Structure and mechanism of a phosphotransferase system glucose transporter.

Glucose is the primary source of energy for many organisms and is efficiently taken up by bacteria through a dedicated transport system that exhibits high specificity. In Escherichia coli, the glucose-specific transporter IICBGlc serves as the major glucose transporter and functions as a component of the phosphoenolpyruvate-dependent phosphotransferase system. Here, we report cryo-electron microscopy (cryo-EM) structures of the glucose-bound IICBGlc protein. The dimeric transporter embedded in lipid nanodiscs was captured in the occluded, inward- and occluded, outward-facing conformations. Together with biochemical and biophysical analyses, and molecular dynamics (MD) simulations, we provide insights into the molecular basis and dynamics for substrate recognition and binding, including the gates regulating the binding sites and their accessibility. By combination of these findings, we present a mechanism for glucose transport across the plasma membrane. Overall, this work provides molecular insights into the structure, dynamics, and mechanism of the IICBGlc transporter in a native-like lipid environment.

Structural insights into the mechanism and dynamics of proteorhodopsin biogenesis and retinal scavenging.

Microbial ion-pumping rhodopsins (MRs) are extensively studied retinal-binding membrane proteins. However, their biogenesis, including oligomerisation and retinal incorporation, remains poorly understood. The bacterial green-light absorbing proton pump proteorhodopsin (GPR) has emerged as a model protein for MRs and is used here to address these open questions using cryo-electron microscopy (cryo-EM) and molecular dynamics (MD) simulations. Specifically, conflicting studies regarding GPR stoichiometry reported pentamer and hexamer mixtures without providing possible assembly mechanisms. We report the pentameric and hexameric cryo-EM structures of a GPR mutant, uncovering the role of the unprocessed N-terminal signal peptide in the assembly of hexameric GPR. Furthermore, certain proteorhodopsin-expressing bacteria lack retinal biosynthesis pathways, suggesting that they scavenge the cofactor from their environment. We shed light on this hypothesis by solving the cryo-EM structure of retinal-free proteoopsin, which together with mass spectrometry and MD simulations suggests that decanoate serves as a temporary placeholder for retinal in the chromophore binding pocket. Further MD simulations elucidate possible pathways for the exchange of decanoate and retinal, offering a mechanism for retinal scavenging. Collectively, our findings provide insights into the biogenesis of MRs, including their oligomeric assembly, variations in protomer stoichiometry and retinal incorporation through a potential cofactor scavenging mechanism.

Light Color-Controlled pH-Adjustment of Aqueous Solutions Using Engineered Proteoliposomes.

Controlling the pH at the microliter scale can be useful for applications in research, medicine, and industry, and therefore represents a valuable application for synthetic biology and microfluidics. The presented vesicular system translates light of different colors into specific pH changes in the surrounding solution. It works with the two light-driven proton pumps bacteriorhodopsin and blue light-absorbing proteorhodopsin Med12, that are oriented in opposite directions in the lipid membrane. A computer-controlled measuring device implements a feedback loop for automatic adjustment and maintenance of a selected pH value. A pH range spanning more than two units can be established, providing fine temporal and pH resolution. As an application example, a pH-sensitive enzyme reaction is presented where the light color controls the reaction progress. In summary, light color-controlled pH-adjustment using engineered proteoliposomes opens new possibilities to control processes at the microliter scale in different contexts, such as in synthetic biology applications.

Structural and biochemical insights into His-tag-induced higher-order oligomerization of membrane proteins by cryo-EM and size exclusion chromatography.

Structural and functional characterization of proteins as well as the design of targeted drugs heavily rely on recombinant protein expression and purification. The polyhistidine-tag (His-tag) is among the most prominent examples of affinity tags used for the isolation of recombinant proteins from their expression hosts. Short peptide tags are commonly considered not to interfere with the structure of the tagged protein and tag removal is frequently neglected. This study demonstrates the formation of higher-order oligomers based on the example of two His-tagged membrane proteins, the dimeric arginine-agmatine antiporter AdiC and the pentameric light-driven proton pump proteorhodopsin. Size exclusion chromatography revealed the formation of tetrameric AdiC and decameric as well as pentadecameric proteorhodopsin through specific interactions between their His-tags. In addition, single particle cryo-electron microscopy (cryo-EM) allowed structural insights into the three-dimensional arrangement of the higher-order oligomers and the underlying His-tag-mediated interactions. These results reinforce the importance of considering the length and removal of affinity purification tags and illustrate how neglect can lead to potential interference with downstream biophysical or biochemical characterization of the target protein.

