The Experts below are selected from a list of 19272 Experts worldwide ranked by ideXlab platform
Stanley J Opella - One of the best experts on this subject based on the ideXlab platform.
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structure determination of membrane proteins in their native phospholipid bilayer environment by rotationally aligned solid state nmr spectroscopy
Accounts of Chemical Research, 2013Co-Authors: Stanley J OpellaAbstract:One of the most important topics in experimental structural biology is determining the structures of membrane proteins. These structures represent one-third of all of the information expressed from a genome, distinguished by their locations within the phospholipid bilayer of cells, organelles, or enveloped viruses. Their highly hydrophobic nature and insolubility in aqueous media means that they require an amphipathic environment. They have unique functions in transport, catalysis, channel formation, and signaling. Researchers are particularly interested in G-protein coupled Receptors (GPCRs) because they modulate many biological processes, and about half of the approximately 800 of these proteins within the human genome are or can be turned into drug Receptors that affect a wide range of diseases. Because of experimental difficulties, researchers have studied membrane proteins using a wide variety of artificial media that mimic membranes, such as mixed organic solvents or detergents. More sophisticated mimics include bilayer discs (bicelles) and the lipid cubic phase (LCP), but both of these contain a very large detergent component, which can disrupt the stability and function of membrane proteins. To have confidence in the resulting structures and their biological functions and to avoid disrupting these delicate proteins, the structures of membrane proteins should be determined in their native environment of liquid crystalline phospholipid bilayers under physiological conditions. This Account describes a recently developed general method for determining the structures of unmodified membrane proteins in phospholipid bilayers by solid-state NMR spectroscopy. Because it relies on the natural, rapid rotational diffusion of these proteins about the bilayer normal, this method is referred to as rotationally aligned (RA) solid-state NMR. This technique elaborates on oriented sample (OS) solid-state NMR, its complementary predecessor. These methods exploit the power of solid-state NMR, which enables researchers to obtain well-resolved spectra from "immobile" membrane proteins in phospholipid bilayers, to separate and measure frequencies that reflect orientations with respect to the bilayer normal, and to make complementary distance measurements. The determination of the structures of several membrane proteins, most prominently the Chemokine Receptor CXCR1, a 350-residue GPCR, has demonstrated this approach.
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structure of the Chemokine Receptor CXCR1 in phospholipid bilayers
Nature, 2012Co-Authors: Sang Ho Park, Fabio Casagrande, Ye Tian, Henry J Nothnagel, Hans Kiefer, Klaus Maier, Anna A De Angelis, Francesca M Marassi, Stanley J OpellaAbstract:NMR spectroscopy is used to determine the three-dimensional structure of the full-length human Chemokine Receptor CXCR1 in phospholipid bilayers under physiological conditions. G-protein-coupled Receptors (GPCRs) are ubiquitous membrane proteins that transduce chemical signals from the outside of a cell, and many of them are drug targets. Sang Ho Park et al. present a new nuclear magnetic resonance spectroscopy method for the study of membrane proteins in phospholipid bilayers and use it to determine the structure of human CXCR1, a high-affinity GPCR for interleukin-8, a major mediator of immune and inflammatory responses. CXCR1 is one of two high-affinity Receptors for the CXC Chemokine interleukin-8 (IL-8), a major mediator of immune and inflammatory responses implicated in many disorders, including tumour growth1,2,3. IL-8, released in response to inflammatory stimuli, binds to the extracellular side of CXCR1. The ligand-activated intracellular signalling pathways result in neutrophil migration to the site of inflammation2. CXCR1 is a class A, rhodopsin-like G-protein-coupled Receptor (GPCR), the largest class of integral membrane proteins responsible for cellular signal transduction and targeted as drug Receptors4,5,6,7. Despite its importance, the molecular mechanism of CXCR1 signal transduction is poorly understood owing to the limited structural information available. Recent structural determination of GPCRs has advanced by modifying the Receptors with stabilizing mutations, insertion of the protein T4 lysozyme and truncations of their amino acid sequences8, as well as addition of stabilizing antibodies and small molecules9 that facilitate crystallization in cubic phase monoolein mixtures10. The intracellular loops of GPCRs are crucial for G-protein interactions11, and activation of CXCR1 involves both amino-terminal residues and extracellular loops2,12,13. Our previous nuclear magnetic resonance studies indicate that IL-8 binding to the N-terminal residues is mediated by the membrane, underscoring the importance of the phospholipid bilayer for physiological activity14. Here we report the three-dimensional structure of human CXCR1 determined by NMR spectroscopy. The Receptor is in liquid crystalline phospholipid bilayers, without modification of its amino acid sequence and under physiological conditions. Features important for intracellular G-protein activation and signal transduction are revealed. The structure of human CXCR1 in a lipid bilayer should help to facilitate the discovery of new compounds that interact with GPCRs and combat diseases such as breast cancer.
