Caroline Koshy - Academia.edu (original) (raw)
Papers by Caroline Koshy
Biophysical Journal, 2013
Sodium-coupled substrate transport plays a central role in many biological processes. However, de... more 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 sodiumbinding site(s) often remains unclear. Several of these structures have the five transmembrane-helix inverted-topology repeat, LeuTlike (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 Km and Kd for sodium, as measured by betaine uptake, tryptophan fluorescence, and 22Na+ 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
The Journal of General Physiology, Feb 6, 2019
Mechanistic understanding of dynamic membrane proteins such as transporters, receptors, and chann... more Mechanistic understanding of dynamic membrane proteins such as transporters, receptors, and channels requires accurate depictions of conformational ensembles, and the manner in which they interchange as a function of environmental factors including substrates, lipids, and inhibitors. Spectroscopic techniques such as electron spin resonance (ESR) pulsed electron-electron double resonance (PELDOR), also known as double electron-electron resonance (DEER), provide a complement to atomistic structures obtained from x-ray crystallography or cryo-EM, since spectroscopic data reflect an ensemble and can be measured in more native solvents, unperturbed by a crystal lattice. However, attempts to interpret DEER data are frequently stymied by discrepancies with the structural data, which may arise due to differences in conditions, the dynamics of the protein, or the flexibility of the attached paramagnetic spin labels. Recently, molecular simulation techniques such as EBMetaD have been developed that create a conformational ensemble matching an experimental distance distribution while applying the minimal possible bias. Moreover, it has been proposed that the work required during an EBMetaD simulation to match an experimentally determined distribution could be used as a metric with which to assign conformational states to a given measurement. Here, we demonstrate the application of this concept for a sodium-coupled transport protein, BetP. Because the probe, protein, and lipid bilayer are all represented in atomic detail, the different contributions to the work, such as the extent of protein backbone movements, can be separated. This work therefore illustrates how ranking simulations based on EBMetaD can help to bridge the gap between structural and biophysical data and thereby enhance our understanding of membrane protein conformational mechanisms.
Biophysical Journal, Feb 1, 2023
Biophysical Journal, Feb 1, 2022
bioRxiv (Cold Spring Harbor Laboratory), Jun 5, 2022
Membrane transporters mediate the passage of molecules across membranes and are essential for cel... more Membrane transporters mediate the passage of molecules across membranes and are essential for cellular function. While the transmembrane region of these proteins is responsible for substrate transport, often the cytoplasmic regions are required for modulating their activity. However, it can be difficult to obtain atomic-resolution descriptions of these autoregulatory domains by classical structural biology techniques if they lack a single, defined structure, as they may not be resolved or may be truncated or modified to facilitate crystallization. The betaine permease, BetP, a homotrimer, is a prominent and well-studied example of a membrane protein whose autoregulation depends on cytoplasmic N-and C-terminal segments. These domains sense and transduce changes in K + concentration and in lipid bilayer properties caused by osmotic stress. However, structural data for these terminal domains is incomplete, which hinders a clear description of the molecular mechanism of autoregulation. Here we used µs-scale molecular simulations of the BetP trimer to compare reported conformations of the 45 amino-acid long C-terminal tails. The simulations provide support for the idea that the conformation derived from EM data represents a stable global orientation of the C-terminal segment under downregulating conditions. The simulations also allow a detailed molecular description of the C-terminal tail dynamics as well as its interactions with lipids, potassium ions, and the cytoplasmic surface of neighboring transporter subunits. Nevertheless, they do not provide information on the N-terminal segment, whose structure was not resolved by the structural studies. We therefore examined the possible interactions of the N-terminal tail by generating de novo models of its structure in the context of the EM-derived structure. The resultant full-length models of the BetP trimer provide a molecular framework for the arrangement of the terminal domains in the downregulated protein. In this framework, each C-terminal tail contacts the neighboring protomer in the clockwise direction (viewed from the cytoplasm), while the N-terminal tails only contact the protomer in the counterclockwise direction. This framework indicates an intricate interplay between the three protomers of BetP and, specifically, a multi-directionality that may facilitate autoregulation of betaine transport. and is also made available for use under a CC0 license.