Peptide transporter structure reveals binding and action mechanism of a potent PEPT1 and PEPT2 inhibitor.

Inhibitors for membrane transporters have been shown to be indispensable as drugs and tool compounds. The proton-dependent oligopeptide transporters PEPT1 and PEPT2 from the SLC15 family play important roles in human and mammalian physiology. With Lys[Z(NO2)]-Val (LZNV), a modified Lys-Val dipeptide, a potent transport inhibitor for PEPT1 and PEPT2 is available. Here we present the crystal structure of the peptide transporter YePEPT in complex with LZNV. The structure revealed the molecular interactions for inhibitor binding and a previously undescribed mostly hydrophobic pocket, the PZ pocket, involved in interaction with LZNV. Comparison with a here determined ligand-free structure of the transporter unveiled that the initially absent PZ pocket emerges through conformational changes upon inhibitor binding. The provided biochemical and structural information constitutes an important framework for the mechanistic understanding of inhibitor binding and action in proton-dependent oligopeptide transporters.

Engineering and functional characterization of a proton-driven ß-lactam antibiotic translocation module for bionanotechnological applications.

Novel approaches in synthetic biology focus on the bottom-up modular assembly of natural, modified natural or artificial components into molecular systems with functionalities not found in nature. A possible application for such techniques is the bioremediation of natural water sources contaminated with small organic molecules (e.g., drugs and pesticides). A simple molecular system to actively accumulate and degrade pollutants could be a bionanoreactor composed of a liposome or polymersome scaffold combined with energizing- (e.g., light-driven proton pump), transporting- (e.g., proton-driven transporter) and degrading modules (e.g., enzyme). This work focuses on the engineering of a transport module specific for ß-lactam antibiotics. We previously solved the crystal structure of a bacterial peptide transporter, which allowed us to improve the affinity for certain ß-lactam antibiotics using structure-based mutagenesis combined with a bacterial uptake assay. We were able to identify specific mutations, which enhanced the affinity of the transporter for antibiotics containing certain structural features. Screening of potential compounds allowed for the identification of a ß-lactam antibiotic ligand with relatively high affinity. Transport of antibiotics was evaluated using a solid-supported membrane electrophysiology assay. In summary, we have engineered a proton-driven ß-lactam antibiotic translocation module, contributing to the growing toolset for bionanotechnological applications.

High-resolution structure of the amino acid transporter AdiC reveals insights into the role of water molecules and networks in oligomerization and substrate binding.

BACKGROUND: The L-arginine/agmatine transporter AdiC is part of the arginine-dependent extreme acid resistance system of the bacterium Escherichia coli and its pathogenic varieties such as strain E. coli O157:H7. At the present time, there is a lack of knowledge concerning the role of water molecules and networks for the structure and function of AdiC, and solute transporters in general. RESULTS: The structure of the L-arginine/agmatine transporter AdiC was determined at 1.7 Å resolution by X-ray crystallography. This high resolution allowed for the identification of numerous water molecules buried in the structure. In combination with molecular dynamics (MD) simulations, we demonstrate that water molecules play an important role for stabilizing the protein and key residues, and act as placeholders for atoms of the AdiC substrates L-arginine and agmatine. MD simulations unveiled flexibility and restrained mobility of gating residues W202 and W293, respectively. Furthermore, a water-filled cavity was identified at the dimer interface of AdiC. The two monomers formed bridging interactions through water-mediated hydrogen bonds. The accessibility and presence of water molecules in this cavity was confirmed with MD simulations. Point mutations disrupting the interfacial water network validated the importance of water molecules for dimer stabilization. CONCLUSIONS: This work gives new insights into the role and importance of water molecules in the L-arginine/agmatine transporter AdiC for protein stabilization and substrate-binding site shaping and as placeholders of substrate atoms. Furthermore, and based on the observed flexibility and restrained mobility of gating residues, a mechanistic role of the gate flexibility in the transport cycle was proposed. Finally, we identified a water-filled cavity at the dimeric interface that contributes to the stability of the amino acid transporter oligomer.