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optimization of purification and refolding of the human Chemokine Receptor CXCR1 improves the stability of proteoliposomes for structure determination
Biochimica et Biophysica Acta, 2012Co-Authors: Sang Ho Park, Fabio Casagrande, Hans Kiefer, Klaus Maier, Mignon Chu, Stanley J OpellaAbstract:The human Chemokine Receptor CXCR1 is a G-protein coupled Receptor that has been successfully expressed in E. coli as inclusion bodies, and purified and refolded in multi-milligram quantities required for structural studies. Expression in E. coli enables selective and uniform isotopic labeling with (13)C and (15)N for NMR studies. Long-term chemical and conformational stability and oligomeric homogeneity of CXCR1 in phospholipid bilayers are crucial for structural studies under physiological conditions. Here we describe substantial refinements in our previously described purification and reconstitution procedures for CXCR1 in phospholipid bilayers. These refinements have led to the preparation of highly purified, completely monomeric, proteoliposome samples that are stable for months at 35°C while subject to the high power radiofrequency irradiations of solid-state NMR experiments. The principal changes from the previously described methods include: 1) ensure that CXCR1 is pure and homogeneously monomeric within the limits of detection (>98%); 2) monitor and control the pH at all times especially following the addition of TCEP, which serves as a reducing agent but also changes the pH; 3) slowly refold CXCR1 with the complete removal of all traces of SDS using a KCl precipitation/dialysis method; and 4) ensure that the molar ratio between the CXCR1 and the phospholipids does not change during refolding and detergent removal. NMR samples prepared with these protocols yield reproducible results over a period of many months at 35°C. This purification and refolding protocol is likely to be applicable with minimal changes to other GPCRs as well as other membrane proteins.
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interactions of interleukin 8 with the human Chemokine Receptor CXCR1 in phospholipid bilayers by nmr spectroscopy
Journal of Molecular Biology, 2011Co-Authors: Sang Ho Park, Fabio Casagrande, Leah Cho, Lauren V Albrecht, Stanley J OpellaAbstract:CXCR1 is a Receptor for the Chemokine interleukin-8 (IL-8), a mediator of immune and inflammatory responses. Strategically located in the cell membrane, CXCR1 binds to IL-8 with high affinity and subsequently transduces a signal across the membrane bilayer to a G-protein-activated second messenger system. Here, we describe NMR studies of the interactions between IL-8 and human CXCR1 in lipid environments. Functional full-length and truncated constructs of CXCR1 and full-length IL-8 were uniformly (15)N-labeled by expression in bacteria followed by purification and refolding. The residues responsible for interactions between IL-8 and the N-terminal domain of CXCR1 were identified by specific chemical shift perturbations of assigned resonances on both IL-8 and CXCR1. Solution NMR signals from IL-8 in q=0.1 isotropic bicelles disappeared completely when CXCR1 in lipid bilayers was added in a 1:1 molar ratio, indicating that binding to the Receptor-containing bilayers immobilizes IL-8 (on the ~10(5) Hz timescale) and broadens the signals beyond detection. The same solution NMR signals from IL-8 were less affected by the addition of N-terminal truncated CXCR1 in lipid bilayers, demonstrating that the N-terminal domain of CXCR1 is mainly responsible for binding to IL-8. The interaction is tight enough to immobilize IL-8 along with the Receptor in phospholipid bilayers and is specific enough to result in well-aligned samples in oriented sample solid-state NMR spectra. A combination of solution NMR and solid-state NMR studies of IL-8 in the presence of various constructs of CXCR1 enables us to propose a model for the multistep binding process.
Sang Ho Park - One of the best experts on this subject based on the ideXlab platform.
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structure of the Chemokine Receptor CXCR1 in phospholipid bilayers
Nature, 2012Co-Authors: Sang Ho Park, Fabio Casagrande, Ye Tian, Henry J Nothnagel, Hans Kiefer, Klaus Maier, Anna A De Angelis, Francesca M Marassi, Stanley J OpellaAbstract:NMR spectroscopy is used to determine the three-dimensional structure of the full-length human Chemokine Receptor CXCR1 in phospholipid bilayers under physiological conditions. G-protein-coupled Receptors (GPCRs) are ubiquitous membrane proteins that transduce chemical signals from the outside of a cell, and many of them are drug targets. Sang Ho Park et al. present a new nuclear magnetic resonance spectroscopy method for the study of membrane proteins in phospholipid bilayers and use it to determine the structure of human CXCR1, a high-affinity GPCR for interleukin-8, a major mediator of immune and inflammatory responses. CXCR1 is one of two high-affinity Receptors for the CXC Chemokine interleukin-8 (IL-8), a major mediator of immune and inflammatory responses implicated in many disorders, including tumour growth1,2,3. IL-8, released in response to inflammatory stimuli, binds to the extracellular side of CXCR1. The ligand-activated intracellular signalling pathways result in neutrophil migration to the site of inflammation2. CXCR1 is a class A, rhodopsin-like G-protein-coupled Receptor (GPCR), the largest class of integral membrane proteins responsible for cellular signal transduction and targeted as drug Receptors4,5,6,7. Despite its importance, the molecular mechanism of CXCR1 signal transduction is poorly understood owing to the limited structural information available. Recent structural determination of GPCRs has advanced by modifying the Receptors with stabilizing mutations, insertion of the protein T4 lysozyme and truncations of their amino acid sequences8, as well as addition of stabilizing antibodies and small molecules9 that facilitate crystallization in cubic phase monoolein mixtures10. The intracellular loops of GPCRs are crucial for G-protein interactions11, and activation of CXCR1 involves both amino-terminal residues and extracellular loops2,12,13. Our previous nuclear magnetic resonance studies indicate that IL-8 binding to the N-terminal residues is mediated by the membrane, underscoring the importance of the phospholipid bilayer for physiological activity14. Here we report the three-dimensional structure of human CXCR1 determined by NMR spectroscopy. The Receptor is in liquid crystalline phospholipid bilayers, without modification of its amino acid sequence and under physiological conditions. Features important for intracellular G-protein activation and signal transduction are revealed. The structure of human CXCR1 in a lipid bilayer should help to facilitate the discovery of new compounds that interact with GPCRs and combat diseases such as breast cancer.
