Betaine Transporter BetP Research Articles

Structural and Functional Basis for Lipid Synergy on the Activity of the Antibacterial Peptide ABC Transporter McjD

The lipid bilayer is a dynamic environment that consists of a mixture of lipids with different properties that regulate the function of membrane proteins; these lipids are either annular, masking the protein hydrophobic surface, or specific lipids, essential for protein function. In this study, using tandem mass spectrometry, we have identified specific lipids associated with the Escherichia coli ABC transporter McjD, which translocates the antibacterial peptide MccJ25. Using non-denaturing mass spectrometry, we show that McjD in complex with MccJ25 survives the gas phase. Partial delipidation of McjD resulted in reduced ATPase activity and thermostability as shown by circular dichroism, both of which could be restored upon addition of defined E. coli lipids. We have resolved a phosphatidylglycerol lipid associated with McjD at 3.4 Å resolution, whereas molecular dynamic simulations carried out in different lipid environments assessed the binding of specific lipids to McjD. Combined, our data show a synergistic effect of zwitterionic and negatively charged lipids on the activity of McjD; the zwitterionic lipids provide structural stability to McjD, whereas the negatively charged lipids are essential for its function.

Carr-Purcell Pulsed Electron Double Resonance with Shaped Inversion Pulses.

Pulsed electron paramagnetic resonance (EPR) spectroscopy allows the determination of distances, in the range of 1.5-8 nm, between two spin-labels attached to macromolecules containing protons. Unfortunately, for hydrophobic lipid-bound or detergent-solubilized membrane proteins, the maximum distance accessible is much lower, because of a strongly reduced coherence time of the electron spins. Here we introduce a pulse sequence, based on a Carr-Purcell decoupling scheme on the observer spin, where each π-pulse is accompanied by a shaped sech/tanh inversion pulse applied to the second spin, to overcome this limitation. This pump/probe excitation scheme efficiently recouples the dipolar interaction, allowing a substantially longer observation time window to be achieved. This increases the upper limit and accuracy of distances that can be determined in membrane protein complexes. We validated the method on a bis-nitroxide model compound and applied this technique to the trimeric betaine transporter BetP. Interprotomer distances as long as 6 nm could be reliably determined, which is impossible with the existing methods.

Substrate-bound outward-open state of the betaine transporter BetP provides insights into Na+ coupling.

The Na(+)-coupled betaine symporter BetP shares a highly conserved fold with other sequence unrelated secondary transporters, for example, with neurotransmitter symporters. Recently, we obtained atomic structures of BetP in distinct conformational states, which elucidated parts of its alternating-access mechanism. Here, we report a structure of BetP in a new outward-open state in complex with an anomalous scattering substrate, adding a fundamental piece to an unprecedented set of structural snapshots for a secondary transporter. In combination with molecular dynamics simulations these structural data highlight important features of the sequential formation of the substrate and sodium-binding sites, in which coordinating water molecules play a crucial role. We observe a strictly interdependent binding of betaine and sodium ions during the coupling process. All three sites undergo progressive reshaping and dehydration during the alternating-access cycle, with the most optimal coordination of all substrates found in the closed state.

Lock and Load: Key Role of the Unique Closed State in Transport Regulation of the Sodium-Coupled Betaine Transporter BetP

The Na+-coupled betaine symporter BetP is regulated by osmotic stress sensing the increasing internal K+ concentration as a direct consequence of hypertonicity by its positively charged C-terminal domain. Recently, we demonstrated that BetP binds K+ cooperatively to its cytoplasmic side between different C-terminal domains within the BetP trimer, while the monomer is not regulated. K+ binding strengthens the helical fold of the C-terminal domain, and together with negatively charged lipids at the trimer center orients it towards the counterclockwise adjacent protomer. The extended C-terminal conformation is assisted by an additional interaction with the N-terminal domain and most crucially with lipids. A recent crystal structure show BetP in a closed state with the C-terminal domain bending back towards its own protomer to interact mainly with lipid head groups at the membrane surface. Thereby, an intratrimeric interaction is inhibited, which allows us to assign the bend-back conformation of the C-terminal domain as the inactive one and the extended conformation as active. A surprising consequence of the inactive C-terminal conformation is the uncoupling of the two sodium sites. Although having all substrates bound BetP cannot any longer isomerize to the inward-facing state because the C-terminal domain together with lipids lock the intracellular gate preventing the opening of the S1 site. Subsequently, all BetP protomers within a trimer remain fully substrate loaded in a closed state with additional betaine molecules sequestered and stored in the second periplasmic (S2) binding site. We propose a trimeric switch model, in which osmotic stress induces a stepwise activation of one protomer after the other by consecutive K+ binding and straightening of the C-terminal helix.

