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The plasma membrane (PM) is composed of a complex lipid mixture that forms heterogeneous membrane environments. Yet, how small-scale lipid organization controls physiological events at the PM remains largely unknown. Here, we show that ORP-related Osh lipid exchange proteins are critical for the synthesis of phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2], a key regulator of dynamic events at the PM. In real-time assays, we find that unsaturated phosphatidylserine (PS) and sterols, both Osh protein ligands, synergistically stimulate phosphatidylinositol 4-phosphate 5-kinase (PIP5K) activity. Biophysical FRET analyses suggest an unconventional co-distribution of unsaturated PS and phosphatidylinositol 4-phosphate (PI4P) species in sterol-containing membrane bilayers. Moreover, using in vivo imaging approaches and molecular dynamics simulations, we show that Osh protein-mediated unsaturated PI4P and PS membrane lipid organization is sensed by the PIP5K specificity loop. Thus, ORP family members create a nanoscale membrane lipid environment that drives PIP5K activity and PI(4,5)P2 synthesis that ultimately controls global PM organization and dynamics.
Cysteine cross-linking in native membranes establishes the transmembrane architecture of Ire1
(2021)
The ER is a key organelle of membrane biogenesis and crucial for the folding of both membrane and secretory proteins. Sensors of the unfolded protein response (UPR) monitor the unfolded protein load in the ER and convey effector functions for maintaining ER homeostasis. Aberrant compositions of the ER membrane, referred to as lipid bilayer stress, are equally potent activators of the UPR. How the distinct signals from lipid bilayer stress and unfolded proteins are processed by the conserved UPR transducer Ire1 remains unknown. Here, we have generated a functional, cysteine-less variant of Ire1 and performed systematic cysteine cross-linking experiments in native membranes to establish its transmembrane architecture in signaling-active clusters. We show that the transmembrane helices of two neighboring Ire1 molecules adopt an X-shaped configuration independent of the primary cause for ER stress. This suggests that different forms of stress converge in a common, signaling-active transmembrane architecture of Ire1.
Complex I couples the free energy released from quinone (Q) reduction to pump protons across the biological membrane in the respiratory chains of mitochondria and many bacteria. The Q reduction site is separated by a large distance from the proton-pumping membrane domain. To address the molecular mechanism of this long-range proton-electron coupling, we perform here full atomistic molecular dynamics simulations, free energy calculations, and continuum electrostatics calculations on complex I from Thermus thermophilus. We show that the dynamics of Q is redox-state-dependent, and that quinol, QH2, moves out of its reduction site and into a site in the Q tunnel that is occupied by a Q analog in a crystal structure of Yarrowia lipolytica. We also identify a second Q-binding site near the opening of the Q tunnel in the membrane domain, where the Q headgroup forms strong interactions with a cluster of aromatic and charged residues, while the Q tail resides in the lipid membrane. We estimate the effective diffusion coefficient of Q in the tunnel, and in turn the characteristic time for Q to reach the active site and for QH2 to escape to the membrane. Our simulations show that Q moves along the Q tunnel in a redox-state-dependent manner, with distinct binding sites formed by conserved residue clusters. The motion of Q to these binding sites is proposed to be coupled to the proton-pumping machinery in complex I.
The structure and flexibility of RNA depends sensitively on the microenvironment. Using pulsed electron-electron double-resonance (PELDOR)/double electron-electron resonance (DEER) spectroscopy combined with advanced labeling techniques, we show that the structure of double-stranded RNA (dsRNA) changes upon internalization into Xenopus lævis oocytes. Compared to dilute solution, the dsRNA A-helix is more compact in cells. We recapitulate this compaction in a densely crowded protein solution. Atomic-resolution molecular dynamics simulations of dsRNA semi-quantitatively capture the compaction, and identify non-specific electrostatic interactions between proteins and dsRNA as a possible driver of this effect.
Maximum likelihood estimates of diffusion coefficients from single-particle tracking experiments
(2021)
Single-molecule localization microscopy allows practitioners to locate and track labeled molecules in biological systems. When extracting diffusion coefficients from the resulting trajectories, it is common practice to perform a linear fit on mean-squared-displacement curves. However, this strategy is suboptimal and prone to errors. Recently, it was shown that the increments between the observed positions provide a good estimate for the diffusion coefficient, and their statistics are well-suited for likelihood-based analysis methods. Here, we revisit the problem of extracting diffusion coefficients from single-particle tracking experiments subject to static noise and dynamic motion blur using the principle of maximum likelihood. Taking advantage of an efficient real-space formulation, we extend the model to mixtures of subpopulations differing in their diffusion coefficients, which we estimate with the help of the expectation–maximization algorithm. This formulation naturally leads to a probabilistic assignment of trajectories to subpopulations. We employ the theory to analyze experimental tracking data that cannot be explained with a single diffusion coefficient. We test how well a dataset conforms to the assumptions of a diffusion model and determine the optimal number of subpopulations with the help of a quality factor of known analytical distribution. To facilitate use by practitioners, we provide a fast open-source implementation of the theory for the efficient analysis of multiple trajectories in arbitrary dimensions simultaneously.