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structure of the Chemokine Receptor CXCR1 in phospholipid bilayers
Nature, 2012Co-Authors: Sang Ho Park, Fabio Casagrande, Ye Tian, Henry J Nothnagel, Hans Kiefer, Klaus Maier, Mignon Chu, Bibhuti B Das, Anna A De AngelisAbstract:CXCR1 is one of two high-affinity Receptors for the CXC Chemokine interleukin-8 (IL-8), a major mediator of immune and inflammatory responses implicated in many disorders, including tumour growth. IL-8, released in response to inflammatory stimuli, binds to the extracellular side of CXCR1. The ligand-activated intracellular signalling pathways result in neutrophil migration to the site of inflammation. CXCR1 is a class A, rhodopsin-like G-protein-coupled Receptor (GPCR), the largest class of integral membrane proteins responsible for cellular signal transduction and targeted as drug Receptors. Despite its importance, the molecular mechanism of CXCR1 signal transduction is poorly understood owing to the limited structural information available. Recent structural determination of GPCRs has advanced by modifying the Receptors with stabilizing mutations, insertion of the protein T4 lysozyme and truncations of their amino acid sequences, as well as addition of stabilizing antibodies and small molecules that facilitate crystallization in cubic phase monoolein mixtures. The intracellular loops of GPCRs are crucial for G-protein interactions, and activation of CXCR1 involves both amino-terminal residues and extracellular loops. Our previous nuclear magnetic resonance studies indicate that IL-8 binding to the N-terminal residues is mediated by the membrane, underscoring the importance of the phospholipid bilayer for physiological activity. Here we report the three-dimensional structure of human CXCR1 determined by NMR spectroscopy. The Receptor is in liquid crystalline phospholipid bilayers, without modification of its amino acid sequence and under physiological conditions. Features important for intracellular G-protein activation and signal transduction are revealed. The structure of human CXCR1 in a lipid bilayer should help to facilitate the discovery of new compounds that interact with GPCRs and combat diseases such as breast cancer.
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optimization of purification and refolding of the human Chemokine Receptor CXCR1 improves the stability of proteoliposomes for structure determination
Biochimica et Biophysica Acta, 2012Co-Authors: Sang Ho Park, Fabio Casagrande, Hans Kiefer, Klaus Maier, Mignon Chu, Stanley J OpellaAbstract:The human Chemokine Receptor CXCR1 is a G-protein coupled Receptor that has been successfully expressed in E. coli as inclusion bodies, and purified and refolded in multi-milligram quantities required for structural studies. Expression in E. coli enables selective and uniform isotopic labeling with (13)C and (15)N for NMR studies. Long-term chemical and conformational stability and oligomeric homogeneity of CXCR1 in phospholipid bilayers are crucial for structural studies under physiological conditions. Here we describe substantial refinements in our previously described purification and reconstitution procedures for CXCR1 in phospholipid bilayers. These refinements have led to the preparation of highly purified, completely monomeric, proteoliposome samples that are stable for months at 35°C while subject to the high power radiofrequency irradiations of solid-state NMR experiments. The principal changes from the previously described methods include: 1) ensure that CXCR1 is pure and homogeneously monomeric within the limits of detection (>98%); 2) monitor and control the pH at all times especially following the addition of TCEP, which serves as a reducing agent but also changes the pH; 3) slowly refold CXCR1 with the complete removal of all traces of SDS using a KCl precipitation/dialysis method; and 4) ensure that the molar ratio between the CXCR1 and the phospholipids does not change during refolding and detergent removal. NMR samples prepared with these protocols yield reproducible results over a period of many months at 35°C. This purification and refolding protocol is likely to be applicable with minimal changes to other GPCRs as well as other membrane proteins.