The SLC6 transporters: perspectives on structure, functions, regulation, and models for transporter dysfunction

The human SLC6 family is composed of approximately 20 structurally related symporters (co-transporters) that use the transmembrane electrochemical gradient to actively import their substrates into cells. Approximately half of the substrates of these transporters are amino acids, with others transporting biogenic amines and/or closely related compounds, such as nutrients and compatible osmolytes. In this short review, five leaders in the field discuss a number of currently important research themes that involve SLC6 transporters, highlighting the integrative role they play across a wide spectrum of different functions. The first essay, by Gary Rudnick, describes the molecular mechanism of their coupled transport which is being progressively better understood based on new crystal structures, functional studies, and modeling. Next, the question of multiple levels of transporter regulation is discussed by Reinhard Krämer, in the context of osmoregulation and stress response by the related bacterial betaine transporter BetP. The role of selected members of the human SLC6 family that function as nutrient amino acid transporters is then reviewed by François Verrey. He discusses how some of these transporters mediate the active uptake of (essential) amino acids into epithelial cells of the gut and the kidney tubule to support systemic amino acid requirements, whereas others are expressed in specific cells to support their specialized metabolism and/or growth. The most extensively studied members of the human SLC6 family are neurotransmitter reuptake transporters, many of which are important drug targets for the treatment of neuropsychiatric disorders. Randy Blakely discusses the role of posttranscriptional modifications of these proteins in regulating transporter subcellular localization and activity state. Finally, Dennis Murphy reviews how natural gene variants and mouse genetic models display consistent behavioral alterations that relate to altered extracellular neurotransmitter levels.

Structural evidence for functional lipid interactions in the betaine transporter BetP

Bilayer lipids contribute to the stability of membrane transporters and are crucially involved in their proper functioning. However, the molecular knowledge of how surrounding lipids affect membrane transport is surprisingly limited and despite its general importance is rarely considered in the molecular description of a transport mechanism. One reason is that only few atomic resolution structures of channels or transporters reveal a functional interaction with lipids, which are difficult to detect in X-ray structures per se. Overcoming these difficulties, we report here on a new structure of the osmotic stress-regulated betaine transporter BetP in complex with anionic lipids. This lipid-associated BetP structure is important in the molecular understanding of osmoregulation due to the strong dependence of activity regulation in BetP on the presence of negatively charged lipids. We detected eight resolved palmitoyl-oleoyl phosphatidyl glycerol (PG) lipids mimicking parts of the membrane leaflets and interacting with key residues in transport and regulation. The lipid-protein interactions observed here in structural detail in BetP provide molecular insights into the role of lipids in osmoregulated secondary transport.

K+-induced conformational changes in the trimeric betaine transporter BetP monitored by ATR-FTIR spectroscopy

The trimeric Na+-coupled betaine symporter BetP from Corynebactrium glutamicum adjusts transport activity according to the external osmolality. BetP senses the increasing internal K+ concentration, which is an immediate consequence of osmotic upshift in C. glutamicum. It is assumed that BetP specifically binds potassium to yet unidentified binding sites, thereby inducing conformational changes resulting in activation. Atomic structures of BetP were obtained in the absence of potassium allowing only a speculative glimpse on a putative mechanism of K+-induced transport activation. The structural data suggest that activation in BetP is crucially linked to its trimeric state involving an interaction network between several arginines and glutamates and aspartates. Here, we describe the effect of K+-induced activation on the specific ionic interaction sites in terminal domains and loops and on the protomer–protomer interactions within the trimer studied by ATR-FTIR spectroscopy. We suggest that arginine and aspartate and/or glutamate residues at the trimeric interface rearrange upon K+-induced activation, although they remain assembled in an interaction network. Our data propose a two-step mechanism comprising first a change in solvent exposure of charged residues and second a modification of their interaction sites in a partner-switching manner. FTIR reveals a higher α-helical content than expected from the X-ray structures that we attribute to the structurally unresolved N-terminal domain modulating regulation. In situ 1H/2H exchange studies point toward an altered exposure of backbone regions to buffer solution upon activation, most likely due to conformational changes in both terminal domains, which further affects ionic interactions within the trimer.

