Elucidating the structural features of ABCA1 in its heterogeneous membrane environment.

ABCA1 plays an integral part in Reverse Cholesterol Transport (RCT) and is critical for maintaining lipid homeostasis. One theory of lipid efflux by the transporter (alternating access) proposes that ABCA1 harbors two different conformations that provide alternate access for lipid binding and release, leading to sequestration via a direct interaction between ABCA1 and its partner, ApoA1. The alternative theory (lateral access) proposes that ABCA1 obtains lipids laterally from the membrane to form a temporary extracellular reservoir containing an isolated pressurized lipid monolayer caused by the net accumulation of lipids in the exofacial leaflet. Recently, a full-length Cryo-EM structure of this 2,261-residue transmembrane protein showed its discreetly folded domains and conformations, as well as detected the presence of a tunnel enclosed within ECDs. While the tunnel was wide enough at the proximal end for accommodating passage of lipids, the distal end displayed substantial narrowing, making it inaccessible for ApoA1. Therefore, this structure was hypothesized to substantiate the lateral access theory, whereby ApoA1 obtained lipids from the proximal end. Utilizing long time-scale multiple replica atomistic molecular dynamics simulations (MDS), we simulated the structure in a heterogeneous lipid environment and found that along with several large conformational changes, the protein widens enough at the distal end of its ECD tunnel to now enable lipid accommodation. In this study we have characterized ABCA1 and the lipid dynamics along with the protein-lipid interactions in the heterogeneous environment, providing novel insights into understanding ABCA1 conformation at an atomistic level.

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Lipid-protein interactions are unique fingerprints for membrane proteins

10.1101/191486 ◽

2017 ◽

Author(s):

Valentina Corradi ◽

Eduardo Mendez-Villuendas ◽

Helgi I. Ingólfsson ◽

Ruo-Xu Gu ◽

Iwona Siuda ◽

...

Keyword(s):

Membrane Proteins ◽

Protein Interactions ◽

Cell Membranes ◽

Spatiotemporal Resolution ◽

Lipid Dynamics ◽

Lipid Species ◽

Lipid Environment ◽

Lipid Protein Interactions ◽

Dynamics Simulations ◽

Asymmetrically Distributed

ABSTRACTCell membranes contain hundreds of different proteins and lipids in an asymmetric arrangement. Understanding the lateral organization principles of these complex mixtures is essential for life and health. However, our current understanding of the detailed organization of cell membranes remains rather elusive, owing to the lack of experimental methods suitable for studying these fluctuating nanoscale assemblies of lipids and proteins with the required spatiotemporal resolution. Here, we use molecular dynamics simulations to characterize the lipid environment of ten 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 leaflets. The simulations detail how each protein modulates its local lipid environment through local lipid composition, thickness, curvature and lipid dynamics. Our results provide a molecular glimpse of the complexity of lipid-protein interactions, with potentially far reaching implications for the overall organization of the cell membrane.

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Multimodal tubulin binding by the yeast kinesin-8, Kip3, underlies its motility and depolymerization

10.1101/2021.10.12.464151 ◽

2021 ◽

Author(s):

Hugo Arellano-Santoyo ◽

Rogelio A Hernandez-Lopez ◽

Emma Stokasimov ◽

Ray YR Wang ◽

David Pellman ◽

...

Keyword(s):

Molecular Dynamics ◽

Single Molecule ◽

Conformational Changes ◽

Intracellular Transport ◽

Structural Features ◽

Cellular Processes ◽

Tubulin Binding ◽

Dynamics Simulations ◽

Spatiotemporal Control ◽

Unique Ability

The microtubule (MT) cytoskeleton is central to cellular processes including axonal growth, intracellular transport, and cell division, all of which rely on precise spatiotemporal control of MT organization. Kinesin-8s play a key role in regulating MT length by combining highly processive directional motility with MT-end disassembly. However, how kinesin-8 switches between these two apparently opposing activities remains unclear. Here, we define the structural features underlying this molecular switch through cryo-EM analysis of the yeast kinesin-8, Kip3 bound to MTs, and molecular dynamics simulations to approximate the complex of Kip3 with the curved tubulin state found at the MT plus-end. By integrating biochemical and single-molecule biophysical assays, we identified specific intra- and intermolecular interactions that modulate processive motility and MT disassembly. Our findings suggest that Kip3 undergoes conformational changes in response to tubulin curvature that underlie its unique ability to interact differently with the MT lattice than with the MT-end.