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the structure of the Chemokine Receptor CXCR1 in phospholipid bilayers and interactions with il 8
Biophysical Journal, 2012Co-Authors: Sang Ho Park, Fabio Casagrande, Ye Tian, Henry J Nothnagel, Francesca M Marassi, Mignon Chu, Bibhuti B Das, Jasmina Racic, Hans KieferAbstract:CXCR1 is a Receptor for the Chemokine interleukin-8 (IL-8), a mediator of immune and inflammatory responses. Strategically located in the cell membrane, CXCR1 binds to IL-8 with high affinity, and subsequently transduces a signal across the membrane bilayer to a G-protein activated second messenger system. Here, we describe the three-dimensional structure of human CXCR1 determined in phospholipid bilayers under physiological conditions by solid-state NMR spectroscopy. We use a five-step approach: (1) prepare a sample of a uniformly 13C/15N labeled CXCR1 in proteoliposomes by bacterial expression, purification, and refolding. (2) Resolve individual signals with MAS solid-state NMR experiments. (3) Assign each signal to a specific residue. (4) Measure two or more orientation-dependent frequencies for each residue and some distances. (5) Calculate de novo three-dimensional structure. The availability of a method for determining the structures of unmodified GPCRs in their native environment of phospholipid bilayers under physiological conditions has the potential to transform the field of structural determination of membrane proteins, and to accelerate the discovery of drugs that interact with membrane-associated Receptors. Progress towards determining the structure and function of IL-8 bound to CXCR1 in membrane environments will be described.
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interactions of interleukin 8 with the human Chemokine Receptor CXCR1 in phospholipid bilayers by nmr spectroscopy
Journal of Molecular Biology, 2011Co-Authors: Sang Ho Park, Fabio Casagrande, Leah Cho, Lauren V Albrecht, Stanley J OpellaAbstract:CXCR1 is a Receptor for the Chemokine interleukin-8 (IL-8), a mediator of immune and inflammatory responses. Strategically located in the cell membrane, CXCR1 binds to IL-8 with high affinity and subsequently transduces a signal across the membrane bilayer to a G-protein-activated second messenger system. Here, we describe NMR studies of the interactions between IL-8 and human CXCR1 in lipid environments. Functional full-length and truncated constructs of CXCR1 and full-length IL-8 were uniformly (15)N-labeled by expression in bacteria followed by purification and refolding. The residues responsible for interactions between IL-8 and the N-terminal domain of CXCR1 were identified by specific chemical shift perturbations of assigned resonances on both IL-8 and CXCR1. Solution NMR signals from IL-8 in q=0.1 isotropic bicelles disappeared completely when CXCR1 in lipid bilayers was added in a 1:1 molar ratio, indicating that binding to the Receptor-containing bilayers immobilizes IL-8 (on the ~10(5) Hz timescale) and broadens the signals beyond detection. The same solution NMR signals from IL-8 were less affected by the addition of N-terminal truncated CXCR1 in lipid bilayers, demonstrating that the N-terminal domain of CXCR1 is mainly responsible for binding to IL-8. The interaction is tight enough to immobilize IL-8 along with the Receptor in phospholipid bilayers and is specific enough to result in well-aligned samples in oriented sample solid-state NMR spectra. A combination of solution NMR and solid-state NMR studies of IL-8 in the presence of various constructs of CXCR1 enables us to propose a model for the multistep binding process.
Amitabha Chattopadhyay - One of the best experts on this subject based on the ideXlab platform.
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conformational plasticity and dynamic interactions of the n terminal domain of the Chemokine Receptor CXCR1
PLOS Computational Biology, 2021Co-Authors: Shalmali Kharche, Amitabha Chattopadhyay, Manali Joshi, Durba SenguptaAbstract:The dynamic interactions between G protein-coupled Receptors (GPCRs) and their cognate protein partners are central to several cell signaling pathways. For example, the association of CXC Chemokine Receptor 1 (CXCR1) with its cognate Chemokine, interleukin-8 (IL8 or CXCL8) initiates pathways leading to neutrophil-mediated immune responses. The N-terminal domain of Chemokine Receptors confers ligand selectivity, but unfortunately the conformational dynamics of this intrinsically disordered region remains unresolved. In this work, we have explored the interaction of CXCR1 with IL8 by microsecond time scale coarse-grain simulations, complemented by atomistic models and NMR chemical shift predictions. We show that the conformational plasticity of the apo-Receptor N-terminal domain is restricted upon ligand binding, driving it to an open C-shaped conformation. Importantly, we corroborated the dynamic complex sampled in our simulations against chemical shift perturbations reported by previous NMR studies and show that the trends are similar. Our results indicate that chemical shift perturbation is often not a reporter of residue contacts in such dynamic associations. We believe our results represent a step forward in devising a strategy to understand intrinsically disordered regions in GPCRs and how they acquire functionally important conformational ensembles in dynamic protein-protein interfaces.