Structural Evidence for Functional Lipid Interactions in the Betaine Transporter BetP

Bilayer lipids contribute greatly to the stability of membrane transporters and are crucially involved in their proper functioning. However, the molecular details of how these lipids affect membrane transport is limited to the few atomic resolution structures of channels and transporters revealing functional lipid interactions. As a consequence, despite their biological importance, bilayer lipid-protein interactions are rarely considered in the molecular description of a transport mechanism, which are difficult to detect in X-ray structures per se. Lipids are often depleted from the detergent-protein complex during isolation or appear too unordered to identify in the crystal. Overcoming these difficulties, we report here on a new structure of the osmotic stress-regulated betaine transporter BetP in complex with anionic lipids. Activity regulation in BetP depends strongly on the presence of negatively charged lipids. We observe eight fully resolved palmitoyl-oleoyl phosphatidyl glycerol (PG) lipids bound to one BetP trimer, mimicking parts of the membrane leaflets. Our data reveal that the PG lipids interact with key residues in transport and regulation. Lipids, while likely being involved in the correct assembly of the BetP trimer also offer important communication sites between the protomers that might be required during stress sensing and transport regulation. The lipid-protein interactions observed in BetP reiterate the influence that the surrounding membrane can have on membrane protein function and urge a more holistic approach towards understanding membrane transport, especially for LeuT-like fold transporters.

Insight into the Mechanism of Substrate Selectivity and Proton-Coupled Transport in BetP

Sodium coupled betaine transporter BetP is a representative member of the BCCT transporter family. Its major function is to accumulate osmolytes in order to prevent cell death upon change in cytoplasmic osmotic pressure. BetP is one of the key models for understanding how the generalized LeuT-fold transporters function, due to the availability of its crystal structure in multiple conformational states. Recent experimental studies have shown that BetP-G153D can transport choline, instead of betaine, under pH gradient across the cellular membrane. Currently no satisfactory explanation exists for the substrate transport mechanism in the context of this dramatic single-site mutation. Aiming to gain better insight into this phenomenon, we have performed several microseconds of molecular dynamics simulations of the BetP-G153D mutant based on an inward-facing crystal structure. The results indicate that the protonation state of the mutation site Asp153 is closely coupled to large-scale gating motion of the entire transporter. Specifically, changing in the protonation state of Asp153 leads to distinct substrate release kinetics via control over conformation fluctuation in transmembrane helix TM6 and rearrangement of interaction network of charged residues at the intracellular side. These results suggest a novel mechanism for substrate transport control in transporters like BetP.

Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP.

Sodium-coupled substrate transport plays a central role in many biological processes. However, despite knowledge of the structures of several sodium-coupled transporters, the location of the sodium-binding site(s) often remains unclear. Several of these structures have the five transmembrane-helix inverted-topology repeat, LeuT-like (FIRL) fold, whose pseudosymmetry has been proposed to facilitate the alternating-access mechanism required for transport. Here, we provide biophysical, biochemical, and computational evidence for the location of the two cation-binding sites in the sodium-coupled betaine symporter BetP. A recent X-ray structure of BetP in a sodium-bound closed state revealed that one of these sites, equivalent to the Na2 site in related transporters, is located between transmembrane helices 1 and 8 of the FIRL-fold; here, we confirm the location of this site by other means. Based on the pseudosymmetry of this fold, we hypothesized that the second site is located between the equivalent helices 6 and 3. Molecular dynamics simulations of the closed-state structure suggest this second sodium site involves two threonine sidechains and a backbone carbonyl from helix 3, a phenylalanine from helix 6, and a water molecule. Mutating the residues proposed to form the two binding sites increased the apparent K(m) and K(d) for sodium, as measured by betaine uptake, tryptophan fluorescence, and (22)Na(+) binding, and also diminished the transient currents measured in proteoliposomes using solid supported membrane-based electrophysiology. Taken together, these results provide strong evidence for the identity of the residues forming the sodium-binding sites in BetP.