Biochimica Et Biophysica Acta - General Subjects, Mar 1, 2015
Structural evidences with functional corroborations have revealed distinct features of lipid-prot... more Structural evidences with functional corroborations have revealed distinct features of lipid-protein interactions especially in channels and receptors. Many membrane embedded transporters are also known to require specific lipids for their functions and for some of them cellular and biochemical data suggest tight regulation by the lipid bilayer. However, molecular details on lipid-protein interactions in transporters are sparse since lipids are either depleted from the detergent solubilized transporters in three-dimensional crystals or not readily resolved in crystal structures. Nevertheless the steady increase in the progress of transporter structure determination contributed more examples of structures with resolved lipids. This review gives an overview on transporter structures in complex with lipids reported to date and discusses commonly encountered difficulties in the identification of functionally significant lipid-protein interactions based on those structures and functional in vitro data. Recent structures provided molecular details into regulation mechanism of transporters by specific lipids. The review highlights common findings and conserved patterns for distantly related transporter families to draw a more general picture on the regulatory role of lipid-protein interactions. Several common themes of the manner in which lipids directly influence membrane-mediated folding, oligomerization and structure stability can be found. Especially for LeuT-like fold transporters similarities in structurally resolved lipid-protein interactions suggest a common way in which transporter conformations are affected by lipids even in evolutionarily distinct transporters. Lipids appear to play an additional role as joints mechanically reinforcing the inverted repeat topology, which is a major determinant in the alternating access mechanism of secondary transporters. This review brings together and adds to the repertoire of knowledge on lipid-protein interactions of functional significance presented in structures of membrane transporters. Knowledge of specific lipid-binding sites and modes of lipid influence on these proteins not only accomplishes the molecular description of transport cycle further, but also sheds light into localization dependent differences of transporter function. This article is part of a Special Issue entitled Structural biochemistry and biophysics of membrane proteins.
Blood Institute. We thank José Faraldo-Gómez and Fabrizio Marinelli for useful discussions. made ... more Blood Institute. We thank José Faraldo-Gómez and Fabrizio Marinelli for useful discussions. made available for use under a CC0 license.
Membrane transporters mediate the passage of molecules across membranes and are essential for cel... more Membrane transporters mediate the passage of molecules across membranes and are essential for cellular function. While the transmembrane region of these proteins is responsible for substrate transport, often the cytoplasmic regions are required for modulating their activity. However, it can be difficult to obtain atomic-resolution descriptions of these autoregulatory domains by classical structural biology techniques if they lack a single, defined structure, as they may not be resolved or may be truncated or modified to facilitate crystallization. The betaine permease, BetP, a homotrimer, is a prominent and well-studied example of a membrane protein whose autoregulation depends on cytoplasmic N- and C-terminal segments. These domains sense and transduce changes in K+ concentration and in lipid bilayer properties caused by osmotic stress. However, structural data for these terminal domains is incomplete, which hinders a clear description of the molecular mechanism of autoregulation. ...
Biophysical Journal, 2022
Sodium-coupled substrate transport plays a central role in many biological processes. However, de... more 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 sodiumbinding site(s) often remains unclear. Several of these structures have the five transmembrane-helix inverted-topology repeat, LeuTlike (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 Km and Kd for sodium, as measured by betaine uptake, tryptophan fluorescence, and 22Na+ 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
Journal of General Physiology, 2019
Mechanistic understanding of dynamic membrane proteins such as transporters, receptors, and chann... more Mechanistic understanding of dynamic membrane proteins such as transporters, receptors, and channels requires accurate depictions of conformational ensembles, and the manner in which they interchange as a function of environmental factors including substrates, lipids, and inhibitors. Spectroscopic techniques such as electron spin resonance (ESR) pulsed electron–electron double resonance (PELDOR), also known as double electron–electron resonance (DEER), provide a complement to atomistic structures obtained from x-ray crystallography or cryo-EM, since spectroscopic data reflect an ensemble and can be measured in more native solvents, unperturbed by a crystal lattice. However, attempts to interpret DEER data are frequently stymied by discrepancies with the structural data, which may arise due to differences in conditions, the dynamics of the protein, or the flexibility of the attached paramagnetic spin labels. Recently, molecular simulation techniques such as EBMetaD have been develope...