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conformational plasticity and dynamic interactions of the n terminal domain of the Chemokine Receptor CXCR1
bioRxiv, 2020Co-Authors: Shalmali Kharche, Amitabha Chattopadhyay, Manali Joshi, Durba SenguptaAbstract:Dynamic interactions between G protein-coupled Receptors (GPCRs) and their cognate protein partners at the membrane interface control several cellular signaling pathways. An important example is the association of CXC Chemokine Receptor 1 (CXCR1) with its cognate Chemokine, interleukin-8 (IL8 or CXCL8) that regulates neutrophil-mediated immune responses. Although the N-terminal domain of the Receptor is known to confer ligand selectivity, the conformational dynamics of this intrinsically disordered region of CXCR1 in particular, and Chemokine Receptors in general, remains unresolved. In this work, we have explored the interaction of CXCR1 with IL8 by microsecond time scale coarse-grain simulations that were validated by atomistic models and NMR chemical shift predictions. We show that the conformational plasticity of the apo -Receptor N-terminal region is restricted upon ligand binding, driving it to an open C-shaped conformation. Importantly, we validated the dynamic complex sampled in our simulations against chemical shift perturbations reported by previous NMR studies. Our results indicate that caution should be exercised when chemical shift perturbation is used as a reporter of residue contacts in such dynamic associations. We believe our results represent a step forward in devising a strategy to understand intrinsically disordered regions in GPCRs and how they acquire functionally important conformational ensembles in dynamic protein-protein interfaces.
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membrane induced organization and dynamics of the n terminal domain of Chemokine Receptor CXCR1 insights from atomistic simulations
Chemistry and Physics of Lipids, 2017Co-Authors: Shalmali Kharche, Manali Joshi, Durba Sengupta, Amitabha ChattopadhyayAbstract:The CXC Chemokine Receptor 1 (CXCR1) is an important member of the G protein-coupled Receptor (GPCR) family in which the extracellular N-terminal domain has been implicated in ligand binding and selectivity. The structure of this domain has not yet been elucidated due to its inherent dynamics, but experimental evidence points toward membrane-dependent organization and dynamics. To gain molecular insight into the interaction of the N-terminal domain with the membrane bilayer, we performed a series of microsecond time scale atomistic simulations of the N-terminal domain of CXCR1 in the presence and absence of POPC bilayers. Our results show that the peptide displays a high propensity to adopt a β-sheet conformation in the presence of the membrane bilayer. The interaction of the peptide with the membrane bilayer was found to be transient in our simulations. Interestingly, a scrambled peptide, containing the same residues in a randomly varying sequence, did not exhibit membrane-modulated structural dynamics. These results suggest that sequence-dependent electrostatics, modulated by the membrane, could play an important role in folding of the N-terminal domain. We believe that our results reinforce the emerging paradigm that cellular membranes could be important modulators of function of G protein-coupled Receptors such as CXCR1.
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organization and dynamics of the n terminal domain of Chemokine Receptor CXCR1 in reverse micelles effect of graded hydration
Journal of Physical Chemistry B, 2013Co-Authors: Arunima Chaudhuri, K. Rajarathnam, Sourav Haldar, Pritam Basu, Mamata Kombrabail, G Krishnamoorthy, Amitabha ChattopadhyayAbstract:Water plays a fundamental role in the folding, structure, dynamics, and function of proteins and peptides. The extracellular N-terminal domain of Chemokine Receptors is crucial in mediating binding affinity, Receptor selectivity, and regulating function. The flexible N-terminal domain becomes ordered in membranes and membrane-mimetic assemblies, thereby indicating that the membrane could play an important role in regulating CXC Chemokine Receptor 1 (CXCR1) function. In view of the role of hydration in lipid-protein interactions in membranes, we explored the organization and dynamics of a 34-mer peptide of the CXCR1 N-terminal domain in reverse micelles by utilizing a combination of fluorescence-based approaches and circular dichroism spectroscopy. Our results show that the secondary structure adopted by the CXCR1 N-domain is critically dependent on hydration. The tryptophan residues of the CXCR1 N-domain experience motional restriction and exhibit red edge excitation shift (REES) upon incorporation in reverse micelles. REES and fluorescence lifetime exhibit reduction with increasing reverse micellar hydration. Time-resolved fluorescence anisotropy measurements reveal the effect of hydration on peptide rotational dynamics. Taken together, these results constitute the first report demonstrating modulation in the organization and dynamics of the N-terminal domain of a Chemokine Receptor in a membrane-like environment of varying hydration. We envisage that these results are relevant in the context of hydration in the function of G protein-coupled Receptors.
