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Intrinsically disordered regions (IDRs) play a pivotal role in organellar remodeling. They transduce signals across membranes, scaffold signaling complexes, and mediate vesicular traffic. Their functions are regulated by constraining conformational ensembles through specific intra- and intermolecular interactions, physical tethering, and posttranslational modifications. The endoplasmic reticulum (ER)-phagy receptor FAM134B/RETREG1, known for its reticulon homology domain (RHD), includes a substantial C-terminal IDR housing the LC3 interacting motif. Beyond engaging the autophagic machinery, the function of the FAM134B-IDR is unclear. Here, we investigate the characteristics of the FAM134B-IDR by extensive modeling and molecular dynamics simulations. We present detailed structural models for the IDR, mapping its conformational landscape in solution and membrane-anchored configurations. Our analysis reveals that depending on the membrane anchor, the IDRs collapse onto the membrane and induce positive membrane curvature to varying degrees. The charge patterns underlying this Janus-like behavior are conserved across other ER-phagy receptors. We found that IDRs alone are sufficient to sense curvature. When combined with RHDs, they intensify membrane remodeling and drive efficient protein clustering, leading to faster budding, thereby amplifying RHD remodeling functions. Our simulations provide a perspective on IDRs of FAM134B, their Janus-like membrane interactions, and the resulting modulatory functions during large-scale ER remodeling., Competing Interests: Competing interests statement:The authors declare no competing interest.

Molecular self-organization driven by concerted many-body interactions produces the ordered structures that define both inanimate and living matter. Here we present an autonomous path sampling algorithm that integrates deep learning and transition path theory to discover the mechanism of molecular self-organization phenomena. The algorithm uses the outcome of newly initiated trajectories to construct, validate and-if needed-update quantitative mechanistic models. Closing the learning cycle, the models guide the sampling to enhance the sampling of rare assembly events. Symbolic regression condenses the learned mechanism into a human-interpretable form in terms of relevant physical observables. Applied to ion association in solution, gas-hydrate crystal formation, polymer folding and membrane-protein assembly, we capture the many-body solvent motions governing the assembly process, identify the variables of classical nucleation theory, uncover the folding mechanism at different levels of resolution and reveal competing assembly pathways. The mechanistic descriptions are transferable across thermodynamic states and chemical space., (© 2023. The Author(s).)

Degradation of the endoplasmic reticulum (ER) via selective autophagy (ER-phagy) is vital for cellular homeostasis. We identify FAM134A/RETREG2 and FAM134C/RETREG3 as ER-phagy receptors, which predominantly exist in an inactive state under basal conditions. Upon autophagy induction and ER stress signal, they can induce significant ER fragmentation and subsequent lysosomal degradation. FAM134A, FAM134B/RETREG1, and FAM134C are essential for maintaining ER morphology in a LC3-interacting region (LIR)-dependent manner. Overexpression of any FAM134 paralogue has the capacity to significantly augment the general ER-phagy flux upon starvation or ER-stress. Global proteomic analysis of FAM134 overexpressing and knockout cell lines reveals several protein clusters that are distinctly regulated by each of the FAM134 paralogues as well as a cluster of commonly regulated ER-resident proteins. Utilizing pro-Collagen I, as a shared ER-phagy substrate, we observe that FAM134A acts in a LIR-independent manner and compensates for the loss of FAM134B and FAM134C, respectively. FAM134C instead is unable to compensate for the loss of its paralogues. Taken together, our data show that FAM134 paralogues contribute to common and unique ER-phagy pathways., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Living cells constantly remodel the shape of their lipid membranes. In the endoplasmic reticulum (ER), the reticulon homology domain (RHD) of the reticulophagy regulator 1 (RETR1/FAM134B) forms dense autophagic puncta that are associated with membrane removal by ER-phagy. In molecular dynamics (MD) simulations, we find that FAM134B-RHD spontaneously forms clusters, driven in part by curvature-mediated attractions. At a critical size, as in a nucleation process, the FAM134B-RHD clusters induce the formation of membrane buds. The kinetics of budding depends sensitively on protein concentration and bilayer asymmetry. Our MD simulations shed light on the role of FAM134B-RHD in ER-phagy and show that membrane asymmetry can be used to modulate the kinetic barrier for membrane remodeling.