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An improved open-channel structure of MscL determined from FRET confocal microscopy and simulation

The Journal of General Physiology ◽

10.1085/jgp.200910376 ◽

2010 ◽

Vol 136 (4) ◽

pp. 483-494 ◽

Cited By ~ 64

Author(s):

Ben Corry ◽

Annette C. Hurst ◽

Prithwish Pal ◽

Takeshi Nomura ◽

Paul Rigby ◽

...

Keyword(s):

Patch Clamp ◽

Conformational Changes ◽

Brownian Dynamics ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Channel Structure ◽

Paramagnetic Resonance ◽

Physiological Processes ◽

Lipid Environment ◽

Dynamics Simulations

Mechanosensitive channels act as molecular transducers of mechanical force exerted on the membrane of living cells by opening in response to membrane bilayer deformations occurring in physiological processes such as touch, hearing, blood pressure regulation, and osmoregulation. Here, we determine the likely structure of the open state of the mechanosensitive channel of large conductance using a combination of patch clamp, fluorescence resonance energy transfer (FRET) spectroscopy, data from previous electron paramagnetic resonance experiments, and molecular and Brownian dynamics simulations. We show that structural rearrangements of the protein can be measured in similar conditions as patch clamp recordings while controlling the state of the pore in its natural lipid environment by modifying the lateral pressure distribution via the lipid bilayer. Transition to the open state is less dramatic than previously proposed, while the N terminus remains anchored at the surface of the membrane where it can either guide the tilt of or directly translate membrane tension to the conformation of the pore-lining helix. Combining FRET data obtained in physiological conditions with simulations is likely to be of great value for studying conformational changes in a range of multimeric membrane proteins.

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Structural features and lipid binding domain of tubulin on biomimetic mitochondrial membranes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1619806114 ◽

2017 ◽

Vol 114 (18) ◽

pp. E3622-E3631 ◽

Cited By ~ 18

Author(s):

David P. Hoogerheide ◽

Sergei Y. Noskov ◽

Daniel Jacobs ◽

Lucie Bergdoll ◽

Vitalii Silin ◽

...

Keyword(s):

Electrochemical Impedance ◽

Physiological Role ◽

Structural Features ◽

Lipid Binding ◽

Binding Domain ◽

Water Soluble ◽

Mitochondrial Outer Membrane ◽

Mitochondrial Membranes ◽

Dynamics Simulations ◽

Integral Proteins

Dimeric tubulin, an abundant water-soluble cytosolic protein known primarily for its role in the cytoskeleton, is routinely found to be associated with mitochondrial outer membranes, although the structure and physiological role of mitochondria-bound tubulin are still unknown. There is also no consensus on whether tubulin is a peripheral membrane protein or is integrated into the outer mitochondrial membrane. Here the results of five independent techniques—surface plasmon resonance, electrochemical impedance spectroscopy, bilayer overtone analysis, neutron reflectometry, and molecular dynamics simulations—suggest that α-tubulin’s amphipathic helix H10 is responsible for peripheral binding of dimeric tubulin to biomimetic “mitochondrial” membranes in a manner that differentiates between the two primary lipid headgroups found in mitochondrial membranes, phosphatidylethanolamine and phosphatidylcholine. The identification of the tubulin dimer orientation and membrane-binding domain represents an essential step toward our understanding of the complex mechanisms by which tubulin interacts with integral proteins of the mitochondrial outer membrane and is important for the structure-inspired design of tubulin-targeting agents.

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Unique structural features of the AIPL1–FKBP domain that support prenyl lipid binding and underlie protein malfunction in blindness

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1704782114 ◽

2017 ◽

Vol 114 (32) ◽

pp. E6536-E6545 ◽

Cited By ~ 7

Author(s):

Ravi P. Yadav ◽

Lokesh Gakhar ◽

Liping Yu ◽

Nikolai O. Artemyev

Keyword(s):