Cryo-EM structure and dynamics of the green-light absorbing proteorhodopsin.

The green-light absorbing proteorhodopsin (GPR) is the archetype of bacterial light-driven proton pumps. Here, we present the 2.9 Å cryo-EM structure of pentameric GPR, resolving important residues of the proton translocation pathway and the oligomerization interface. Superposition with the structure of a close GPR homolog and molecular dynamics simulations reveal conformational variations, which regulate the solvent access to the intra- and extracellular half channels harbouring the primary proton donor E109 and the proposed proton release group E143. We provide a mechanism for the structural rearrangements allowing hydration of the intracellular half channel, which are triggered by changing the protonation state of E109. Functional characterization of selected mutants demonstrates the importance of the molecular organization around E109 and E143 for GPR activity. Furthermore, we present evidence that helices involved in the stabilization of the protomer interfaces serve as scaffolds for facilitating the motion of the other helices. Combined with the more constrained dynamics of the pentamer compared to the monomer, these observations illustrate the previously demonstrated functional significance of GPR oligomerization. Overall, this work provides molecular insights into the structure, dynamics and function of the proteorhodopsin family that will benefit the large scientific community employing GPR as a model protein.

The Heavy Chain 4F2hc Modulates the Substrate Affinity and Specificity of the Light Chains LAT1 and LAT2.

The human L-type amino acid transporters LAT1 and LAT2 mediate the transport of amino acids and amino acid derivatives across plasma membranes in a sodium-independent, obligatory antiport mode. In mammalian cells, LAT1 and LAT2 associate with the type-II membrane N-glycoprotein 4F2hc to form heteromeric amino acid transporters (HATs). The glycosylated ancillary protein 4F2hc is known to be important for successful trafficking of the unglycosylated transporters to the plasma membrane. The heavy (i.e., 4F2hc) and light (i.e., LAT1 and LAT2) chains belong to the solute carrier (SLC) families SLC3 and SLC7, and are covalently linked by a conserved disulfide bridge. Overexpression, absence, or malfunction of certain HATs is associated with human diseases and HATs are therefore considered therapeutic targets. Here, we present a comparative, functional characterization of the HATs 4F2hc-LAT1 and 4F2hc-LAT2, and their light chains LAT1 and LAT2. For this purpose, the HATs and the light chains were expressed in the methylotrophic yeast Pichia pastoris and a radiolabel transport assay was established. Importantly and in contrast to mammalian cells, P. pastoris has proven useful as eukaryotic expression system to successfully express human LAT1 and LAT2 in the plasma membrane without the requirement of co-expressed trafficking chaperone 4F2hc. Our results show a novel function of the heavy chain 4F2hc that impacts transport by modulating the substrate affinity and specificity of corresponding LATs. In addition, the presented data confirm that the light chains LAT1 and LAT2 constitute the substrate-transporting subunits of the HATs, and that light chains are also functional in the absence of the ancillary protein 4F2hc.

Engineering and Production of the Light-Driven Proton Pump Bacteriorhodopsin in 2D Crystals for Basic Research and Applied Technologies.

The light-driven proton pump bacteriorhodopsin (BR) from the extreme halophilic archaeon Halobacterium salinarum is a retinal-binding protein, which forms highly ordered and thermally stable 2D crystals in native membranes (termed purple membranes). BR and purple membranes (PMs) have been and are still being intensively studied by numerous researchers from different scientific disciplines. Furthermore, PMs are being successfully used in new, emerging technologies such as bioelectronics and bionanotechnology. Most published studies used the wild-type form of BR, because of the intrinsic difficulty to produce genetically modified versions in purple membranes homologously. However, modification and engineering is crucial for studies in basic research and, in particular, to tailor BR for specific applications in applied sciences. We present an extensive and detailed protocol ranging from the genetic modification and cultivation of H. salinarum to the isolation, and biochemical, biophysical and functional characterization of BR and purple membranes. Pitfalls and problems of the homologous expression of BR versions in H. salinarum are discussed and possible solutions presented. The protocol is intended to facilitate the access to genetically modified BR versions for researchers of different scientific disciplines, thus increasing the application of this versatile biomaterial.

