Regulatory properties and interaction of the C- and N-terminal domains of BetP, an osmoregulated betaine transporter from Corynebacterium glutamicum

The glycine betaine carrier BetP from Corynebacterium glutamicum responds to changes in external osmolality by regulation of its transport activity, and the C-terminal domain was previously identified to be involved in this process. Here we investigate the structural requirements of the C-terminal domain for osmoregulation as well as interacting domains that are relevant for intramolecular signal transduction in response to osmotic stress. For this purpose, we applied a proline scanning approach and amino acid replacements other than proline in selected positions. To analyze the impact of the surrounding membrane, BetP mutants were studied in both C. glutamicum and Escherichia coli, which strongly differ in their phospholipid composition. A region of approximately 25 amino acid residues within the C-terminal domain with a high propensity for alpha-helical structure was found to be essential in terms of its conformational properties for osmodependent regulation. The size of this region was larger in E. coli membranes than in the highly negatively charged C. glutamicum membranes. As a novel aspect of BetP regulation, interaction of the C-terminal domain with one of the cytoplasmic loops as well as with the N-terminal domain was shown to be involved in osmosensing and/or osmoregulation. These results support a functional model of BetP activation that involves the C-terminal domain shifting from interaction with the membrane to interaction with intramolecular domains in response to osmotic stress.     

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.     

Dynamic lipid interactions in the plasma membrane Na+,K+-ATPase

The function of ion-transporting Na⁺,K⁺-ATPases depends on the surrounding lipid environment in biological membranes. Two established lipid-interaction sites A and B within the transmembrane domain have been observed to induce protein activation and stabilization, respectively. In addition, lipid-mediated inhibition has been assigned to a site C, but with the exact location not experimentally confirmed. Also, possible effects on lipid interactions by disease mutants dwelling in the membrane-protein interface remain relatively uncharacterized. We simulated human Na⁺,K⁺-ATPase α₁β₁FXYD homology models in E1 and E2 states in an asymmetric, multicomponent plasma membrane to determine both wild-type and disease mutant lipid-protein interactions. The simulated wild-type lipid interactions at the established sites A and B were in agreement with experimental results thereby confirming the membrane-protein model system. The less well-characterized, proposed inhibitory site C was dominated by lipids lacking inhibitory properties. Instead, two sites hosting inhibitory lipids were identified at the extracellular side and also a cytoplasmic CHL-binding site that provide putative alternative locations of Na⁺,K⁺-ATPase inhibition. Three disease mutations, Leu302Arg, Glu840Arg and Met859Arg resided in the lipid-protein interface and caused drastic changes in the lipid interactions. The simulation results show that lipid interactions to the human Na⁺,K⁺-ATPase α₁β₁FXYD protein in the plasma membrane are highly state-dependent and can be disturbed by disease mutations located in the lipid interface, which can open up for new venues to understand genetic disorders.     

Stimulus analysis of BetP activation under in vivo conditions

The secondary active, Na⁺ coupled glycine betaine carrier BetP from Corynebacterium glutamicum BetP was shown to harbor two different functions, transport catalysis (betaine uptake) and stimulus sensing, as well as activity regulation in response to hyperosmotic stress. By analysis in a reconstituted system, the rise in the cytoplasmic K⁺ concentration was identified as a primary stimulus for BetP activation. We have now studied regulation of BetP in vivo by independent variation of both the cytoplasmic K⁺ concentration and the transmembrane osmotic gradient. The rise in internal K⁺ was found to be necessary but not sufficient for BetP activation in cells. In addition hyperosmotic stress is required for full transport activity in cells, but not in proteoliposomes. This second stimulus of BetP could be mimicked in cells by the addition of the amphiphile tetracaine which hints to a relationship of this type of stimulus to a change in membrane properties. Determination of the molecular activity of BetP in both cells and proteoliposomes provided experimental evidence that in proteoliposomes BetP exists in a pre-stimulated condition and reaches full activity already in response to the K⁺ stimulus.     

Hyperosmotic Stress Allosterically Reconfigures Betaine Binding Pocket in BetP

The transporter BetP in C. glutamicum is essential in maintaining bacterial cell viability during hyperosmotic stress and functions by co-transporting betaine and Na⁺ into bacterial cells. Hyperosmotic stress leads to increased intracellular K⁺ concentrations which in turn promotes betaine binding. While structural details of multiple end state conformations of BetP have provided high resolution snapshots, how K⁺ sensing by the C-terminal domain is allosterically relayed to the betaine binding site is not well understood. In this study, we describe conformational dynamics in solution of BetP using amide hydrogen/deuterium exchange mass spectrometry. These reveal how K⁺ alters conformation of the disordered C- and N-terminal domains to allosterically reconfigure transmembrane helices 3, 8, and 10 to enhance betaine interactions. A map of the betaine binding site, at near single amino acid resolution, reveals a critical extrahelical H-bond mediated by TM3 with betaine.     