Binding Pocket ◽

Structural Features ◽

Biochemical Properties ◽

Lipid Binding ◽

Severe Form ◽

Nmr Studies ◽

Interacting Protein ◽

Aryl Hydrocarbon ◽

Dynamics Simulations ◽

Fkbp Domain

FKBP-domain proteins (FKBPs) are pivotal modulators of cellular signaling, protein folding, and gene transcription. Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is a distinctive member of the FKBP superfamily in terms of its biochemical properties, and it plays an important biological role as a chaperone of phosphodiesterase 6 (PDE6), an effector enzyme of the visual transduction cascade. Malfunction of mutant AIPL1 proteins triggers a severe form of Leber congenital amaurosis and leads to blindness. The mechanism underlying the chaperone activity of AIPL1 is largely unknown, but involves the binding of isoprenyl groups on PDE6 to the FKBP domain of AIPL1. We solved the crystal structures of the AIPL1–FKBP domain and its pathogenic mutant V71F, both in the apo form and in complex with isoprenyl moieties. These structures reveal a module for lipid binding that is unparalleled within the FKBP superfamily. The prenyl binding is enabled by a unique “loop-out” conformation of the β4-α1 loop and a conformational “flip-out” switch of the key W72 residue. A second major conformation of apo AIPL1–FKBP was identified by NMR studies. This conformation, wherein W72 flips into the ligand-binding pocket and renders the protein incapable of prenyl binding, is supported by molecular dynamics simulations and appears to underlie the pathogenicity of the V71F mutant. Our findings offer critical insights into the mechanisms that underlie AIPL1 function in health and disease, and highlight the structural and functional diversity of the FKBPs.

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The membrane anchor of the transcriptional activator SREBP is characterized by intrinsic conformational flexibility

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1513782112 ◽

2015 ◽

Vol 112 (40) ◽

pp. 12390-12395 ◽

Cited By ~ 12

Author(s):

Rasmus Linser ◽

Nicola Salvi ◽

Rodolfo Briones ◽

Petra Rovó ◽

Bert L. de Groot ◽

...

Keyword(s):

Stress Responses ◽

Chemical Shifts ◽

Transmembrane Protein ◽

Regulatory Element ◽

Conformational Flexibility ◽

Therapeutic Interventions ◽

Solvent Exposure ◽

Element Binding Protein ◽

Lipid Environment ◽

Dynamics Simulations

Regulated intramembrane proteolysis (RIP) is a conserved mechanism crucial for numerous cellular processes, including signaling, transcriptional regulation, axon guidance, cell adhesion, cellular stress responses, and transmembrane protein fragment degradation. Importantly, it is relevant in various diseases including Alzheimer’s disease, cardiovascular diseases, and cancers. Even though a number of structures of different intramembrane proteases have been solved recently, fundamental questions concerning mechanistic underpinnings of RIP and therapeutic interventions remain. In particular, this includes substrate recognition, what properties render a given substrate amenable for RIP, and how the lipid environment affects the substrate cleavage. Members of the sterol regulatory element-binding protein (SREBP) family of transcription factors are critical regulators of genes involved in cholesterol/lipid homeostasis. After site-1 protease cleavage of the inactive SREBP transmembrane precursor protein, RIP of the anchor intermediate by site-2 protease generates the mature transcription factor. In this work, we have investigated the labile anchor intermediate of SREBP-1 using NMR spectroscopy. Surprisingly, NMR chemical shifts, site-resolved solvent exposure, and relaxation studies show that the cleavage site of the lipid-signaling protein intermediate bears rigid α-helical topology. An evolutionary conserved motif, by contrast, interrupts the secondary structure ∼9–10 residues C-terminal of the scissile bond and acts as an inducer of conformational flexibility within the carboxyl-terminal transmembrane region. These results are consistent with molecular dynamics simulations. Topology, stability, and site-resolved dynamics data suggest that the cleavage of the α-helical substrate in the case of RIP may be associated with a hinge motion triggered by the molecular environment.

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A Topological Data Analytic Approach for Discovering Biophysical Signatures in Protein Dynamics

10.1101/2021.07.28.454240 ◽

2021 ◽

Author(s):

Wai Shing Tang ◽

Gabriel Monteiro da Silva ◽

Henry Kirveslahti ◽

Erin Skeens ◽

Bibo Feng ◽

...

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulations ◽

Conformational Changes ◽

Protein Structures ◽

Structural Features ◽

Biologically Relevant ◽

Visual Identification ◽

Topological Features ◽

Protein Ensembles ◽

Dynamics Simulations

Identifying structural differences among proteins can be a non-trivial task. When contrasting ensembles of protein structures obtained from molecular dynamics simulations, biologically-relevant features can be easily overshadowed by spurious fluctuations. Here, we present SINATRA Pro, a computational pipeline designed to robustly identify topological differences between two sets of protein structures. Algorithmically, SINATRA Pro works by first taking in the 3D atomic coordinates for each protein snapshot and summarizing them according to their underlying topology. Statistically significant topological features are then projected back onto an user-selected representative protein structure, thus facilitating the visual identification of biophysical signatures of different protein ensembles. We assess the ability of SINATRA Pro to detect minute conformational changes in five independent protein systems of varying complexities. In all test cases, SINATRA Pro identifies known structural features that have been validated by previous experimental and computational studies, as well as novel features that are also likely to be biologically-relevant according to the literature. These results highlight SINATRA Pro as a promising method for facilitating the non-trivial task of pattern recognition in trajectories resulting from molecular dynamics simulations, with substantially increased resolution.