Cryo-electron microscopic and X-ray crystallographic analysis of the light-driven proton pump proteorhodopsin reveals a pentameric assembly.

The green-light absorbing proteorhodopsin (GPR) is the prototype of bacterial light-driven proton pumps. It has been the focus of continuous research since its discovery 20 years ago and has sparked the development and application of various biophysical techniques. However, a certain controversy and ambiguity about the oligomeric assembly of GPR still remains. We present here the first tag-free purification of pentameric GPR. The combination of ion exchange and size exclusion chromatography yields homogeneous and highly pure untagged pentamers from GPR overexpressing Escherichia coli. The presented purification procedure provides native-like protein and excludes the need for affinity purification tags. Importantly, three-dimensional protein crystals of GPR were successfully grown and analyzed by X-ray crystallography. These results together with data from single particle cryo-electron microscopy provide direct evidence for the pentameric stoichiometry of purified GPR.

Effects of Mutations and Ligands on the Thermostability of the l-Arginine/Agmatine Antiporter AdiC and Deduced Insights into Ligand-Binding of Human l-Type Amino Acid Transporters.

The l-arginine/agmatine transporter AdiC is a prokaryotic member of the SLC7 family, which enables pathogenic enterobacteria to survive the extremely acidic gastric environment. Wild-type AdiC from Escherichia coli, as well as its previously reported point mutants N22A and S26A, were overexpressed homologously and purified to homogeneity. A size-exclusion chromatography-based thermostability assay was used to determine the melting temperatures (Tms) of the purified AdiC variants in the absence and presence of the selected ligands l-arginine (Arg), agmatine, l-arginine methyl ester, and l-arginine amide. The resulting Tms indicated stabilization of AdiC variants upon ligand binding, in which Tms and ligand binding affinities correlated positively. Considering results from this and previous studies, we revisited the role of AdiC residue S26 in Arg binding and proposed interactions of the α-carboxylate group of Arg exclusively with amide groups of the AdiC backbone. In the context of substrate binding in the human SLC7 family member l-type amino acid transporter-1 (LAT1; SLC7A5), an analogous role of S66 in LAT1 to S26 in AdiC is discussed based on homology modeling and amino acid sequence analysis. Finally, we propose a binding mechanism for l-amino acid substrates to LATs from the SLC7 family.

High-Resolution Imaging and Multiparametric Characterization of Native Membranes by Combining Confocal Microscopy and an Atomic Force Microscopy-Based Toolbox.

To understand how membrane proteins function requires characterizing their structure, assembly, and inter- and intramolecular interactions in physiologically relevant conditions. Conventionally, such multiparametric insight is revealed by applying different biophysical methods. Here we introduce the combination of confocal microscopy, force-distance curve-based (FD-based) atomic force microscopy (AFM), and single-molecule force spectroscopy (SMFS) for the identification of native membranes and the subsequent multiparametric analysis of their membrane proteins. As a well-studied model system, we use native purple membrane from Halobacterium salinarum, whose membrane protein bacteriorhodopsin was His-tagged to bind nitrilotriacetate (NTA) ligands. First, by confocal microscopy we localize the extracellular and cytoplasmic surfaces of purple membrane. Then, we apply AFM to image single bacteriorhodopsins approaching sub-nanometer resolution. Afterwards, the binding of NTA ligands to bacteriorhodopsins is localized and quantified by FD-based AFM. Finally, we apply AFM-based SMFS to characterize the (un)folding of the membrane protein and to structurally map inter- and intramolecular interactions. The multimethodological approach is generally applicable to characterize biological membranes and membrane proteins at physiologically relevant conditions.

Electron crystallography reveals that substrate release from the PTS IIC glucose transporter is coupled to a subtle conformational change.

The phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) is a structurally and functionally complex system that mediates sugar uptake in bacteria. Besides several soluble subunits, the glucose-specific PTS includes the integral membrane protein IICB that couples the transmembrane transport of glucose to its phosphorylation. Here, we used electron crystallography of sugar-embedded tubular crystals of the glucose-specific IIC transport domain from Escherichia coli (ecIICglc) to visualize the structure of the transporter in the presence and absence of its substrate. Using an in vivo transport assay and binding competition experiments, we first established that, while it transports d-glucose, ecIICglc does not bind l-glucose. We then determined the projection structure of ecIICglc from tubular crystals embedded in d- and l-glucose and found a subtle conformational change. From comparison of the ecIICglc projection maps with crystal structures of other IIC transporters, we can deduce that the transporter adopts an inward-facing conformation, and that the maps in the presence and absence of the substrate reflect the transporter before and after release of the transported glucose into the cytoplasm. The transition associated with substrate release appears to require a subtle structural rearrangement in the region that includes hairpin 1.

Detecting Ligand-Binding Events and Free Energy Landscape while Imaging Membrane Receptors at Subnanometer Resolution.

Force-distance curve-based atomic force microscopy has emerged into a sophisticated technique for imaging cellular membranes and for detecting specific ligand-binding events of native membrane receptors. However, so far the resolution achieved has been insufficient to structurally map ligand-binding sites onto membrane proteins. Here, we introduce experimental and theoretical approaches for overcoming this limitation. To establish a structurally and functionally well-defined reference sample, we engineer a ligand-binding site to the light-driven proton pump bacteriorhodopsin of purple membrane. Functionalizing the AFM stylus with an appropriate linker-system tethering the ligand and optimizing the AFM conditions allows for imaging the engineered bacteriorhodopsin at subnanometer resolution while structurally mapping the specific ligand-receptor binding events. Improved data analysis allows reconstructing the ligand-binding free energy landscape from the experimental data, thus providing thermodynamic and kinetic insight into the ligand-binding process. The nanoscopic method introduced is generally applicable for imaging receptors in native membranes at subnanometer resolution and for systematically mapping and quantifying the free energy landscape of ligand binding.

Insights into the molecular basis for substrate binding and specificity of the wild-type L-arginine/agmatine antiporter AdiC.

Pathogenic enterobacteria need to survive the extreme acidity of the stomach to successfully colonize the human gut. Enteric bacteria circumvent the gastric acid barrier by activating extreme acid-resistance responses, such as the arginine-dependent acid resistance system. In this response, l-arginine is decarboxylated to agmatine, thereby consuming one proton from the cytoplasm. In Escherichia coli, the l-arginine/agmatine antiporter AdiC facilitates the export of agmatine in exchange of l-arginine, thus providing substrates for further removal of protons from the cytoplasm and balancing the intracellular pH. We have solved the crystal structures of wild-type AdiC in the presence and absence of the substrate agmatine at 2.6-Å and 2.2-Å resolution, respectively. The high-resolution structures made possible the identification of crucial water molecules in the substrate-binding sites, unveiling their functional roles for agmatine release and structure stabilization, which was further corroborated by molecular dynamics simulations. Structural analysis combined with site-directed mutagenesis and the scintillation proximity radioligand binding assay improved our understanding of substrate binding and specificity of the wild-type l-arginine/agmatine antiporter AdiC. Finally, we present a potential mechanism for conformational changes of the AdiC transport cycle involved in the release of agmatine into the periplasmic space of E. coli.

Purification of Human and Mammalian Membrane Proteins Expressed in Xenopus laevis Frog Oocytes for Structural Studies.

This protocol describes the isolation of recombinant human and mammalian membrane proteins expressed in Xenopus laevis frog oocytes for structural studies. The cDNA-derived cRNA of the desired genes is injected into several hundreds of oocytes, which are incubated for several days to allow protein expression. Recombinant proteins are then purified via affinity chromatography. The novelty of this method comes from the design of a plasmid that produces multi-tagged proteins and, most importantly, the development of a protocol for efficiently discarding lipids, phospholipids, and lipoproteins from the oocyte egg yolk, which represent the major contaminants in protein purifications. Thus, the high protein purity and good yield obtained from this method allows protein structure determination by transmission electron microscopy of single detergent-solubilized protein particles and of 2D crystals of membrane protein embedded in lipid bilayers. Additionally, a radiotracer assay for functional analysis of the expressed target proteins in oocytes is described. Overall, this method is a valuable option for structural studies of mammalian and particularly human proteins, for which other expression systems often fail.

