RESUMO
The SLC4 family of secondary bicarbonate transporters is responsible for the transport of HCO3-, CO32-, Cl-, Na+, K+, NH3, and H+, which are necessary for regulation of pH and ion homeostasis. They are widely expressed in numerous tissues throughout the body and function in different cell types with different membrane properties. Potential lipid roles in SLC4 function have been reported in experimental studies, focusing mostly on two members of the family: AE1 (Cl-/HCO3- exchanger) and NBCe1 (Na+-CO32-cotransporter). Previous computational studies of the outward-facing state of AE1 with model lipid membranes revealed enhanced protein-lipid interactions between cholesterol (CHOL) and phosphatidylinositol bisphosphate (PIP2). However, the protein-lipid interactions in other members of the family and other conformation states are still poorly understood and this precludes the detailed studies of a potential regulatory role for lipids in the SLC4 family. In this work, we performed coarse-grained and atomistic molecular dynamics simulations on three members of the SLC4 family with different transport modes: AE1, NBCe1, and NDCBE (an Na+-CO32-/Cl- exchanger), in model HEK293 membranes consisting of CHOL, PIP2, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin. The recently resolved inward-facing state of AE1 was also included in the simulations. Lipid-protein contact analysis of the simulated trajectories was performed with the ProLint server, which provides a multitude of visualization tools for illustration of areas of enhanced lipid-protein contact and identification of putative lipid binding sites within the protein matrix. We observed enrichment of CHOL and PIP2 around all proteins with subtle differences in their distribution depending on the protein type and conformation state. Putative binding sites were identified for CHOL, PIP2, phosphatidylcholine, and sphingomyelin in the three studied proteins, and their potential roles in the SLC4 transport function, conformational transition, and protein dimerization are discussed.
Assuntos
Simulação de Dinâmica Molecular , Ligação Proteica , Simportadores de Sódio-Bicarbonato/metabolismo , Simportadores de Sódio-Bicarbonato/química , Humanos , Colesterol/metabolismo , Colesterol/químicaRESUMO
The functional activity of membrane proteins is carried out in a complex lipid environment. Increasingly, it is becoming clear that lipids are an important player in regulating or generally modulating their activity. A routinely used method to gain insight into this interplay between lipids and proteins are Molecular Dynamics (MD) simulations, since they allow us to study interactions at atomic or near-atomic detail as a function of time. A major bottleneck, however, is analyzing and visualizing lipid-protein interactions, which, in practice, is a time-demanding task. Here, we present ProLint (www.prolint.ca), a webserver that completely automates analysis of MD generated files and visualization of lipid-protein interactions. Analysis is modular allowing users to select their preferred method, and visualization is entirely interactive through custom built applications that enable a detailed qualitative and quantitative exploration of lipid-protein interactions. ProLint also includes a database of published MD results that have been processed through the ProLint workflow and can be visualized by anyone regardless of their level of experience with MD. The automated analysis, feature-rich visualization, database integration, and open-source distribution with an easy to install process, will allow ProLint to become a routine workflow in lipid-protein interaction studies.
Assuntos
Lipídeos/química , Proteínas de Membrana/química , Simulação de Dinâmica Molecular , Software , Colesterol/química , Internet , Fosfatidilinositóis/química , Receptores Acoplados a Proteínas G/química , Receptor Smoothened/químicaRESUMO
Membrane lipids interact with proteins in a variety of ways, ranging from providing a stable membrane environment for proteins to being embedded in to detailed roles in complicated and well-regulated protein functions. Experimental and computational advances are converging in a rapidly expanding research area of lipid-protein interactions. Experimentally, the database of high-resolution membrane protein structures is growing, as are capabilities to identify the complex lipid composition of different membranes, to probe the challenging time and length scales of lipid-protein interactions, and to link lipid-protein interactions to protein function in a variety of proteins. Computationally, more accurate membrane models and more powerful computers now enable a detailed look at lipid-protein interactions and increasing overlap with experimental observations for validation and joint interpretation of simulation and experiment. Here we review papers that use computational approaches to study detailed lipid-protein interactions, together with brief experimental and physiological contexts, aiming at comprehensive coverage of simulation papers in the last five years. Overall, a complex picture of lipid-protein interactions emerges, through a range of mechanisms including modulation of the physical properties of the lipid environment, detailed chemical interactions between lipids and proteins, and key functional roles of very specific lipids binding to well-defined binding sites on proteins. Computationally, despite important limitations, molecular dynamics simulations with current computer power and theoretical models are now in an excellent position to answer detailed questions about lipid-protein interactions.