The interaction of the Bax C-terminal domain with negatively charged lipids modifies the secondary structure and changes its way of insertion into membranes

Fourier transform infrared spectroscopy (FTIR) was used to study the secondary structure of peptides which imitate the amino acid sequences of the C-terminal domain of the pro-apoptotic protein Bax (Bax-C) when incorporated into different lipid vesicles with or without negatively charged phospholipids. The infrared spectroscopy results showed that while the beta-sheet components are predominant in the membrane-free Bax-C secondary structure as well as in the presence of phosphatidylcholine vesicles, the peptide changes its secondary structure in the presence of negatively charged membranes, including phospholipids such as phosphatidylglycerol or phosphatidylinositol, depending on both the lipid composition and their molar ratio. The negative charges in the model membrane surface caused a marked change from beta-sheet to alpha-helix structure. Moreover, using attenuated total reflection infrared spectroscopy (ATR-FTIR), we investigated the orientation of Bax-C alpha-helical structures with respect to the normal to the internal reflection element. The orientation of Bax-C in membranes was also affected by negatively charged lipids, the presence of phosphatidylglycerol reduced the angle it forms with the normal to the germanium plate from 45 degrees in phosphatidylcholine to 27 degrees in phosphatidylglycerol vesicles. These results highlight the importance of lipid-protein interaction for the correct folding of membrane proteins and they suggest that the C-terminal domain of Bax will only span membranes with a net negative charge in their surface.     

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.     

Bacterial osmosensing: roles of membrane structure and electrostatics in lipid-protein and protein-protein interactions

Bacteria act to maintain their hydration when the osmotic pressure of their environment changes. When the external osmolality decreases (osmotic downshift), mechanosensitive channels are activated to release low molecular weight osmolytes (and hence water) from the cytoplasm. Upon osmotic upshift, osmoregulatory transporters are activated to import osmolytes (and hence water). Osmoregulatory channels and transporters sense and respond to osmotic stress via different mechanisms. Mechanosensitive channel MscL senses the increasing tension in the membrane and appears to gate when the lateral pressure in the acyl chain region of the lipids drops below a threshold value. Transporters OpuA, BetP and ProP are activated when increasing external osmolality causes threshold ionic concentrations in excess of about 0.05 M to be reached in the proteoliposome lumen. The threshold activation concentrations for the OpuA transporter are strongly dependent on the fraction of anionic lipids that surround the cytoplasmic face of the protein. The higher the fraction of anionic lipids, the higher the threshold ionic concentrations. A similar trend is observed for the BetP transporter. The lipid dependence of osmotic activation of OpuA and BetP suggests that osmotic signals are transmitted to the protein via interactions between charged osmosensor domains and the ionic headgroups of the lipids in the membrane. The charged, C-terminal domains of BetP and ProP are important for osmosensing. The C-terminal domain of ProP participates in homodimeric coiled-coil formation and it may interact with the membrane lipids and soluble protein ProQ. The activation of ProP by lumenal, macromolecular solutes at constant ionic strength indicates that its structure and activity may also respond to macromolecular crowding. This excluded volume effect may restrict the range over which the osmosensing domain can electrostatically interact. A simplified version of the dissociative double layer theory is used to explain the activation of the transporters by showing how changes in ion concentration could modulate interactions between charged osmosensor domains and charged lipid or protein surfaces. Importantly, the relatively high ionic concentrations at which osmosensors become activated at different surface charge densities compare well with the predicted dependence of 'critical' ion concentrations on surface charge density. The critical ion concentrations represent transitions in Maxwellian ionic distributions at which the surface potential reaches 25.7 mV for monovalent ions. The osmosensing mechanism is qualitatively described as an "ON/OFF switch" representing thermally relaxed and electrostatically locked protein conformations.     