Engineering a Chemical Switch into the Light-driven Proton Pump Proteorhodopsin by Cysteine Mutagenesis and Thiol Modification.

For applications in synthetic biology, for example, the bottom-up assembly of biomolecular nanofactories, modules of specific and controllable functionalities are essential. Of fundamental importance in such systems are energizing modules, which are able to establish an electrochemical gradient across a vesicular membrane as an energy source for powering other modules. Light-driven proton pumps like proteorhodopsin (PR) are excellent candidates for efficient energy conversion. We have extended the versatility of PR by implementing an on/off switch based on reversible chemical modification of a site-specifically introduced cysteine residue. The position of this cysteine residue in PR was identified by structure-based cysteine mutagenesis combined with a proton-pumping assay using E. coli cells overexpressing PR and PR proteoliposomes. The identified PR mutant represents the first light-driven proton pump that can be chemically switched on/off depending on the requirements of the molecular system.

Role of electrostatic interactions for ligand recognition and specificity of peptide transporters.

BACKGROUND: Peptide transporters are membrane proteins that mediate the cellular uptake of di- and tripeptides, and of peptidomimetic drugs such as ß-lactam antibiotics, antiviral drugs and antineoplastic agents. In spite of their high physiological and pharmaceutical importance, the molecular recognition by these transporters of the amino acid side chains of short peptides and thus the mechanisms for substrate binding and specificity are far from being understood. RESULTS: The X-ray crystal structure of the peptide transporter YePEPT from the bacterium Yersinia enterocolitica together with functional studies have unveiled the molecular bases for recognition, binding and specificity of dipeptides with a charged amino acid residue at the N-terminal position. In wild-type YePEPT, the significant specificity for the dipeptides Asp-Ala and Glu-Ala is defined by electrostatic interaction between the in the structure identified positively charged Lys314 and the negatively charged amino acid side chain of these dipeptides. Mutagenesis of Lys314 into the negatively charged residue Glu allowed tuning of the substrate specificity of YePEPT for the positively charged dipeptide Lys-Ala. Importantly, molecular insights acquired from the prokaryotic peptide transporter YePEPT combined with mutagenesis and functional uptake studies with human PEPT1 expressed in Xenopus oocytes also allowed tuning of human PEPT1's substrate specificity, thus improving our understanding of substrate recognition and specificity of this physiologically and pharmaceutically important peptide transporter. CONCLUSION: This study provides the molecular bases for recognition, binding and specificity of peptide transporters for dipeptides with a charged amino acid residue at the N-terminal position.

2D and 3D crystallization of the wild-type IIC domain of the glucose PTS transporter from Escherichia coli.

The bacterial phosphoenolpyruvate: sugar phosphotransferase system serves the combined uptake and phosphorylation of carbohydrates. This structurally and functionally complex system is composed of several conserved functional units that, through a cascade of phosphorylated intermediates, catalyze the transfer of the phosphate moiety from phosphoenolpyruvate to the substrate, which is bound to the integral membrane domain IIC. The wild-type glucose-specific IIC domain (wt-IIC(glc)) of Escherichia coli was cloned, overexpressed and purified for biochemical and functional characterization. Size-exclusion chromatography and scintillation-proximity binding assays showed that purified wt-IIC(glc) was homogenous and able to bind glucose. Crystallization was pursued following two different approaches: (i) reconstitution of wt-IIC(glc) into a lipid bilayer by detergent removal through dialysis, which yielded tubular 2D crystals, and (ii) vapor-diffusion crystallization of detergent-solubilized wt-IIC(glc), which yielded rhombohedral 3D crystals. Analysis of the 2D crystals by cryo-electron microscopy and the 3D crystals by X-ray diffraction indicated resolutions of better than 6Å and 4Å, respectively. Furthermore, a complete X-ray diffraction data set could be collected and processed to 3.93Å resolution. These 2D and 3D crystals of wt-IIC(glc) lay the foundation for the determination of the first structure of a bacterial glucose-specific IIC domain.