The EMBO Journal, 2011
BetP is an Na þ-coupled betaine-specific transporter of the betaine-choline-carnitine (BCC) trans... more BetP is an Na þ-coupled betaine-specific transporter of the betaine-choline-carnitine (BCC) transporter family involved in the response to hyperosmotic stress. The crystal structure of BetP revealed an overall fold of two inverted structurally related repeats (LeuT-fold) that BetP shares with other sequence-unrelated Na þ-coupled symporters. Numerous structures of LeuT-fold transporters in distinct conformational states have contributed substantially to our understanding of the alternating access mechanism of transport. Nevertheless, coupling of substrate and cotransported ion fluxes has not been structurally corroborated to the same extent. We converted BetP by a singlepoint mutation-glycine to aspartate-into an H þ-coupled choline-specific transporter and solved the crystal structure of this mutant in complex with choline. The structure of BetP-G153D demonstrates a new inward-facing open conformation for BetP. Choline binding to a location close to the second, low-affinity sodium-binding site (Na2) of LeuT-fold transporters is facilitated by the introduced aspartate. Our data confirm the importance of a cationbinding site in BetP, playing a key role in a proposed molecular mechanism of Na þ and H þ coupling in BCC transporters.
The glycine betaine symporter BetP regulates the osmotic stress response of Corynebacterium gluta... more The glycine betaine symporter BetP regulates the osmotic stress response of Corynebacterium glutamicum, a soil bacterium used extensively in biotechnology. Although BetP is a homotrimer, biochemical studies have shown that each protomer is able to transport its substrate independently. Crystallographic structures of BetP have been determined in several conformations, seemingly capturing outward-open, inward-open and occluded states, both loaded with the substrate and in the apo form. However, it has been challenging to establish a correspondence between each of these structures and specific states in the mechanism of the transporter under more physiological conditions. To this end, we examined the dynamics of spin-labelled BetP using pulsed electron-electron double resonance (PELDOR) under different stimuli. We then carried out molecular simulations of structures of the BetP monomer to interpret the PELDOR data, using the enhanced-sampling methodology EBMetaD [Marinelli & Faraldo-Go...
Biophysical Journal, 2013
Bilayer lipids contribute to the stability of membrane transporters and are crucially involved in... more 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 stressregulated 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.
Biophysical Journal, 2013
Sodium-coupled substrate transport plays a central role in many biological processes. However, de... more 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 sodiumbinding site(s) often remains unclear. Several of these structures have the five transmembrane-helix inverted-topology repeat, LeuTlike (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 Km and Kd for sodium, as measured by betaine uptake, tryptophan fluorescence, and 22Na+ 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
The Journal of General Physiology, Feb 6, 2019
Mechanistic understanding of dynamic membrane proteins such as transporters, receptors, and chann... more Mechanistic understanding of dynamic membrane proteins such as transporters, receptors, and channels requires accurate depictions of conformational ensembles, and the manner in which they interchange as a function of environmental factors including substrates, lipids, and inhibitors. Spectroscopic techniques such as electron spin resonance (ESR) pulsed electron-electron double resonance (PELDOR), also known as double electron-electron resonance (DEER), provide a complement to atomistic structures obtained from x-ray crystallography or cryo-EM, since spectroscopic data reflect an ensemble and can be measured in more native solvents, unperturbed by a crystal lattice. However, attempts to interpret DEER data are frequently stymied by discrepancies with the structural data, which may arise due to differences in conditions, the dynamics of the protein, or the flexibility of the attached paramagnetic spin labels. Recently, molecular simulation techniques such as EBMetaD have been developed that create a conformational ensemble matching an experimental distance distribution while applying the minimal possible bias. Moreover, it has been proposed that the work required during an EBMetaD simulation to match an experimentally determined distribution could be used as a metric with which to assign conformational states to a given measurement. Here, we demonstrate the application of this concept for a sodium-coupled transport protein, BetP. Because the probe, protein, and lipid bilayer are all represented in atomic detail, the different contributions to the work, such as the extent of protein backbone movements, can be separated. This work therefore illustrates how ranking simulations based on EBMetaD can help to bridge the gap between structural and biophysical data and thereby enhance our understanding of membrane protein conformational mechanisms.
