes unbiased conformational sampling easier to achieve than with simplified bilayers.

Characterization of the multidrug efflux transporter styMdtM from Salmonella enterica serovar Typhi.

Salmonellae are foodborne pathogens and the major cause of gastroenteritis in humans. Salmonellae express multidrug efflux transporters that play a key role in their drug resistance, which is becoming an increasing problem for therapeutic intervention. Despite their biomedical importance, the mechanisms underlying substrate transport by multidrug efflux transporters remain poorly understood. Here, we describe the first characterization of a multidrug transporter belonging to the major facilitator superfamily from the genus Salmonella. We show that several clinical Salmonella Typhi (S. Typhi) isolates constitutively express the styMdtM (STY4874) gene, which encodes a known multidrug-resistance (MDR) transporter. Guided by the structure of the Escherichia coli (E. coli) homolog, we studied two residues critical for substrate transport, Asp25 and Arg111. Mutation of Asp25 to glutamate did not affect the transport function of styMdtM, whereas mutation to alanine reduced its transport activity, suggesting that a negative charge at this position is critical for substrate translocation across the membrane. Substrate-affinity measurements by intrinsic fluorescence spectroscopy showed that the Asp25Ala mutant retained its capacity to bind substrate, albeit at a lower level. Mutation of Arg111 to alanine resulted in a decrease in secondary structure content of the transporter, and mutation to lysine completely destabilized the structure of the transporter. A homology model of styMdtM suggests that Arg111 is important for stabilizing the transmembrane domain by mediating necessary interactions between neighboring helices. Together, our studies provide new structural and mechanistic insights into the Salmonella MDR transporter styMdtM.

Function Trumps Form in Two Sugar Symporters, LacY and vSGLT.

Active transport of sugars into bacteria occurs through symporters driven by ion gradients. LacY is the most well-studied proton sugar symporter, whereas vSGLT is the most characterized sodium sugar symporter. These are members of the major facilitator (MFS) and the amino acid-Polyamine organocation (APS) transporter superfamilies. While there is no structural homology between these transporters, they operate by a similar mechanism. They are nano-machines driven by their respective ion electrochemical potential gradients across the membrane. LacY has 12 transmembrane helices (TMs) organized in two 6-TM bundles, each containing two 3-helix TM repeats. vSGLT has a core structure of 10 TM helices organized in two inverted repeats (TM 1-5 and TM 6-10). In each case, a single sugar is bound in a central cavity and sugar selectivity is determined by hydrogen- and hydrophobic- bonding with side chains in the binding site. In vSGLT, the sodium-binding site is formed through coordination with carbonyl- and hydroxyl-oxygens from neighboring side chains, whereas in LacY the proton (H3O+) site is thought to be a single glutamate residue (Glu325). The remaining challenge for both transporters is to determine how ion electrochemical potential gradients drive uphill sugar transport.

Harnessing Environmental Ca2+ for Extracellular Protein Thermostabilization.

Ca2+ is the third-most prevalent metal ion in the environment. EF hands are common Ca2+-binding motifs found in both extracellular and intracellular proteins of eukaryotes and prokaryotes. Cytoplasmic EF hand proteins often mediate allosteric control of signal transduction pathway components in response to intracellular Ca2+ concentration fluctuations by coupling Ca2+ binding to changes in protein structure. We show that an extracellular structural Ca2+-binding site mediates protein thermostabilization by such conformational coupling as well. Binding Ca2+ to the EF hand of the extracellular (periplasmic) Escherichia coli glucose-galactose binding protein thermostabilizes this protein by ∼17 K relative to its Ca2+-free form. Using statistical thermodynamic analysis of a fluorescent conjugate of ecGGBP that reports simultaneously on ligand binding and multiple conformational states, we found that its Ca2+-mediated stabilization is determined by conformational coupling mechanisms in two independent conformational exchange reactions. Binding to folded and unfolded states determines the maximum Ca2+-mediated stability. A disorder → order transition accompanies the formation of the Ca2+ complex in the folded state and dictates the minimum Ca2+ concentration at which the Ca2+-bound state becomes dominant. Similar transitions also encode the structural changes necessary for Ca2+-mediated control elements in signal transduction pathways. Ca2+-mediated thermostabilization and allosteric control, therefore, share a fundamental conformational coupling mechanism, which may have implications for the evolution of EF hands.

Plant glucose transporter structure and function.