Cells maintain membrane fluidity by regulating lipid saturation, but the molecular mechanisms of this homeoviscous adaptation remain poorly understood. We have reconstituted the core machinery for regulating lipid saturation in baker’s yeast to study its molecular mechanism. By combining molecular dynamics simulations with experiments, we uncover a remarkable sensitivity of the transcriptional regulator Mga2 to the abundance, position, and configuration of double bonds in lipid acyl chains, and provide insights into the molecular rules of membrane adaptation. Our data challenge the prevailing hypothesis that membrane fluidity serves as the measured variable for regulating lipid saturation. Rather, we show that Mga2 senses the molecular lipid-packing density in a defined region of the membrane. Our findings suggest that membrane property sensors have evolved remarkable sensitivities to highly specific aspects of membrane structure and dynamics, thus paving the way toward the development of genetically encoded reporters for such properties in the future.
Proton-powered c-ring rotation in mitochondrial ATP synthase is crucial to convert the transmembrane protonmotive force into torque to drive the synthesis of ATP. Capitalizing on recent cryo-EM structures, we aim at a structural and energetic understanding of how functional directional rotation is achieved. We performed multi-microsecond atomistic simulations to determine the free energy profiles along the c-ring rotation angle before and after the arrival of a new proton. Our results reveal that rotation proceeds by dynamic sliding of the ring over the a-subunit surface, during which interactions with conserved polar residues stabilize distinct intermediates. Ordered water chains line up for a Grotthuss-type proton transfer in one of these intermediates. After proton transfer, a high barrier prevents backward rotation and an overall drop in free energy favors forward rotation, ensuring the directionality of c-ring rotation required for the thermodynamically disfavored ATP synthesis. The essential arginine of the a-subunit stabilizes the rotated configuration through a salt-bridge with the c-ring. Overall, we describe a complete mechanism for the rotation step of the ATP synthase rotor, thereby illuminating a process critical to all life at atomic resolution.
Proton-powered c-ring rotation in mitochondrial ATP synthase is crucial to convert the transmembrane protonmotive force into torque to drive the synthesis of ATP. Capitalizing on recent cryo-EM structures, we aim at a structural and energetic understanding of how functional directional rotation is achieved. We performed multi-microsecond atomistic simulations to determine the free energy profiles along the c-ring rotation angle before and after the arrival of a new proton. Our results reveal that rotation proceeds by dynamic sliding of the ring over the a-subunit surface, during which interactions with conserved polar residues stabilize distinct intermediates. Ordered water chains line up for a Grotthuss-type proton transfer in one of these intermediates. After proton transfer, a high barrier prevents backward rotation and an overall drop in free energy favors forward rotation, ensuring the directionality of c-ring rotation required for the thermodynamically disfavored ATP synthesis. The essential arginine of the a-subunit stabilizes the rotated configuration through a salt-bridge with the c-ring. Overall, we describe a complete mechanism for the rotation step of the ATP synthase rotor, thereby illuminating a process critical to all life at atomic resolution.
Cryo-electron tomography (cryo-ET) is a powerful method to elucidate subcellular architecture and to structurally analyse biomolecules in situ by subtomogram averaging (STA). Specimen thickness is a key factor affecting cryo-ET data quality. Cells that are too thick for transmission imaging can be thinned by cryo-focused-ion-beam (cryo-FIB) milling. However, optimal specimen thickness for cryo-ET on lamellae has not been systematically investigated. Furthermore, the ions used to ablate material can cause damage in the lamellae, thereby reducing STA resolution. Here, we systematically benchmark the resolution depending on lamella thickness and the depth of the particles within the sample. Up to ca. 180 nm, lamella thickness does not negatively impact resolution. This shows that there is no need to generate very thin lamellae and thickness can be chosen such that it captures major cellular features. Furthermore, we show that gallium-ion-induced damage extends to depths of up to 30 nm from either lamella surface.
Cryo-electron tomography (CryoET) resolves individual macromolecules inside living cells. However, the complex composition and high density of cells challenge the faithful identification of features in tomograms. Here, we capitalize on recent advances in electron tomography and demonstrate that 3D template matching (TM) localizes a wide range of structures inside crowded eukaryotic cells with confidence 10 to 100-fold above the noise level. We establish a TM pipeline with systematically tuned parameters for automated, objective and comprehensive feature identification. High-fidelity and high-confidence localizations of nuclear pore complexes, vaults, ribosomes, proteasomes, lipid membranes and microtubules, and individual subunits, demonstrate that TM is generic. We resolve ~100-kDa proteins, connect the functional states of complexes to their cellular localization, and capture vaults carrying ribosomal cargo in situ. By capturing individual molecular events inside living cells with defined statistical confidence, high-confidence TM greatly speeds up the CryoET workflow and sets the stage for visual proteomics.