Alternating-access mechanism in conformationally asymmetric trimers of the betaine transporter BetP

Betaine and Na(+) symport has been extensively studied in the osmotically regulated transporter BetP from Corynebacterium glutamicum, a member of the betaine/choline/carnitine transporter family, which shares the conserved LeuT-like fold of two inverted structural repeats. BetP adjusts its transport activity by sensing the cytoplasmic K(+) concentration as a measure for hyperosmotic stress via the osmosensing carboxy-terminal domain. BetP needs to be in a trimeric state for communication between individual protomers through several intratrimeric interaction sites. Recently, crystal structures of inward-facing BetP trimers have contributed to our understanding of activity regulation on a molecular level. Here we report new crystal structures, which reveal two conformationally asymmetric BetP trimers, capturing among them three distinct transport states. We observe a total of four new conformations at once: an outward-open apo and an outward-occluded apo state, and two closed transition states--one in complex with betaine and one substrate-free. On the basis of these new structures, we identified local and global conformational changes in BetP that underlie the molecular transport mechanism, which partially resemble structural changes observed in other sodium-coupled LeuT-like fold transporters, but show differences we attribute to the osmolytic nature of betaine, the exclusive substrate specificity and the regulatory properties of BetP.

Conformational changes of the betaine transporter BetP from Corynebacterium glutamicum studied by pulse EPR spectroscopy

The betaine transporter BetP from Corynebacterium glutamicum is activated by hyperosmotic stress critically depending on the presence and integrity of its sensory C-terminal domain. The conformational properties of the trimeric BetP reconstituted in liposomes in the inactive state and during osmotic activation were investigated by site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. Comparison of intra- and intermolecular inter spin distance distributions obtained by double electron–electron resonance (DEER) EPR with the crystal structure of BetP by means of a rotamer library analysis suggest a rotation of BetP protomers within the trimer by about 15° as compared to the X-ray structure. Furthermore, we observed conformational changes upon activation of BetP, which are reflected in changes of the distances between positions 545 and 589 of different protomers in the trimer. Introduction of proline at positions 550 and 572, both leading to BetP variants with a permanent (low level) transport activity, caused changes of the DEER data similar to those observed for the activated and inactivated state, respectively. This indicates that not only displacements of the C-terminal domain in general but also concomitant interactions of its primary structure with surrounding protein domains and/or lipids are crucial for the activity regulation of BetP.

Role of Bundle Helices in a Regulatory Crosstalk in the Trimeric Betaine Transporter BetP

The Na +-coupled betaine symporter BetP regulates transport activity in response to hyperosmotic stress only in its trimeric state, suggesting a regulatory crosstalk between individual protomers. BetP shares the overall fold of two inverted structurally related five-transmembrane (TM) helix repeats with the sequence-unrelated Na +-coupled symporters LeuT, vSGLT, and Mhp1, which are neither trimeric nor regulated in transport activity. Conformational changes characteristic for this transporter fold involve the two first helices of each repeat, which form a four-TM-helix bundle. Here, we identify two ionic networks in BetP located on both sides of the membrane that might be responsible for BetP's unique regulatory behavior by restricting the conformational flexibility of the four-TM-helix bundle. The cytoplasmic ionic interaction network links both first helices of each repeat in one protomer to the osmosensing C-terminal domain of the adjacent protomer. Moreover, the periplasmic ionic interaction network conformationally locks the four-TM-helix bundle between the same neighbor protomers. By a combination of site-directed mutagenesis, cross-linking, and betaine uptake measurements, we demonstrate how conformational changes in individual bundle helices are transduced to the entire bundle by specific inter-helical interactions. We suggest that one purpose of bundle networking is to assist crosstalk between protomers during transport regulation by specifically modulating the transition from outward-facing to inward-facing state.