The effect of surface charge density on valinomycin-K+ complex formation in model membranes

The model membrane approach was used to investigate the surface charge effect on the ion-antibiotic complexation process. Mixed monolayers of valinomycin and lipids were spread on subphases containing K⁺ or Na⁺. The surface charge density was modified by spreading ionizable valinomycin analogs on aqueous subphases of different pH or by changing the nature of the lipid (neutral, negatively charged) in the mixed film. Surface pressure and surface potential measurements demonstrated that a neutral lipid (phosphatidylcholine) or positively charged valinomycin analogs didn't enhance the antibiotic complexing capacity. However, a maximal complexation is reached for a critical lipid concentration in the valinomycin-phosphatidylserine mixed film. The role of the surface charge on the valinomycin complexing properties was examined in terms of the Gouy-Chapman theory. As a consequence of the negative charge of the lipid monolayer, the K⁺ concentration near the surface is larger than the bulk concentration, by a Boltzmann factor. A good agreement was observed between the experimental results and the theoretical predictions. Conductance measurements of asymmetric bilayers containing a neutral lipid (egg lecithin) on one side and a negatively charged lipid (phosphatidylserine) on the other, confirm the role of the surface charge. Indeed, addition of K⁺ to the neutral side of the bilayer containing valinomycin had no effect on the conductance whereas addition of K⁺ to the charged side of the bilayer caused a 80-fold conductance increase.     

Structural asymmetry in a trimeric Na+/betaine symporter, BetP, from Corynebacterium glutamicum

The Na⁺-coupled symporter BetP catalyzes the uptake of the compatible solute betaine in the soil bacterium Corynebacterium glutamicum. BetP also senses hyperosmotic stress and regulates its own activity in response to stress level. We determined a three-dimensional (3D) map (at 8 Å in-plane resolution) of a constitutively active mutant of BetP in a C. glutamicum membrane environment by electron cryomicroscopy of two-dimensional crystals. The map shows that the constitutively active mutant, which lacks the C-terminal domain involved in osmosensing, is trimeric like wild-type BetP. Recently, we reported the X-ray crystal structure of BetP at 3.35 Å, in which all three protomers displayed a substrate-occluded state. Rigid-body fitting of this trimeric structure to the 3D map identified the periplasmic and cytoplasmic sides of the membrane. Fitting of an X-ray monomer to the individual protomer maps allowed assignment of transmembrane helices and of the substrate pathway, and revealed differences in trimer architecture from the X-ray structure in the tilt angle of each protomer with respect to the membrane. The three protomer maps showed pronounced differences around the substrate pathway, suggesting three different conformations within the same trimer. Two of those protomer maps closely match those of the atomic structures of the outward-facing and inward-facing states of the hydantoin transporter Mhp1, suggesting that the BetP protomer conformations reflect key states of the transport cycle. Thus, the asymmetry in the two-dimensional maps may reflect cooperativity of conformational changes within the BetP trimer, which potentially increases the rate of glycine betaine uptake.     

NMR Characterization and Membrane Interactions of the Loop Region of Kindlin-3 F1 Subdomain

Kindlins-1,2 and 3 are FERM domain-containing cytosolic proteins involved in the activation and regulation of integrin-mediated cell adhesion. Apart from binding to integrin β cytosolic tails, kindlins and the well characterized integrin-activator talin bind membrane phospholipids. The ubiquitin-like F1 sub-domain of the FERM domain of talin contains a short loop that binds to the lipid membrane. By contrast, the F1 sub-domain of kindlins contains a long loop demonstrated binding to the membrane. Here, we report structural characterization and lipid interactions of the 83-residue F1 loop of kindlin-3 using NMR and optical spectroscopy methods. NMR studies demonstrated that the F1 loop of kindlin-3 is globally unfolded but stretches of residues assuming transient helical conformations could be detected in aqueous solution. We mapped membrane binding interactions of the F1 loop with small unilamellar vesicles (SUVs) containing either zwitterionic lipids or negatively charged lipids using ¹⁵N-¹H HSQC titrations. These experiments revealed that the F1 loop of kindlin-3 preferentially interacted with the negatively charged SUVs employing almost all of the residues. By contrast, only fewer residues appeared to be interacted with SUVs containing neutral lipids. Further, CD and NMR data suggested stabilization of helical conformations and predominant resonance perturbations of the F1 loop in detergent containing solutions. Conformations of an isolated N-terminal peptide fragment, or EK21, of the F1 loop, containing a poly-Lys sequence motif, important for membrane interactions, were also investigated in detergent solutions. EK21 adopted a rather extended or β-type conformations in complex with negatively charged SDS micelles. To our knowledge, this is the first report describing the conformations and residue-specific interactions of kindlin F1 loop with lipids. These data therefore provide important insights into the interactions of kindlin FERM domain with membrane lipids that contribute toward the integrin activating property.     