Autophagosomes form de novo in a manner that is incompletely understood. Particularly enigmatic are autophagy-related protein 9 (Atg9)-containing vesicles that are required for autophagy machinery assembly but do not supply the bulk of the autophagosomal membrane. In this study, we reconstituted autophagosome nucleation using recombinant components from yeast. We found that Atg9 proteoliposomes first recruited the phosphatidylinositol 3-phosphate kinase complex, followed by Atg21, the Atg2-Atg18 lipid transfer complex, and the E3-like Atg12-Atg5-Atg16 complex, which promoted Atg8 lipidation. Furthermore, we found that Atg2 could transfer lipids for Atg8 lipidation. In selective autophagy, these reactions could potentially be coupled to the cargo via the Atg19-Atg11-Atg9 interactions. We thus propose that Atg9 vesicles form seeds that establish membrane contact sites to initiate lipid transfer from compartments such as the endoplasmic reticulum., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Misfolded luminal endoplasmic reticulum (ER) proteins undergo ER-associated degradation (ERAD-L): They are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome. ERAD-L is mediated by the Hrd1 complex (composed of Hrd1, Hrd3, Der1, Usa1, and Yos9), but the mechanism of retrotranslocation remains mysterious. Here, we report a structure of the active Hrd1 complex, as determined by cryo-electron microscopy analysis of two subcomplexes. Hrd3 and Yos9 jointly create a luminal binding site that recognizes glycosylated substrates. Hrd1 and the rhomboid-like Der1 protein form two "half-channels" with cytosolic and luminal cavities, respectively, and lateral gates facing one another in a thinned membrane region. These structures, along with crosslinking and molecular dynamics simulation results, suggest how a polypeptide loop of an ERAD-L substrate moves through the ER membrane., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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.

We investigate system-size effects on the rotational diffusion of membrane proteins and other membrane-embedded molecules in molecular dynamics simulations. We find that the rotational diffusion coefficient slows down relative to the infinite-system value by a factor of one minus the ratio of protein and box areas. This correction factor follows from the hydrodynamics of rotational flows under periodic boundary conditions and is rationalized in terms of Taylor-Couette flow. For membrane proteins like transporters, channels, or receptors in typical simulation setups, the protein-covered area tends to be relatively large, requiring a significant finite-size correction. Molecular dynamics simulations of the protein adenine nucleotide translocase (ANT1) and of a carbon nanotube porin in lipid membranes show that the hydrodynamic finite-size correction for rotational diffusion is accurate in standard-use cases. The dependence of the rotational diffusion on box size can be used to determine the membrane viscosity.

Eukaryotic cells face the challenge of maintaining the complex composition of several coexisting organelles. The molecular mechanisms underlying the homeostasis of subcellular membranes and their adaptation during stress are only now starting to emerge. Here, we discuss three membrane property sensors of the endoplasmic reticulum (ER), namely OPI1, MGA2, and IRE1, each controlling a large cellular program impacting the lipid metabolic network. OPI1 coordinates the production of membrane and storage lipids, MGA2 regulates the production of unsaturated fatty acids required for membrane biogenesis, and IRE1 controls the unfolded protein response (UPR) to adjust ER size, protein folding, and the secretory capacity of the cell. Although these proteins use remarkably distinct sensing mechanisms, they are functionally connected via the ER membrane and cooperate to maintain membrane homeostasis. As a rationalization of the recently described mechanism of UPR activation by lipid bilayer stress, we propose that IRE1 can sense the protein-to-lipid ratio in the ER membrane to ensure a balanced production of membrane proteins and lipids., (Copyright © 2018 Elsevier Inc. All rights reserved.)