The role of trimerization in the osmoregulated betaine transporter BetP

The osmoregulated betaine transporter BetP is a stable trimer. Structural studies have shown that individual protomers can adopt distinct transport conformations, implying a functional role for the trimeric state in transport, although the role of trimerization in regulation is not yet understood. We designed putative monomeric mutants by molecular-dynamics simulations and in silico alanine-scanning mutagenesis. Several mutants including BetP-W101A/T351A were monomeric in detergent as well as in the membrane, as shown by blue native gel electrophoresis, crosslinking and electron microscopy. This monomeric form retains the ability to accumulate betaine, but is no longer regulated by hyperosmotic shock.

Computational and Experimental Studies of Substrate Binding, Conformational Change and Importance of the Trimeric State in the Glycine Betaine Transporter BetP

The glycine betaine/sodium symporter BetP responds to changes in external osmolality by regulation of its transport activity. A recent X-ray structure of BetP confirms that it is a homotrimer and in this structure each protomer adopts an identical conformation, in which the pathway is occluded from both sides. Despite the availability of a wealth of experimental data for BetP, the structures of the alternate states (e.g., open to the outside of the cell), molecular mechanisms of substrate and Na+ binding and transport, as well as the functional implications of the trimeric state remain poorly understood. To address these questions, we carried out computational studies using a range of techniques to derive hypotheses that were then tested experimentally. First, to identify structural features of the alternate states, we developed a procedure for flexible fitting of the X-ray structure of BetP into a lower-resolution cryo-EM map of BetP in a more native lipid environment, in which the three protomers have different conformations. These results suggest that: (i) the protomers adopt distinct conformational states relevant to the transport cycle; and (ii) there is conformational coupling between the protomers. Second, we performed all-atom molecular dynamics simulations and in silico alanine scanning of BetP trimers in order to identify interface residues crucial for maintaining the trimeric state. Mutations of these residues to alanine were introduced experimentally revealing that the isolated monomers are functional, and that the trimeric state is important for the regulation and higher activity of the protein. Finally, using molecular modeling and biochemical experiments we identified two Na+ binding sites in BetP that could not be resolved in the 3.35 A resolution X-ray structure.

The BCCT family of carriers: from physiology to crystal structure

Increases in the environmental osmolarity are key determinants for the growth of microorganisms. To ensure a physiologically acceptable level of cellular hydration and turgor at high osmolarity, many bacteria accumulate compatible solutes. Osmotically controlled uptake systems allow the scavenging of these compounds from scarce environmental sources as effective osmoprotectants. A number of these systems belong to the BCCT family (betaine-choline-carnitine-transporter), sodium- or proton-coupled transporters (e.g. BetP and BetT respectively) that are ubiquitous in microorganisms. The BCCT family also contains CaiT, an L-carnitine/γ-butyrobetaine antiporter that is not involved in osmotic stress responses. The glycine betaine transporter BetP from Corynebacterium glutamicum is a representative for osmoregulated symporters of the BCCT family and functions both as an osmosensor and osmoregulator. The crystal structure of BetP in an occluded conformation in complex with its substrate glycine betaine and two crystal structures of CaiT in an inward-facing open conformation in complex with L-carnitine and γ-butyrobetaine were reported recently. These structures and the wealth of biochemical data on the activity control of BetP in response to osmotic stress enable a correlation between the sensing of osmotic stress by a transporter protein with the ensuing regulation of transport activity. Molecular determinants governing the high-affinity binding of the compatible solutes by BetP and CaiT, the coupling in symporters and antiporters, and the osmoregulatory properties are discussed in detail for BetP and various BCCT carriers.