Membrane surface charge modulates lipoprotein complex forming capability of peptides derived from the C-terminal domain of apolipoprotein E

Apolipoprotein E (apoE) plays a major role in the transport and metabolism of lipid by acting as a ligand for low density lipoprotein-receptors. The amphipathic helical regions of its C-terminal domain are necessary for the lipoprotein binding and assembly of nascent lipoprotein particles. Lipoproteins in the plasma are known to possess a net negative charge, determined by both its protein and lipid components, which regulates the metabolism of lipoproteins. The role of membrane surface charge on the interaction of apoE has not been studied previously. Also the importance of individual amphipathic helical regions of its C-terminal domain in binding to negatively charged lipid membrane is not addressed. In this study we have compared the interaction of four peptide segments of apoE C-terminal domain (apoE_(202-223), apoE_(223-244), apoE_(245-266), and apoE_(268-289)) with zwitterionic and negatively charged model membranes by employing UV-visible and fluorescence spectroscopy, circular dichroism, and native PAGE analysis. Our results show that the peptide sequence 202-223, 245-266 and 268-289 of apoE has higher affinity towards negatively charged lipid membrane and are independently capable of forming lipoprotein particles of 17 ± 2 nm Stokes diameter. The results suggest that surface charge of lipoprotein regulates its metabolism possibly by modulating the recruitment of apoE on its surface.     

Surface Charges of the Membrane Crucially Affect Regulation of Na,K-ATPase by Phospholemman (FXYD1)

The human α₁/His₁₀-β₁ isoform of Na,K-ATPase has been reconstituted as a complex with and without FXYD1 into proteoliposomes of various lipid compositions in order to study the effect of the regulatory subunit on the half-saturating Na⁺ concentration (K _1/2) of Na⁺ ions for activation of the ion pump. It has been shown that the fraction of negatively charged lipid in the bilayer crucially affects the regulatory properties. At low concentrations of the negatively charged lipid DOPS (<10 %), FXYD1 increases K _1/2 of Na⁺ ions for activation of the ion pump. Phosphorylation of FXYD1 by protein kinase A at Ser68 abrogates this effect. Conversely, for proteoliposomes made with high concentrations of DOPS (>10 %), little or no effect of FXYD1 on the K _1/2 of Na⁺ ions is observed. Depending on ionic strength and lipid composition of the proteoliposomes, FXYD1 can alter the K _1/2 of Na⁺ ions by up to twofold. We propose possible molecular mechanisms to explain the regulatory effects of FXYD1 and the influence of charged lipid and protein phosphorylation. In particular, the positively charged C-terminal helix of FXYD1 appears to be highly mobile and may interact with the cytoplasmic N domain of the α-subunit, the interaction being strongly affected by phosphorylation at Ser68 and the surface charge of the membrane.     

Membrane Targeting of the Spir·Formin Actin Nucleator Complex Requires a Sequential Handshake of Polar Interactions

Spir and formin (FMN)-type actin nucleators initiate actin polymerization at vesicular membranes necessary for long range vesicular transport processes. Here we studied in detail the membrane binding properties and protein/protein interactions that govern the assembly of the membrane-associated Spir·FMN complex. Using biomimetic membrane models we show that binding of the C-terminal Spir-2 FYVE-type zinc finger involves both the presence of negatively charged lipids and hydrophobic contributions from the turret loop that intrudes the lipid bilayer. In solution, we uncovered a yet unknown intramolecular interaction between the Spir-2 FYVE-type domain and the N-terminal kinase non-catalytic C-lobe domain (KIND) that could not be detected in the membrane-bound state. Interestingly, we found that the intramolecular Spir-2 FYVE/KIND and the trans-regulatory Fmn-2-FSI/Spir-2-KIND interactions are competitive. We therefore characterized co-expressed Spir-2 and Fmn-2 fluorescent protein fusions in living cells by fluorescence cross-correlation spectroscopy. The data corroborate a model according to which Spir-2 exists in two different states, a cytosolic monomeric conformation and a membrane-bound state in which the KIND domain is released and accessible for subsequent Fmn-2 recruitment. This sequence of interactions mechanistically couples membrane binding of Spir to the recruitment of FMN, a pivotal step for initiating actin nucleation at vesicular membranes.     