Assuntos
Membrana Celular/química , Membrana Celular/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Simulação por Computador , Humanos , Canais Iônicos/química , Canais Iônicos/metabolismo , Modelos Biológicos , Modelos Moleculares , Simulação de Acoplamento Molecular , Conformação Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismoRESUMO
G protein-coupled receptors (GPCRs) are membrane-bound proteins that depend on their lipid environment to carry out their physiological function. Combined efforts from many theoretical and experimental studies on the lipid-protein interaction profile of several GPCRs hint at an intricate relationship of these receptors with their surrounding membrane environment, with several lipids emerging as particularly important. Using coarse-grained molecular dynamics simulations, we explore the lipid-protein interaction profiles of 28 different GPCRs, spanning different levels of classification and conformational states and totaling to 1 ms of simulation time. We find a close relationship with lipids for all GPCRs simulated, in particular, cholesterol and phosphatidylinositol phosphate (PIP) lipids, but the number, location, and estimated strength of these interactions is dependent on the specific GPCR as well as its conformational state. Although both cholesterol and PIP lipids bind specifically to GPCRs, they utilize distinct mechanisms. Interactions with PIP lipids are mediated by charge-charge interactions with intracellular loop residues and stabilized by one or both of the transmembrane helices linked by the loop. Interactions with cholesterol, on the other hand, are mediated by a hydrophobic environment, usually made up of residues from more than one helix, capable of accommodating its ring structure and stabilized by interactions with aromatic and charged/polar residues. Cholesterol binding to GPCRs occurs in a small number of sites, some of which (like the binding site on the extracellular side of transmembrane 6/7) are shared among many class A GPCRs. Combined with a thorough investigation of the local membrane structure, our results provide a detailed picture of GPCR-lipid interactions. Additionally, we provide an accompanying website to interactively explore the lipid-protein interaction profile of all GPCRs simulated to facilitate analysis and comparison of our data.
Assuntos
Simulação de Dinâmica Molecular , Receptores Acoplados a Proteínas G , Sítios de Ligação , Proteínas de Ligação ao GTP , Lipídeos , Ligação Proteica , Receptores Acoplados a Proteínas G/metabolismoRESUMO
The SLC4 family of secondary bicarbonate transporters is responsible for the transport of HCO 3 -, CO 3 2- , Cl - , Na + , K + , NH 3 and H + necessary for regulation of pH and ion homeostasis. They are widely expressed in numerous tissues throughout the body and function in different cell types with different membrane properties. Potential lipid roles in SLC4 function have been reported in experimental studies, focusing mostly on two members of the family: AE1 (Cl - /HCO 3 - exchanger) and NBCe1 (Na + -CO 3 2- cotransporter). Previous computational studies of the outward facing (OF) state of AE1 with model lipid membranes revealed enhanced protein-lipid interactions between cholesterol (CHOL) and phosphatidylinositol bisphosphate (PIP2). However, the protein-lipid interactions in other members of the family and other conformation states are still poorly understood and this precludes the detailed studies of a potential regulatory role for lipids in the SLC4 family. In this work, we performed multiple 50 µs coarse-grained molecular dynamics simulations on three members of the SLC4 family with different transport modes: AE1, NBCe1 and NDCBE (a Na + -CO 3 2- /Cl - exchanger), in model HEK293 membranes consisting of CHOL, PIP2, phosphatidylcholine (POPC), phosphatidylethanolamine (POPE), phosphatidylserine (POPS), and sphingomyelin (POSM). The recently resolved inward-facing (IF) state of AE1 was also included in the simulations. Lipid-protein contact analysis of the simulated trajectories was performed with the ProLint server, which provides a multitude of visualization tools for illustration of areas of enhanced lipid-protein contact and identification of putative lipid binding sites within the protein matrix. We observed enrichment of CHOL and PIP2 around all proteins with subtle differences in their distribution depending on the protein type and conformation state. Putative binding sites were