The carbohydrate D-glucose is the main source of energy in living organisms. In contrast to animals, as well as most fungi, bacteria, and archaea, plants are capable to synthesize a surplus of sugars characterizing them as autothrophic organisms. Thus, plants are de facto the source of all food on earth, either directly or indirectly via feed to livestock. Glucose is stored as polymeric glucan, in animals as glycogen and in plants as starch. Despite serving a general source for metabolic energy and energy storage, glucose is the main building block for cellulose synthesis and represents the metabolic starting point of carboxylate- and amino acid synthesis. Finally yet importantly, glucose functions as signalling molecule conveying the plant metabolic status for adjustment of growth, development, and survival. Therefore, cell-to-cell and long-distance transport of photoassimilates/sugars throughout the plant body require the fine-tuned activity of sugar transporters facilitating the transport across membranes. The functional plant counterparts of the animal sodium/glucose transporters (SGLTs) are represented by the proton-coupled sugar transport proteins (STPs) of the plant monosaccharide transporter(-like) family (MST). In the framework of this special issue on "Glucose Transporters in Health and Disease," this review gives an overview of the function and structure of plant STPs in comparison to the respective knowledge obtained with the animal Na+-coupled glucose transporters (SGLTs).

Structural Basis for Blocking Sugar Uptake into the Malaria Parasite Plasmodium falciparum.

Plasmodium species, the causative agent of malaria, rely on glucose for energy supply during blood stage. Inhibition of glucose uptake thus represents a potential strategy for the development of antimalarial drugs. Here, we present the crystal structures of PfHT1, the sole hexose transporter in the genome of Plasmodium species, at resolutions of 2.6 Å in complex with D-glucose and 3.7 Å with a moderately selective inhibitor, C3361. Although both structures exhibit occluded conformations, binding of C3361 induces marked rearrangements that result in an additional pocket. This inhibitor-binding-induced pocket presents an opportunity for the rational design of PfHT1-specific inhibitors. Among our designed C3361 derivatives, several exhibited improved inhibition of PfHT1 and cellular potency against P. falciparum, with excellent selectivity to human GLUT1. These findings serve as a proof of concept for the development of the next-generation antimalarial chemotherapeutics by simultaneously targeting the orthosteric and allosteric sites of PfHT1.

Monoclonal antibody 4B1 influences the pKa of Glu325 in lactose permease (LacY) from Escherichia coli: evidence from SEIRAS.

The monoclonal antibody 4B1 binds to a conformational epitope on the periplasmic side of lactose permease (LacY) of Escherichia coli and inhibits H+ /lactose symport and lactose efflux under nonenergized conditions. At the same time, ligand binding and translocation reactions that do not involve net H+ translocation remain unaffected by 4B1. In this study, surface-enhanced infrared absorption spectroscopy applied to the immobilized LacY was used to study the pH-dependent changes in LacY and to access in situ the effect of the 4B1 antibody on the pKa of Glu325, the primary functional H+ -binding site in LacY. A small shift of the pK value from 10.5 to 9.5 was identified that can be corroborated with the inactivation of LacY upon 4B1 binding.

Identification and Expression Analysis of the SWEET Gene Family from Poa pratensis Under Abiotic Stresses.

The sugars will eventually be exported transporters (SWEET) gene family is a glycoprotein gene family that can regulate the transport of sugar in plants and plays an important role in plant growth and development, as well as in response to environmental stress. In this study, Kentucky bluegrass (cv. Baron) seedlings were grown in various treatments, including heavy metal cadmium, salt, drought, cold, and heat stress for 6 h, 24 h, 48 h, and 7 day. The relative expression of the identified PpSWEET genes in Kentucky bluegrass was measured. The results showed there were a total of 13 SWEET genes, which could be divided into four clades by phylogenetic analysis. Most PpSWEET genes are alkali proteins with seven transmembrane helices. Moreover, almost all PpSWEET proteins possess similar conserved motifs and active sites. In addition, an analysis of the relative expression of PpSWEET genes under various stress treatments indicated that PpSWEET12 and PpSWEET15 had very high expression under the five types of stress, meaning they can be used as important candidate genes for studying responses to environmental stresses of turfgrass. Furthermore, certain genes only showed changes in expression under one or two specific stress treatments. This study provides important insight into the SWEET gene family in Kentucky bluegrass and its functional roles in responses to various environmental stresses.

Cell-free expression tools to study co-translational folding of alpha helical membrane transporters.

Most helical membrane proteins fold co-translationally during unidirectional polypeptide elongation by the ribosome. Studies thus far, however, have largely focussed on refolding full-length proteins from artificially induced denatured states that are far removed from the natural co-translational process. Cell-free translation offers opportunities to remedy this deficit in folding studies and has previously been used for membrane proteins. We exploit this cell-free approach to develop tools to probe co-translational folding. We show that two transporters from the ubiquitous Major Facilitator Superfamily can successfully insert into a synthetic bilayer without the need for translocon insertase apparatus that is essential in vivo. We also assess the cooperativity of domain insertion, by expressing the individual transporter domains cell-free. Furthermore, we manipulate the cell-free reaction to pause and re-start protein synthesis at specific points in the protein sequence. We find that full-length protein can still be made when stalling after the first N terminal helix has inserted into the bilayer. However, stalling after the first three helices have exited the ribosome cannot be successfully recovered. These three helices cannot insert stably when ribosome-bound during co-translational folding, as they require insertion of downstream helices.