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Molecular Basis of Glucose Transport Mechanism in Plants

10.26434/chemrxiv.8049848.v1 ◽

2019 ◽

Cited By ~ 2

Author(s):

Balaji Selvam ◽

Ya-Chi Yu ◽

Liqing Chen ◽

Diwakar Shukla

Keyword(s):

Molecular Dynamics ◽

Free Energy ◽

Molecular Dynamics Simulations ◽

Conformational Changes ◽

Transport Mechanism ◽

Conformational Dynamics ◽

Site Directed Mutagenesis ◽

Free Energy Barrier ◽

Transport Cycle ◽

Dynamics Simulations

<p>The SWEET family belongs to a class of transporters in plants that undergoes large conformational changes to facilitate transport of sugar molecules across the cell membrane. However, the structures of their functionally relevant conformational states in the transport cycle have not been reported. In this study, we have characterized the conformational dynamics and complete transport cycle of glucose in OsSWEET2b transporter using extensive molecular dynamics simulations. Using Markov state models, we estimated the free energy barrier associated with different states as well as 1 for the glucose the transport mechanism. SWEETs undergoes structural transition to outward-facing (OF), Occluded (OC) and inward-facing (IF) and strongly support alternate access transport mechanism. The glucose diffuses freely from outside to inside the cell without causing major conformational changes which means that the conformations of glucose unbound and bound snapshots are exactly same for OF, OC and IF states. We identified a network of hydrophobic core residues at the center of the transporter that restricts the glucose entry to the cytoplasmic side and act as an intracellular hydrophobic gate. The mechanistic predictions from molecular dynamics simulations are validated using site-directed mutagenesis experiments. Our simulation also revealed hourglass like intermediate states making the pore radius narrower at the center. This work provides new fundamental insights into how substrate-transporter interactions actively change the free energy landscape of the transport cycle to facilitate enhanced transport activity.</p>

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pH-Dependent Thermal Stability of Vibrio cholerae L-asparaginase

Protein and Peptide Letters ◽

10.2174/0929866526666190617092944 ◽

2019 ◽

Vol 26 (10) ◽

pp. 743-750 ◽

Cited By ~ 1

Author(s):

Remya Radha ◽

Sathyanarayana N. Gummadi

Keyword(s):

Vibrio Cholerae ◽

Conformational Changes ◽

Kinetic Studies ◽

Structural Features ◽

Structure Stability ◽

Kcal Mole ◽

Scanning Calorimetry ◽

Wide Range ◽

Ph Dependent ◽

Ph Range

Background:pH is one of the decisive macromolecular properties of proteins that significantly affects enzyme structure, stability and reaction rate. Change in pH may protonate or deprotonate the side group of aminoacid residues in the protein, thereby resulting in changes in chemical and structural features. Hence studies on the kinetics of enzyme deactivation by pH are important for assessing the bio-functionality of industrial enzymes. L-asparaginase is one such important enzyme that has potent applications in cancer therapy and food industry.Objective:The objective of the study is to understand and analyze the influence of pH on deactivation and stability of Vibrio cholerae L-asparaginase.Methods:Kinetic studies were conducted to analyze the effect of pH on stability and deactivation of Vibrio cholerae L-asparaginase. Circular Dichroism (CD) and Differential Scanning Calorimetry (DSC) studies have been carried out to understand the pH-dependent conformational changes in the secondary structure of V. cholerae L-asparaginase.Results:The enzyme was found to be least stable at extreme acidic conditions (pH< 4.5) and exhibited a gradual increase in melting temperature from 40 to 81 °C within pH range of 4.0 to 7.0. Thermodynamic properties of protein were estimated and at pH 7.0 the protein exhibited ΔG37of 26.31 kcal mole-1, ΔH of 204.27 kcal mole-1 and ΔS of 574.06 cal mole-1 K-1.Conclusion:The stability and thermodynamic analysis revealed that V. cholerae L-asparaginase was highly stable over a wide range of pH, with the highest stability in the pH range of 5.0–7.0.

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