These findings establish plant minimal START proteins as bona fide lipid-binding proteins that utilize a bipartite ligand-binding mechanism coupled with membrane-responsive gating dynamics, collectively suggesting a membrane-guided gating mechanism for lipid exchange.
It is suggested that bacterial START domains participate in a remarkably broad range of biological processes-including small-molecule binding, metabolic regulation, enzymatic catalysis, and stress adaptation-rather than traditional lipid transport.
Ece Aslan, Ksenia I. Lubova, Alexander Speer et al.· FEMS Microbiology Reviews· 0 citations
Membrane association of intrinsically disordered proteins (IDPs) mediates various cellular functions including membrane remodeling and signal transduction. Whereas membrane association through amphipathic helices and polybasic motifs is well understood, sequence determinants for the insertion of aromatic residues into the membrane hydrophobic core are still poorly characterized. Here, we decipher the sequence code for membrane insertion of aromatic-centered motifs. For an initial set of 10 9-residue aromatic-centered sequences, all-atom molecular dynamics simulations and the positioning of proteins in membranes (PPM) method produced very similar membrane insertion propensities. Applying PPM to a full library of 1.2 × 106 sequences with an F, W, or Y residue flanked by L, R, G, N, or E at four positions on either side, we found that aliphatic (L) and basic (R) residues favor membrane insertion, whereas acidic (E) and polar (N) residues disfavor it. Guided by these rules, we developed a mathematical model dubbed AroMIP (Aromatic Membrane Insertion Predictor) to predict the membrane insertion propensities of aromatic-centered motifs. AroMIP achieves 91.2%, 92.0%, and 99.7% accuracies for F-, W-, and Y-centered motifs, respectively, in disordered regions of the human proteome and is available as a web server at https://zhougroup-uic.github.io/AroMIP/. The present work provides the sequence basis and a mechanistic understanding of how IDPs employ aromatic-centered motifs to drive membrane insertion, and enriches the tools for the study of IDP-membrane association.
Membrane recruitment is a fundamental regulator of protein function. However, the allosteric mechanisms by which lipid binding controls protein activity remain poorly understood. In autophagy, the ubiquitin-like protein LC3 is lipid-anchored to autophagosomes, where it is essential for receptor recruitment and vesicle formation. While LC3-receptor interactions are structurally well defined, how membrane engagement governs LC3 functional dynamics has remained enigmatic. Here, we uncover that membrane binding triggers a major conformational transition in LC3, exposing functional pockets that are occluded in its cytosolic form. We demonstrate that this shift is mediated by dynamic coupling between the allosteric site (α3-loop5-β3-loop6) and the functional binding pockets. To conclusively test this mechanism, we utilised an ensemble-based protein design strategy guided by molecular dynamics to engineer the allosteric site. From a series of mutants, two variants emerged that stabilized LC3 conformation in either active or inactive state on the membrane. X-ray crystal structures of mutant LC3, biophysical assays, super-resolution microscopy, and TEM confirmed that the activated allosteric site mutant facilitates receptor binding and cargo capture. In contrast, the inactive variant is functionally inert on the membrane. Our work identifies a fundamental lipid-triggered allosteric site in LC3 that is critical for autophagy regulation and broader implications of membrane-dependent reprogrammable protein activities. The allosteric mechanisms by which lipid binding controls protein activity remain poorly understood. Here, the authors identify a lipid-triggered allosteric site in LC3 that remodels its receptor-binding interface upon membrane interaction. This site can be engineered to precisely tune LC3-LIR interactions, offering a new strategy to regulate selective autophagy.
Deepanshi Gahlot, Jesu Castin, S. Mathur et al.· Nature Communications· 0 citations
Organelle membrane identity is encoded by protein and lipid composition1. Human cells produce thousands of distinct lipid species for this purpose2,3. Yet, the precise molecular functions of most lipids remain unknown, owing to limited methods for in situ investigations of individual lipid species. Here, we use minimally modified bifunctional lipid probes to detect and functionally characterize lipid-protein interactions across time and subcellular compartments. We leverage this approach to map time-resolved protein interactomes of individual lipids representing major membrane lipid classes as they are transported through the organelle system of human cells. We identify hundreds of unique lipid-protein interaction candidates, measure lipid-induced protein abundance changes, and distinguish between directly and indirectly interacting subunits of associated protein complexes. Informed by this multimodal dataset, we interrogate the lipid specificity of membrane organizing complexes, in particular the selective co-association of phosphatidylethanolamine with the ancestral subunits of the nuclear pore (NDC1) and MICOS (MIC60) complexes. Using super-resolution microscopy and molecular dynamics simulations, we demonstrate that phosphatidylethanolamine stabilizes both NDC1 and MIC60 in highly curved membrane nanodomains, indicating that major membrane organizing complexes are positioned by selective lipid-protein interactions. Altogether, our experimental strategy provides a blueprint for discovering and characterizing cellular functions of individual lipids.
Katelyn C. Cook, Karen Palacio-Rodríguez, Kristin Böhlig et al.· bioRxiv· 0 citations
Regarded as one of the most complex membrane protein folds, CLCs form a large family of membrane proteins that function as anion channels and secondary active anion/proton transporters. Despite low sequence similarity, available structures are remarkably similar, maintaining the same inverted topology and fold, with subunits assembled as dimers in wild-type structures. Because of these strong structural features, CLC-ec1, a prokaryotic homolog from E. coli, has become a highly valuable model system for studying membrane protein folding and oligomerization assembly. Associating via a membrane-embedded dimerization interface, the subunits participate in a dynamic equilibrium between monomers and dimers in lipid bilayers, enabling investigation of the physical driving forces underlying the formation of stable membrane complexes. Like soluble protein assembly, studies indicate that CLC-ec1 dimerization is driven by a solvophobic force arising from the free energy gained by burying lipid bilayer defects. Dimerization stability is influenced by lipid composition and pH, which, in turn, provide a mechanism for functional regulation through oligomerization. In this review, I provide an overview of how the exploration of CLC folding and oligomerization advances our understanding of membrane protein self-assembly in general and the role of oligomerization in regulating function.
Janice L. Robertson· Chemical Reviews· 0 citations
The model provides a computationally efficient framework for studying conformational ensembles and assembly of proteins at bilayer–water interfaces and is validated against Wimley–White free energies of transfer of hydrophobic peptides and against an experimentally refined conformational ensemble of a flexible membrane receptor.
Riccardo Saltutti, G. Tesei· bioRxiv· 0 citations
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