identified for CHOL, PIP2, POPC, and POSM in the three studied proteins and their potential roles in the SLC4 transport function, conformational transition and protein dimerization were discussed. Statement of significance: The SLC4 protein family is involved in critical physiological processes like pH and blood pressure regulation and maintenance of ion homeostasis. Its members can be found in various tissues. A number of studies suggest possible lipid regulation of the SLC4 function. However, the protein-lipid interactions in the SLC4 family are still poorly understood. Here we make use of long coarse-grained molecular dynamics simulations to assess the protein-lipid interactions in three SLC4 proteins with different transport modes, AE1, NBCe1, and NDCBE. We identify putative lipid binding sites for several lipid types of potential mechanistic importance, discuss them in the framework of the known experimental data and provide a necessary basis for further studies on lipid regulation of SLC4 function.
RESUMO
Recent advances in computational approaches and their integration into structural biology enable tackling increasingly complex questions. Here, we discuss several key areas, highlighting breakthroughs and remaining challenges. Theoretical modeling has provided tools to accurately predict and design protein structures on a scale currently difficult to achieve using experimental approaches. Molecular Dynamics simulations have become faster and more precise, delivering actionable information inaccessible by current experimental methods. Virtual screening workflows allow a high-throughput approach to discover ligands that bind and modulate protein function, while Machine Learning methods enable the design of proteins with new functionalities. Integrative structural biology combines several of these approaches, pushing the frontiers of structural and functional characterization to ever larger systems, advancing towards a complete understanding of the living cell. These breakthroughs will accelerate and significantly impact diverse areas of science.
Assuntos
Biologia Computacional , Proteínas , Proteínas/química , Simulação de Dinâmica MolecularRESUMO
The limited availability of cytokines in solid tumours hinders maintenance of the antitumour activity of chimeric antigen receptor (CAR) T cells. Cytokine receptor signalling pathways in CAR T cells can be activated by transgenic expression or injection of cytokines in the tumour, or by engineering the activation of cognate cytokine receptors. However, these strategies are constrained by toxicity arising from the activation of bystander cells, by the suboptimal biodistribution of the cytokines and by downregulation of the cognate receptor. Here we show that replacement of the extracellular domains of heterodimeric cytokine receptors in T cells with two leucine zipper motifs provides optimal Janus kinase/signal transducer and activator of transcription signalling. Such chimeric cytokine receptors, which can be generated for common γ-chain receptors, interleukin-10 and -12 receptors, enabled T cells to survive cytokine starvation without induction of autonomous cell growth, and augmented the effector function of CAR T cells in vitro in the setting of chronic antigen exposure and in human tumour xenografts in mice. As a modular design, leucine zippers can be used to generate constitutively active cytokine receptors in effector immune cells.
RESUMO
Cell membranes contain hundreds of different proteins and lipids in an asymmetric arrangement. Our current understanding of the detailed organization of cell membranes remains rather elusive, because of the challenge to study fluctuating nanoscale assemblies of lipids and proteins with the required spatiotemporal resolution. Here, we use molecular dynamics simulations to characterize the lipid environment of 10 different membrane proteins. To provide a realistic lipid environment, the proteins are embedded in a model plasma membrane, where more than 60 lipid species are represented, asymmetrically distributed between the leaflets. The simulations detail how each protein modulates its local lipid environment in a unique way, through enrichment or depletion of specific lipid components, resulting in thickness and curvature gradients. Our results provide a molecular glimpse of the complexity of lipid-protein interactions, with potentially far-reaching implications for our understanding of the overall organization of real cell membranes.