We critically test and validate the CS-Rosetta methodology for de novo structure prediction of α-helical membrane proteins (MPs) from NMR data, such as chemical shifts and NOE distance restraints. By systematically reducing the number and types of NOE restraints, we focus on determining the regime in which MP structures can be reliably predicted and pinpoint the boundaries of the approach. Five MPs of known structure were used as test systems, phototaxis sensory rhodopsin II (pSRII), a subdomain of pSRII, disulfide binding protein B (DsbB), microsomal prostaglandin E2 synthase-1 (mPGES-1), and translocator protein (TSPO). For pSRII and DsbB, where NMR and X-ray structures are available, resolution-adapted structural recombination (RASREC) CS-Rosetta yields structures that are as close to the X-ray structure as the published NMR structures if all available NMR data are used to guide structure prediction. For mPGES-1 and Bacillus cereus TSPO, where only X-ray crystal structures are available, highly accurate structures are obtained using simulated NMR data. One main advantage of RASREC CS-Rosetta is its robustness with respect to even a drastic reduction of the number of NOEs. Close-to-native structures were obtained with one randomly picked long-range NOEs for every 14, 31, 38, and 8 residues for full-length pSRII, the pSRII subdomain, TSPO, and DsbB, respectively, in addition to using chemical shifts. For mPGES-1, atomically accurate structures could be predicted even from chemical shifts alone. Our results show that atomic level accuracy for helical membrane proteins is achievable with CS-Rosetta using very sparse NOE restraint sets to guide structure prediction. Proteins 2017; 85:812-826. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Maintaining a fluid bilayer is essential for cell signaling and survival. Lipid saturation is a key factor determining lipid packing and membrane fluidity, and it must be tightly controlled to guarantee organelle function and identity. A dedicated eukaryotic mechanism of lipid saturation sensing, however, remains elusive. Here we show that Mga2, a transcription factor conserved among fungi, acts as a lipid-packing sensor in the ER membrane to control the production of unsaturated fatty acids. Systematic mutagenesis, molecular dynamics simulations, and electron paramagnetic resonance spectroscopy identify a pivotal role of the oligomeric transmembrane helix (TMH) of Mga2 for intra-membrane sensing, and they show that the lipid environment controls the proteolytic activation of Mga2 by stabilizing alternative rotational orientations of the TMH region. This work establishes a eukaryotic strategy of lipid saturation sensing that differs significantly from the analogous bacterial mechanism relying on hydrophobic thickness., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Carbon nanotubes can serve as simple nonpolar water channels. Here we report computer simulations exploring the relationship between the mechanical properties of such channels and their interaction with water. We show that on one hand, increasing the flexibility of the carbon nanotubes increases their apparent hydrophobic character, while on the other hand the presence of water inside the channel makes them more resistant to radial collapse. We quantify the effect of increasing flexibility on the hydrophobicity of the nanotube water channel. We also show that flexibility impedes water transport across the nanotube channel by increasing the free-energy barriers to such motion. Conversely, the presence of water inside the nanotube is shown to affect the energetics of radial collapse in a water nanotube, an ostensibly mechanical property. We quantify the magnitude of the effect and show that it arises from the formation of energetically favorable low-dimensional water structures inside the nanotube such as one-dimensional wires and two-dimensional sheets. [ABSTRACT FROM AUTHOR]

The formation of vesicles is essential for many biological processes, in particular for the trafficking of membrane proteins within cells. The Endosomal Sorting Complex Required for Transport (ESCRT) directs membrane budding away from the cytosol. Unlike other vesicle formation pathways, the ESCRT-mediated budding occurs without a protein coat. Here, we propose a minimal model of ESCRT-induced vesicle budding. Our model is based on recent experimental observations from direct fluorescence microscopy imaging that show ESCRT proteins colocalized only in the neck region of membrane buds. The model, cast in the framework of membrane elasticity theory, reproduces the experimentally observed vesicle morphologies with physically meaningful parameters. In this parameter range, the minimum energy configurations of the membrane are coatless buds with ESCRTs localized in the bud neck, consistent with experiment. The minimum energy configurations agree with those seen in the fluorescence images, with respect to both bud shapes and ESCRT protein localization. On the basis of our model, we identify distinct mechanistic pathways for the ESCRT-mediated budding process. The bud size is determined by membrane material parameters, explaining the narrow yet different bud size distributions in vitro and in vivo. Our membrane elasticity model thus sheds light on the energetics and possible mechanisms of ESCRT-induced membrane budding. [ABSTRACT FROM AUTHOR]

Water molecules confined to nonpolar pores and cavities of nanoscopic dimensions exhibit highly unusual properties. Water fill- ing is strongly cooperative, with the possible coexistence of filled and empty states and sensitivity to small perturbations of the pore polar- ity and solvent conditions. Confined water molecules form tightly hydrogen-bonded wires or clusters. The weak attractions to the confining wall, combined with strong interactions between water molecules, permit exceptionally rapid water flow, exceeding expec- tations from macroscopic hydrodynamics by several orders of mag- nitude. The proton mobility along 1D water wires also substantially exceeds that in the bulk. Proteins appear to exploit these unusual properties of confined water in their biological function (e.g., to en- sure rapid water flow in aquaporins or to gate proton flow in proton pumps and enzymes). The unusual properties of water in nonpolar confinement are also relevant to the design of novel nanofluidic and molecular separation devices or fuel cells. [ABSTRACT FROM AUTHOR]

Most membrane proteins are modified by covalent addition of complex sugars through N- and O-glycosylation. Unlike proteins, glycans do not typically adopt specific secondary structures and remain very mobile, shielding potentially large fractions of protein surface. High glycan conformational freedom hinders complete structural elucidation of glycoproteins. Computer simulations may be used to model glycosylated proteins but require hundreds of thousands of computing hours on supercomputers, thus limiting routine use. Here, we describe GlycoSHIELD, a reductionist method that can be implemented on personal computers to graft realistic ensembles of glycan conformers onto static protein structures in minutes. Using molecular dynamics simulation, small-angle X-ray scattering, cryoelectron microscopy, and mass spectrometry, we show that this open-access toolkit provides enhanced models of glycoprotein structures. Focusing on N-cadherin, human coronavirus spike proteins, and gamma-aminobutyric acid receptors, we show that GlycoSHIELD can shed light on the impact of glycans on the conformation and activity of complex glycoproteins. [Display omitted] • GlycoSHIELD predicts morphology and span of glycan shields on protein surface • Predicts glycan impact on protein conformation and surface accessibility • GlycoSHIELD models are consistent with cryo-EM datasets • Enhances the interpretation of experimental density maps GlycoSHIELD and its associated library of precomputed glycan conformers enable the generation of realistic glycosylated protein structure models at a fraction of the computing cost of conventional molecular dynamics simulations. [ABSTRACT FROM AUTHOR]