Biophysical Journal, Feb 1, 2023
Biophysical Journal, Feb 1, 2022
bioRxiv (Cold Spring Harbor Laboratory), Jun 5, 2022
Membrane transporters mediate the passage of molecules across membranes and are essential for cel... more Membrane transporters mediate the passage of molecules across membranes and are essential for cellular function. While the transmembrane region of these proteins is responsible for substrate transport, often the cytoplasmic regions are required for modulating their activity. However, it can be difficult to obtain atomic-resolution descriptions of these autoregulatory domains by classical structural biology techniques if they lack a single, defined structure, as they may not be resolved or may be truncated or modified to facilitate crystallization. The betaine permease, BetP, a homotrimer, is a prominent and well-studied example of a membrane protein whose autoregulation depends on cytoplasmic N-and C-terminal segments. These domains sense and transduce changes in K + concentration and in lipid bilayer properties caused by osmotic stress. However, structural data for these terminal domains is incomplete, which hinders a clear description of the molecular mechanism of autoregulation. Here we used µs-scale molecular simulations of the BetP trimer to compare reported conformations of the 45 amino-acid long C-terminal tails. The simulations provide support for the idea that the conformation derived from EM data represents a stable global orientation of the C-terminal segment under downregulating conditions. The simulations also allow a detailed molecular description of the C-terminal tail dynamics as well as its interactions with lipids, potassium ions, and the cytoplasmic surface of neighboring transporter subunits. Nevertheless, they do not provide information on the N-terminal segment, whose structure was not resolved by the structural studies. We therefore examined the possible interactions of the N-terminal tail by generating de novo models of its structure in the context of the EM-derived structure. The resultant full-length models of the BetP trimer provide a molecular framework for the arrangement of the terminal domains in the downregulated protein. In this framework, each C-terminal tail contacts the neighboring protomer in the clockwise direction (viewed from the cytoplasm), while the N-terminal tails only contact the protomer in the counterclockwise direction. This framework indicates an intricate interplay between the three protomers of BetP and, specifically, a multi-directionality that may facilitate autoregulation of betaine transport. and is also made available for use under a CC0 license.
Biochimica Et Biophysica Acta - General Subjects, Mar 1, 2015
Structural evidences with functional corroborations have revealed distinct features of lipid-prot... more Structural evidences with functional corroborations have revealed distinct features of lipid-protein interactions especially in channels and receptors. Many membrane embedded transporters are also known to require specific lipids for their functions and for some of them cellular and biochemical data suggest tight regulation by the lipid bilayer. However, molecular details on lipid-protein interactions in transporters are sparse since lipids are either depleted from the detergent solubilized transporters in three-dimensional crystals or not readily resolved in crystal structures. Nevertheless the steady increase in the progress of transporter structure determination contributed more examples of structures with resolved lipids. This review gives an overview on transporter structures in complex with lipids reported to date and discusses commonly encountered difficulties in the identification of functionally significant lipid-protein interactions based on those structures and functional in vitro data. Recent structures provided molecular details into regulation mechanism of transporters by specific lipids. The review highlights common findings and conserved patterns for distantly related transporter families to draw a more general picture on the regulatory role of lipid-protein interactions. Several common themes of the manner in which lipids directly influence membrane-mediated folding, oligomerization and structure stability can be found. Especially for LeuT-like fold transporters similarities in structurally resolved lipid-protein interactions suggest a common way in which transporter conformations are affected by lipids even in evolutionarily distinct transporters. Lipids appear to play an additional role as joints mechanically reinforcing the inverted repeat topology, which is a major determinant in the alternating access mechanism of secondary transporters. This review brings together and adds to the repertoire of knowledge on lipid-protein interactions of functional significance presented in structures of membrane transporters. Knowledge of specific lipid-binding sites and modes of lipid influence on these proteins not only accomplishes the molecular description of transport cycle further, but also sheds light into localization dependent differences of transporter function. This article is part of a Special Issue entitled Structural biochemistry and biophysics of membrane proteins.
Blood Institute. We thank José Faraldo-Gómez and Fabrizio Marinelli for useful discussions. made ... more Blood Institute. We thank José Faraldo-Gómez and Fabrizio Marinelli for useful discussions. made available for use under a CC0 license.