The properties and contribution of the Corynebacterium glutamicum MscS variant to fine-tuning of osmotic adaptation

Based on sequence similarity, the mscCG gene product of Corynebacterium glutamicum belongs to the family of MscS-type mechanosensitive channels. In order to investigate the physiological significance of MscCG in response to osmotic shifts in detail, we studied its properties using both patch-clamp techniques and betaine efflux kinetics. After heterologous expression in an E scherichia coli strain devoid of mechanosensitive channels, in patch-clamp analysis of giant E. coli spheroplasts MscCG showed the typical pressure dependent gating behavior of a stretch-activated channel with a current/voltage dependence indicating a strongly rectifying behavior. Apart from that, MscCG is characterized by significant functional differences with respect to conductance, ion selectivity and desensitation behavior as compared to MscS from E. coli. Deletion and complementation studies in C. glutamicum showed a significant contribution of MscCG to betaine efflux in response to hypoosmotic conditions. A detailed analysis of concomitant betaine uptake (by the betaine transporter BetP) and efflux (by MscCG) under hyperosmotic conditions indicates that MscCG may act in osmoregulation in C. glutamicum by fine-tuning the steady state concentration of compatible solutes in the cytoplasm which are accumulated in response to hyperosmotic stress.

Regulative interactions of the osmosensing C-terminal domain in the trimeric glycine betaine transporter BetP from Corynebacterium glutamicum

Activation of the osmoregulated trimeric betaine transporter BetP from Corynebacterium glutamicum was shown to depend mainly on the correct folding and integrity of its 55 amino acid long, partly alpha-helical C-terminal domain. Reorientation of the three C-terminal domains in the BetP trimer indicates different lipid-protein and protein-protein interactions of the C-terminal domain during osmoregulation. A regulation mechanism is suggested where this domain switches the transporter from the inactive to the active state. Interpretation of recently obtained electron and X-ray crystallography data of BetP led to a structure-function based model of C-terminal molecular switching involved in osmoregulation.

The role of lipids and salts in two-dimensional crystallization of the glycine–betaine transporter BetP from Corynebacterium glutamicum

The osmoregulated and chill-sensitive glycine–betaine transporter (BetP) from Corynebacterium glutamicum was reconstituted into lipids to form two-dimensional (2D) crystals. The sensitivity of BetP partly bases on its interaction with lipids. Here we demonstrate that lipids and salts influence crystal morphology and crystallinity of a C-terminally truncated BetP. The salt type and concentration during crystallization determined whether crystals grew in the form of planar-tubes, sheets or vesicles, while the lipid type influenced crystal packing and order. Three different lipid preparations for 2D crystallization were compared. Only the use of lipids extracted from C. glutamicum cells led to the formation of large, well-ordered crystalline areas. To understand the lipid-derived influence on crystallinity, lipid extracts from different stages of the crystallization process were analyzed by quantitative multiple-precursor ion scanning mass spectroscopy (MS). Results show that BetP has a preference for fatty acid moieties 16:0–18:1, and that a phosphatidyl glycerol (PG) 16:0–18:1 rich preparation prevents formation of pseudo crystals.

Osmolality, Temperature, and Membrane Lipid Composition Modulate the Activity of Betaine Transporter BetP in Corynebacterium glutamicum

The gram-positive soil bacterium Corynebacterium glutamicum, a major amino acid-producing microorganism in biotechnology, is equipped with several osmoregulated uptake systems for compatible solutes, which is relevant for the physiological response to osmotic stress. The most significant carrier, BetP, is instantly activated in response to an increasing cytoplasmic K(+) concentration. Importantly, it is also activated by chill stress independent of osmotic stress. We show that the activation of BetP by both osmotic stress and chill stress is altered in C. glutamicum cells grown at and adapted to low temperatures. BetP from cold-adapted cells is less sensitive to osmotic stress. In order to become susceptible for chill activation, cold-adapted cells in addition needed a certain amount of osmotic stimulation, indicating that there is cross talk of these two types of stimuli at the level of BetP activity. We further correlated the change in BetP regulation properties in cells grown at different temperatures to changes in the lipid composition of the plasma membrane. For this purpose, the glycerophospholipidome of C. glutamicum grown at different temperatures was analyzed by mass spectrometry using quantitative multiple precursor ion scanning. The molecular composition of glycerophospholipids was strongly affected by the growth temperature. The modulating influence of membrane lipid composition on BetP function was further corroborated by studying the influence of artificial modulation of membrane dynamics by local anesthetics and the lack of a possible influence of internally accumulated betaine on BetP activity.