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membrane interaction of the n terminal domain of Chemokine Receptor CXCR1
Biochimica et Biophysica Acta, 2010Co-Authors: Sourav Haldar, K. Rajarathnam, H Raghuraman, Trishool Namani, Amitabha ChattopadhyayAbstract:The N-terminal domain of Chemokine Receptors constitutes one of the two critical ligand binding sites, and plays important roles by mediating binding affinity, Receptor selectivity, and regulating function. In this work, we monitored the organization and dynamics of a 34-mer peptide of the CXC Chemokine Receptor 1 (CXCR1) N-terminal domain and its interaction with membranes by utilizing a combination of fluorescence-based approaches and surface pressure measurements. Our results show that the CXCR1 N-domain 34-mer peptide binds vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and upon binding, the tryptophan residues of the peptide experience motional restriction and exhibit red edge excitation shift (REES) of 19 nm. These results are further supported by increase in fluorescence anisotropy and mean fluorescence lifetime upon membrane binding. These results constitute one of the first reports demonstrating membrane interaction of the N-terminal domain of CXCR1 and gain relevance in the context of the emerging role of cellular membranes in Chemokine signaling.
Fabio Casagrande - One of the best experts on this subject based on the ideXlab platform.
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structure of the Chemokine Receptor CXCR1 in phospholipid bilayers
Nature, 2012Co-Authors: Sang Ho Park, Fabio Casagrande, Ye Tian, Henry J Nothnagel, Hans Kiefer, Klaus Maier, Anna A De Angelis, Francesca M Marassi, Stanley J OpellaAbstract:NMR spectroscopy is used to determine the three-dimensional structure of the full-length human Chemokine Receptor CXCR1 in phospholipid bilayers under physiological conditions. G-protein-coupled Receptors (GPCRs) are ubiquitous membrane proteins that transduce chemical signals from the outside of a cell, and many of them are drug targets. Sang Ho Park et al. present a new nuclear magnetic resonance spectroscopy method for the study of membrane proteins in phospholipid bilayers and use it to determine the structure of human CXCR1, a high-affinity GPCR for interleukin-8, a major mediator of immune and inflammatory responses. CXCR1 is one of two high-affinity Receptors for the CXC Chemokine interleukin-8 (IL-8), a major mediator of immune and inflammatory responses implicated in many disorders, including tumour growth1,2,3. IL-8, released in response to inflammatory stimuli, binds to the extracellular side of CXCR1. The ligand-activated intracellular signalling pathways result in neutrophil migration to the site of inflammation2. CXCR1 is a class A, rhodopsin-like G-protein-coupled Receptor (GPCR), the largest class of integral membrane proteins responsible for cellular signal transduction and targeted as drug Receptors4,5,6,7. Despite its importance, the molecular mechanism of CXCR1 signal transduction is poorly understood owing to the limited structural information available. Recent structural determination of GPCRs has advanced by modifying the Receptors with stabilizing mutations, insertion of the protein T4 lysozyme and truncations of their amino acid sequences8, as well as addition of stabilizing antibodies and small molecules9 that facilitate crystallization in cubic phase monoolein mixtures10. The intracellular loops of GPCRs are crucial for G-protein interactions11, and activation of CXCR1 involves both amino-terminal residues and extracellular loops2,12,13. Our previous nuclear magnetic resonance studies indicate that IL-8 binding to the N-terminal residues is mediated by the membrane, underscoring the importance of the phospholipid bilayer for physiological activity14. Here we report the three-dimensional structure of human CXCR1 determined by NMR spectroscopy. The Receptor is in liquid crystalline phospholipid bilayers, without modification of its amino acid sequence and under physiological conditions. Features important for intracellular G-protein activation and signal transduction are revealed. The structure of human CXCR1 in a lipid bilayer should help to facilitate the discovery of new compounds that interact with GPCRs and combat diseases such as breast cancer.
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structure of the Chemokine Receptor CXCR1 in phospholipid bilayers
Nature, 2012Co-Authors: Sang Ho Park, Fabio Casagrande, Ye Tian, Henry J Nothnagel, Hans Kiefer, Klaus Maier, Mignon Chu, Bibhuti B Das, Anna A De AngelisAbstract:CXCR1 is one of two high-affinity Receptors for the CXC Chemokine interleukin-8 (IL-8), a major mediator of immune and inflammatory responses implicated in many disorders, including tumour growth. IL-8, released in response to inflammatory stimuli, binds to the extracellular side of CXCR1. The ligand-activated intracellular signalling pathways result in neutrophil migration to the site of inflammation. CXCR1 is a class A, rhodopsin-like G-protein-coupled Receptor (GPCR), the largest class of integral membrane proteins responsible for cellular signal transduction and targeted as drug Receptors. Despite its importance, the molecular mechanism of CXCR1 signal transduction is poorly understood owing to the limited structural information available. Recent structural determination of GPCRs has advanced by modifying the Receptors with stabilizing mutations, insertion of the protein T4 lysozyme and truncations of their amino acid sequences, as well as addition of stabilizing antibodies and small molecules that facilitate crystallization in cubic phase monoolein mixtures. The intracellular loops of GPCRs are crucial for G-protein interactions, and activation of CXCR1 involves both amino-terminal residues and extracellular loops. Our previous nuclear magnetic resonance studies indicate that IL-8 binding to the N-terminal residues is mediated by the membrane, underscoring the importance of the phospholipid bilayer for physiological activity. Here we report the three-dimensional structure of human CXCR1 determined by NMR spectroscopy. The Receptor is in liquid crystalline phospholipid bilayers, without modification of its amino acid sequence and under physiological conditions. Features important for intracellular G-protein activation and signal transduction are revealed. The structure of human CXCR1 in a lipid bilayer should help to facilitate the discovery of new compounds that interact with GPCRs and combat diseases such as breast cancer.