Membrane transporters mediate the passage of molecules across membranes and are essential for cel... more Membrane transporters mediate the passage of molecules across membranes and are essential for cellular function. While the transmembrane region of these proteins is responsible for substrate transport, often the cytoplasmic regions are required for modulating their activity. However, it can be difficult to obtain atomic-resolution descriptions of these autoregulatory domains by classical structural biology techniques if they lack a single, defined structure, as they may not be resolved or may be truncated or modified to facilitate crystallization. The betaine permease, BetP, a homotrimer, is a prominent and well-studied example of a membrane protein whose autoregulation depends on cytoplasmic N- and C-terminal segments. These domains sense and transduce changes in K+ concentration and in lipid bilayer properties caused by osmotic stress. However, structural data for these terminal domains is incomplete, which hinders a clear description of the molecular mechanism of autoregulation. ...
Biophysical Journal, 2022
Sodium-coupled substrate transport plays a central role in many biological processes. However, de... more 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 sodiumbinding site(s) often remains unclear. Several of these structures have the five transmembrane-helix inverted-topology repeat, LeuTlike (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 Km and Kd for sodium, as measured by betaine uptake, tryptophan fluorescence, and 22Na+ 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
Journal of General Physiology, 2019
Mechanistic understanding of dynamic membrane proteins such as transporters, receptors, and chann... more Mechanistic understanding of dynamic membrane proteins such as transporters, receptors, and channels requires accurate depictions of conformational ensembles, and the manner in which they interchange as a function of environmental factors including substrates, lipids, and inhibitors. Spectroscopic techniques such as electron spin resonance (ESR) pulsed electron–electron double resonance (PELDOR), also known as double electron–electron resonance (DEER), provide a complement to atomistic structures obtained from x-ray crystallography or cryo-EM, since spectroscopic data reflect an ensemble and can be measured in more native solvents, unperturbed by a crystal lattice. However, attempts to interpret DEER data are frequently stymied by discrepancies with the structural data, which may arise due to differences in conditions, the dynamics of the protein, or the flexibility of the attached paramagnetic spin labels. Recently, molecular simulation techniques such as EBMetaD have been develope...
The EMBO Journal, 2011
BetP is an Na þ-coupled betaine-specific transporter of the betaine-choline-carnitine (BCC) trans... more BetP is an Na þ-coupled betaine-specific transporter of the betaine-choline-carnitine (BCC) transporter family involved in the response to hyperosmotic stress. The crystal structure of BetP revealed an overall fold of two inverted structurally related repeats (LeuT-fold) that BetP shares with other sequence-unrelated Na þ-coupled symporters. Numerous structures of LeuT-fold transporters in distinct conformational states have contributed substantially to our understanding of the alternating access mechanism of transport. Nevertheless, coupling of substrate and cotransported ion fluxes has not been structurally corroborated to the same extent. We converted BetP by a singlepoint mutation-glycine to aspartate-into an H þ-coupled choline-specific transporter and solved the crystal structure of this mutant in complex with choline. The structure of BetP-G153D demonstrates a new inward-facing open conformation for BetP. Choline binding to a location close to the second, low-affinity sodium-binding site (Na2) of LeuT-fold transporters is facilitated by the introduced aspartate. Our data confirm the importance of a cationbinding site in BetP, playing a key role in a proposed molecular mechanism of Na þ and H þ coupling in BCC transporters.
The glycine betaine symporter BetP regulates the osmotic stress response of Corynebacterium gluta... more The glycine betaine symporter BetP regulates the osmotic stress response of Corynebacterium glutamicum, a soil bacterium used extensively in biotechnology. Although BetP is a homotrimer, biochemical studies have shown that each protomer is able to transport its substrate independently. Crystallographic structures of BetP have been determined in several conformations, seemingly capturing outward-open, inward-open and occluded states, both loaded with the substrate and in the apo form. However, it has been challenging to establish a correspondence between each of these structures and specific states in the mechanism of the transporter under more physiological conditions. To this end, we examined the dynamics of spin-labelled BetP using pulsed electron-electron double resonance (PELDOR) under different stimuli. We then carried out molecular simulations of structures of the BetP monomer to interpret the PELDOR data, using the enhanced-sampling methodology EBMetaD [Marinelli & Faraldo-Go...
Biophysical Journal, 2013
Bilayer lipids contribute to the stability of membrane transporters and are crucially involved in... more 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 stressregulated 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.