Transporters of glucose and other carbohydrates in bacteria.

Glucose arguably is the most important energy carrier, carbon source for metabolites and building block for biopolymers in all kingdoms of life. The proper function of animal organs and tissues depends on the continuous supply of glucose from the bloodstream. Most animals can resorb only a small number of monosaccharides, mostly glucose, galactose and fructose, while all other sugars oligosaccharides and dietary fibers are degraded and metabolized by the microbiota of the lower intestine. Bacteria, in contrast, are omnivorous. They can import and metabolize structurally different sugars and, as a consortium of different species, utilize almost any sugar, sugar derivative and oligosaccharide occurring in nature. Bacteria have membrane transport systems for the uptake of sugars against steep concentration gradients energized by ATP, the proton motive force and the high energy glycolytic intermediate phosphoenolpyruvate (PEP). Different uptake mechanisms and the broad range of overlapping substrate specificities allow bacteria to quickly adapt to and colonize changing environments. Here, we review the structures and mechanisms of bacterial representatives of (i) ATP-dependent cassette (ABC) transporters, (ii) major facilitator (MFS) superfamily proton symporters, (iii) sodium solute symporters (SSS) and (iv) enzyme II integral membrane subunits of the bacterial PEP-dependent phosphotransferase system (PTS). We give a short overview on the distribution of transporter genes and their phylogenetic relationship in different bacterial species. Some sugar transporters are hijacked for import of bacteriophage DNA and antibacterial toxins (bacteriocins) and they facilitate the penetration of polar antibiotics. Finally, we describe how the expression and activity of certain sugar transporters are controlled in response to the availability of sugars and how the presence and uptake of sugars may affect pathogenicity and host-microbiota interactions.

Diversity in kinetics correlated with structure in nano body-stabilized LacY.

The structure of lactose permease, stabilized in a periplasmic open conformation by two Gly to Trp replacements (LacYww) and complexed with a nanobody directed against this conformation, provides the highest resolution structure of the symporter. The nanobody binds in a different manner than two other nanobodies made against the same mutant, which also bind to the same general region on the periplasmic side. This region of the protein may represent an immune hotspot. The CDR3 loop of the nanobody is held by hydrogen bonds in a conformation that partially blocks access to the substrate-binding site. As a result, kon and koff for galactoside binding to either LacY or the double mutant complexed with the nanobody are lower than for the other two LacY/nanobody complexes though the Kd values are similar, reflecting the fact that the nanobodies rigidify structures along the pathway. While the wild-type LacY/nanobody complex clearly stabilizes a similar 'extracellular open' conformation in solution, judged by binding kinetics, the complex with wild-type LacY did not yet crystallize, suggesting the nanobody does not bind strongly enough to shift the equilibrium to stabilize a periplasmic side-open conformation suitable for crystallization. However, the similarity of the galactoside binding kinetics for the nanobody-bound complexes with wild type LacY and with LacYWW indicates that they have similar structures, showing that the reported co-structures reliably show nanobody interactions with LacY.

Structural and Functional Adaptability of Sucrose and Lactose Permeases from Escherichia coli to the Membrane Lipid Composition.

The lipid environment in which membrane proteins are embedded can influence their structure and function. Lipid-protein interactions and lipid-induced conformational changes necessary for protein function remain intractable in vivo using high-resolution techniques. Using Escherichia coli strains in which the normal phospholipid composition can be altered or foreign lipids can be introduced, we established the importance of membrane lipid composition for the proper folding, assembly, and function of E. coli lactose (LacY) and sucrose (CscB) permeases. However, the molecular mechanism underlying the lipid dependence for active transport remains unknown. Herein, we demonstrate that the structure and function of CscB and LacY can be modulated by the composition of the lipid environment. Using a combination of assays (transport activity of the substrate, protein topology, folding, and assembly into the membrane), we found that alterations in the membrane lipid composition lead to lipid-dependent structural changes in CscB and LacY. These changes affect the orientation of residues involved in LacY proton translocation and impact the rates of protonation and deprotonation of E325 by affecting the arrangement of transmembrane domains in the vicinity of the R302-E325 charge pair. Furthermore, the structural changes caused by changes in membrane lipid composition can be altered by a single-point mutation, highlighting the adaptability of these transporters to their environment. Altogether, our results demonstrate that direct interactions between a protein and its lipid environment uniquely contribute to membrane protein organization and function. Because members of the major facilitator superfamily present with well-conserved functional architecture, we anticipate that our findings can be extrapolated to other membrane protein transporters.