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optimization of purification and refolding of the human Chemokine Receptor CXCR1 improves the stability of proteoliposomes for structure determination
Biochimica et Biophysica Acta, 2012Co-Authors: Sang Ho Park, Fabio Casagrande, Hans Kiefer, Klaus Maier, Mignon Chu, Stanley J OpellaAbstract:The human Chemokine Receptor CXCR1 is a G-protein coupled Receptor that has been successfully expressed in E. coli as inclusion bodies, and purified and refolded in multi-milligram quantities required for structural studies. Expression in E. coli enables selective and uniform isotopic labeling with (13)C and (15)N for NMR studies. Long-term chemical and conformational stability and oligomeric homogeneity of CXCR1 in phospholipid bilayers are crucial for structural studies under physiological conditions. Here we describe substantial refinements in our previously described purification and reconstitution procedures for CXCR1 in phospholipid bilayers. These refinements have led to the preparation of highly purified, completely monomeric, proteoliposome samples that are stable for months at 35°C while subject to the high power radiofrequency irradiations of solid-state NMR experiments. The principal changes from the previously described methods include: 1) ensure that CXCR1 is pure and homogeneously monomeric within the limits of detection (>98%); 2) monitor and control the pH at all times especially following the addition of TCEP, which serves as a reducing agent but also changes the pH; 3) slowly refold CXCR1 with the complete removal of all traces of SDS using a KCl precipitation/dialysis method; and 4) ensure that the molar ratio between the CXCR1 and the phospholipids does not change during refolding and detergent removal. NMR samples prepared with these protocols yield reproducible results over a period of many months at 35°C. This purification and refolding protocol is likely to be applicable with minimal changes to other GPCRs as well as other membrane proteins.
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the structure of the Chemokine Receptor CXCR1 in phospholipid bilayers and interactions with il 8
Biophysical Journal, 2012Co-Authors: Sang Ho Park, Fabio Casagrande, Ye Tian, Henry J Nothnagel, Francesca M Marassi, Mignon Chu, Bibhuti B Das, Jasmina Racic, Hans KieferAbstract:CXCR1 is a Receptor for the Chemokine interleukin-8 (IL-8), a mediator of immune and inflammatory responses. Strategically located in the cell membrane, CXCR1 binds to IL-8 with high affinity, and subsequently transduces a signal across the membrane bilayer to a G-protein activated second messenger system. Here, we describe the three-dimensional structure of human CXCR1 determined in phospholipid bilayers under physiological conditions by solid-state NMR spectroscopy. We use a five-step approach: (1) prepare a sample of a uniformly 13C/15N labeled CXCR1 in proteoliposomes by bacterial expression, purification, and refolding. (2) Resolve individual signals with MAS solid-state NMR experiments. (3) Assign each signal to a specific residue. (4) Measure two or more orientation-dependent frequencies for each residue and some distances. (5) Calculate de novo three-dimensional structure. The availability of a method for determining the structures of unmodified GPCRs in their native environment of phospholipid bilayers under physiological conditions has the potential to transform the field of structural determination of membrane proteins, and to accelerate the discovery of drugs that interact with membrane-associated Receptors. Progress towards determining the structure and function of IL-8 bound to CXCR1 in membrane environments will be described.
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interactions of interleukin 8 with the human Chemokine Receptor CXCR1 in phospholipid bilayers by nmr spectroscopy
Journal of Molecular Biology, 2011Co-Authors: Sang Ho Park, Fabio Casagrande, Leah Cho, Lauren V Albrecht, Stanley J OpellaAbstract:CXCR1 is a Receptor for the Chemokine interleukin-8 (IL-8), a mediator of immune and inflammatory responses. Strategically located in the cell membrane, CXCR1 binds to IL-8 with high affinity and subsequently transduces a signal across the membrane bilayer to a G-protein-activated second messenger system. Here, we describe NMR studies of the interactions between IL-8 and human CXCR1 in lipid environments. Functional full-length and truncated constructs of CXCR1 and full-length IL-8 were uniformly (15)N-labeled by expression in bacteria followed by purification and refolding. The residues responsible for interactions between IL-8 and the N-terminal domain of CXCR1 were identified by specific chemical shift perturbations of assigned resonances on both IL-8 and CXCR1. Solution NMR signals from IL-8 in q=0.1 isotropic bicelles disappeared completely when CXCR1 in lipid bilayers was added in a 1:1 molar ratio, indicating that binding to the Receptor-containing bilayers immobilizes IL-8 (on the ~10(5) Hz timescale) and broadens the signals beyond detection. The same solution NMR signals from IL-8 were less affected by the addition of N-terminal truncated CXCR1 in lipid bilayers, demonstrating that the N-terminal domain of CXCR1 is mainly responsible for binding to IL-8. The interaction is tight enough to immobilize IL-8 along with the Receptor in phospholipid bilayers and is specific enough to result in well-aligned samples in oriented sample solid-state NMR spectra. A combination of solution NMR and solid-state NMR studies of IL-8 in the presence of various constructs of CXCR1 enables us to propose a model for the multistep binding process.