Membrane binding properties of IRSp53-missing in metastasis domain (IMD) protein

The 53-kDa insulin receptor substrate protein (IRSp53) organizes the actin cytoskeleton in response to stimulation of small GTPases, promoting the formation of cell protrusions such as filopodia and lamellipodia. IMD is the N-terminal 250 amino acid domain (IRSp53/MIM Homology Domain) of IRSp53 (also called I-BAR), which can bind to negatively charged lipid molecules. Overexpression of IMD induces filopodia formation in cells and purified IMD assembles finger-like protrusions in reconstituted lipid membranes. IMD was shown by several groups to bundle actin filaments, but other groups showed that it also binds to membranes. IMD binds to negatively charged lipid molecules with preference to clusters of PI(4,5)P₂. Here, we performed a range of different in vitro fluorescence experiments to determine the binding properties of the IMD to phospholipids. We used different constructs of large unilamellar vesicles (LUVETs), containing neutral or negatively charged phospholipids. We found that IMD has a stronger binding interaction with negatively charged PI(4,5)P₂ or PS lipids than PS/PC or neutral PC lipids. The equilibrium dissociation constant for the IMD–lipid interaction falls into the 78–170 μM range for all the lipids tested. The solvent accessibility of the fluorescence labels on the IMD during its binding to lipids is also reduced as the lipids become more negatively charged. Actin affects the IMD–lipid interaction, depending on its polymerization state. Monomeric actin partially disrupts the binding, while filamentous actin can further stabilize the IMD–lipid interaction.     

The (non)specificity in the lipid-requirement of calcium- and (sodium plus potassium)-transporting adenosine triphosphatases

The lipid requirement of alkali cation transporting ATPases has been investigated by studying the effect of well controlled modifications of the endogenous lipids on the activity of the enzymes. In principle, two different approaches can be distinguished. Firstly, complete delipidation with concomitant loss of activity followed by reconstitution of the system with well-defined lipids. A second approach involves specific enzymatic degradations or detergent mediated replacements, both selectively modifying the endogenous lipid complement. Each of these strategies has advantages and disadvantages, which have given rise to many conflicting opinions regarding the question whether a specificity in the lipid requirements of the various ATPases does exist. Only recently, it was established that the (Ca²⁺ + Mg²⁺)-ATPase in the sarcoplasmic reticulum does not have any particular specificity in its lipid requirement. Almost every amphiphile, including a variety of (non-ionic) detergents, can fulfil its needs in this respect. On the other hand, the analogous enzyme found in the erythrocyte membrane requires the presence of either mono- or diacylglycerophospholipids. In the native situation, however, it is only that fraction of the total $\text{glycero}$-phospholipid complement of the membrane that forms part of the inner half of the lipid bilayer that is actually involved. The most pronounced specificity in lipid requirement seems to exist for the (Na⁺ + K⁺)-ATPase system. Reactivation studies strongly suggest an absolute requirement for negatively charged phospholipids. Quite interestingly, individual differences may exist among various membrane species as to which of the endogenous negatively charged glycero-phospholipids is actually involved, e.g. phosphatidylserine in the erythrocyte membrane and phosphatidylinositol in rabbit kidney microsomes, respectively. Furthermore, it is suggested by experiments involving phospholipases that only a small fraction of the total activating phospholipid is involved; this fraction may be rather closely associated with the (Na⁺ + K⁺)- ATPase protein.     

Molecular dynamics simulation of cation–phospholipid clustering in phospholipid bilayers: Possible role in stalk formation during membrane fusion

In this study, we performed all-atom long-timescale molecular dynamics simulations of phospholipid bilayers incorporating three different proportions of negatively charged lipids in the presence of K⁺, Mg^2 +, and Ca^2 + ions to systemically determine how membrane properties are affected by cations and lipid compositions. Our simulations revealed that the binding affinity of Ca^2 + ions with lipids is significantly stronger than that of K⁺ and Mg^2 + ions, regardless of the composition of the lipid bilayer. The binding of Ca^2 + ions to the lipids resulted in bilayers having smaller lateral areas, greater thicknesses, greater order, and slower rotation of their lipid head groups, relative to those of corresponding K⁺- and Mg^2 +-containing systems. The Ca^2 + ions bind preferentially to the phosphate groups of the lipids. The complexes formed between the cations and the lipids further assembled to form various multiple-cation-centered clusters in the presence of anionic lipids and at higher ionic strength—most notably for Ca^2 +. The formation of cation–lipid complexes and clusters dehydrated and neutralized the anionic lipids, creating a more-hydrophobic environment suitable for membrane aggregation. We propose that the formation of Ca^2 +–phospholipid clusters across apposed lipid bilayers can work as a “cation glue” to adhere apposed membranes together, providing an adequate configuration for stalk formation during membrane fusion.