Summary PglK is a lipid-linked oligosaccharide (LLO) flippase essential for asparagine-linked protein glycosylation in Campylobacter jejuni. Previously we have proposed a non-alternating-access LLO translocation mechanism, where postulated outward-facing states play a primary role. To investigate this unusual mechanistic proposal, we have determined a high-resolution structure of PglK that displays an outward semi-occluded state with the two nucleotide binding domains forming an asymmetric closed dimer with two bound ATPγS molecules. Based on this structure, we performed extensive molecular dynamics simulations to investigate LLO recognition and flipping. Our results suggest that PglK may employ a "substrate-hunting" mechanism to locally increase the LLO concentration and facilitate its jump into the translocation pathway, for which sugars from the LLO head group are essential. We further conclude that the release of LLO to the outside occurs before ATP hydrolysis and is followed by the closing of the periplasmic cavity of PglK. Graphical Abstract Highlights • An outward semi-occluded structure of PglK with ATPγS-bound is shown • Molecular dynamics simulations show a recognition site for LLO • Sugars from the LLO head group are essential for translocation • LLO release is a pre-hydrolysis event followed by quick closure of the cavity An outward semi-occluded structure of PglK with ATPγS bound together with molecular dynamics simulations describe the mode of LLO recognition and flipping. Transfer of LLO into the translocation pathway occurs after a surface recognition step, followed by closing of the periplasmic cavity. [ABSTRACT FROM AUTHOR]

The T cell receptor (TCR) expressed by T lymphocytes initiates protective immune responses to pathogens and tumors. To explore the structural basis of how TCR signaling is initiated when the receptor binds to peptide-loaded major histocompatibility complex (pMHC) molecules, we used cryogenic electron microscopy to determine the structure of a tumor-reactive TCRαβ/CD3δγε 2 ζ 2 complex bound to a melanoma-specific human class I pMHC at 3.08 Å resolution. The antigen-bound complex comprises 11 subunits stabilized by multivalent interactions across three structural layers, with clustered membrane-proximal cystines stabilizing the CD3-εδ and CD3-εγ heterodimers. Extra density sandwiched between transmembrane helices reveals the involvement of sterol lipids in TCR assembly. The geometry of the pMHC/TCR complex suggests that efficient TCR scanning of pMHC requires accurate pre-positioning of T cell and antigen-presenting cell membranes. Comparisons of the ligand-bound and unliganded receptors, along with molecular dynamics simulations, indicate that TCRs can be triggered in the absence of spontaneous structural rearrangements. [Display omitted] • Connecting peptides create rigid links to stabilize membrane-bound TCR subunits • Sterol lipid contributes to transmembrane assembly of the TCR complex • A pMHC/TCR structure highlights the cooperative nature of antigen recognition • TCR signaling may be triggered without spontaneous structural rearrangements Cryo-EM structural analysis of a T cell receptor (TCR) complex bound to a tumor-specific human class I pMHC indicates the functional impact of connecting peptides and sterol lipid for complex assembly and suggests that TCR signaling may be triggered without spontaneous structural rearrangements. [ABSTRACT FROM AUTHOR]

Summary: The ESCRT-I and ESCRT-II supercomplex induces membrane buds that invaginate into the lumen of endosomes, a process central to the lysosomal degradation of ubiquitinated membrane proteins. The solution conformation of the membrane-budding ESCRT-I-II supercomplex from yeast was refined against small-angle X-ray scattering (SAXS), single-molecule Förster resonance energy transfer (smFRET), and double electron-electron resonance (DEER) spectra. These refinements yielded an ensemble of 18 ESCRT-I-II supercomplex structures that range from compact to highly extended. The crescent shapes of the ESCRT-I-II supercomplex structures provide the basis for a detailed mechanistic model, in which ESCRT-I-II stabilizes membrane buds and coordinates cargo sorting by lining the pore of the nascent bud necks. The hybrid refinement used here is general and should be applicable to other dynamic multiprotein assmeblies. [ABSTRACT FROM AUTHOR]