Durba Sengupta - One of the best experts on this subject based on the ideXlab platform.
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conformational plasticity and dynamic interactions of the n terminal domain of the Chemokine Receptor CXCR1
PLOS Computational Biology, 2021Co-Authors: Shalmali Kharche, Amitabha Chattopadhyay, Manali Joshi, Durba SenguptaAbstract:The dynamic interactions between G protein-coupled Receptors (GPCRs) and their cognate protein partners are central to several cell signaling pathways. For example, the association of CXC Chemokine Receptor 1 (CXCR1) with its cognate Chemokine, interleukin-8 (IL8 or CXCL8) initiates pathways leading to neutrophil-mediated immune responses. The N-terminal domain of Chemokine Receptors confers ligand selectivity, but unfortunately the conformational dynamics of this intrinsically disordered region remains unresolved. In this work, we have explored the interaction of CXCR1 with IL8 by microsecond time scale coarse-grain simulations, complemented by atomistic models and NMR chemical shift predictions. We show that the conformational plasticity of the apo-Receptor N-terminal domain is restricted upon ligand binding, driving it to an open C-shaped conformation. Importantly, we corroborated the dynamic complex sampled in our simulations against chemical shift perturbations reported by previous NMR studies and show that the trends are similar. Our results indicate that chemical shift perturbation is often not a reporter of residue contacts in such dynamic associations. We believe our results represent a step forward in devising a strategy to understand intrinsically disordered regions in GPCRs and how they acquire functionally important conformational ensembles in dynamic protein-protein interfaces.
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conformational plasticity and dynamic interactions of the n terminal domain of the Chemokine Receptor CXCR1
bioRxiv, 2020Co-Authors: Shalmali Kharche, Amitabha Chattopadhyay, Manali Joshi, Durba SenguptaAbstract:Dynamic interactions between G protein-coupled Receptors (GPCRs) and their cognate protein partners at the membrane interface control several cellular signaling pathways. An important example is the association of CXC Chemokine Receptor 1 (CXCR1) with its cognate Chemokine, interleukin-8 (IL8 or CXCL8) that regulates neutrophil-mediated immune responses. Although the N-terminal domain of the Receptor is known to confer ligand selectivity, the conformational dynamics of this intrinsically disordered region of CXCR1 in particular, and Chemokine Receptors in general, remains unresolved. In this work, we have explored the interaction of CXCR1 with IL8 by microsecond time scale coarse-grain simulations that were validated by atomistic models and NMR chemical shift predictions. We show that the conformational plasticity of the apo -Receptor N-terminal region is restricted upon ligand binding, driving it to an open C-shaped conformation. Importantly, we validated the dynamic complex sampled in our simulations against chemical shift perturbations reported by previous NMR studies. Our results indicate that caution should be exercised when chemical shift perturbation is used as a reporter of residue contacts in such dynamic associations. We believe our results represent a step forward in devising a strategy to understand intrinsically disordered regions in GPCRs and how they acquire functionally important conformational ensembles in dynamic protein-protein interfaces.
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membrane induced organization and dynamics of the n terminal domain of Chemokine Receptor CXCR1 insights from atomistic simulations
Chemistry and Physics of Lipids, 2017Co-Authors: Shalmali Kharche, Manali Joshi, Durba Sengupta, Amitabha ChattopadhyayAbstract:The CXC Chemokine Receptor 1 (CXCR1) is an important member of the G protein-coupled Receptor (GPCR) family in which the extracellular N-terminal domain has been implicated in ligand binding and selectivity. The structure of this domain has not yet been elucidated due to its inherent dynamics, but experimental evidence points toward membrane-dependent organization and dynamics. To gain molecular insight into the interaction of the N-terminal domain with the membrane bilayer, we performed a series of microsecond time scale atomistic simulations of the N-terminal domain of CXCR1 in the presence and absence of POPC bilayers. Our results show that the peptide displays a high propensity to adopt a β-sheet conformation in the presence of the membrane bilayer. The interaction of the peptide with the membrane bilayer was found to be transient in our simulations. Interestingly, a scrambled peptide, containing the same residues in a randomly varying sequence, did not exhibit membrane-modulated structural dynamics. These results suggest that sequence-dependent electrostatics, modulated by the membrane, could play an important role in folding of the N-terminal domain. We believe that our results reinforce the emerging paradigm that cellular membranes could be important modulators of function of G protein-coupled Receptors such as CXCR1.