Membranes are functionalized by a proteolipid code.

Membranes are protein and lipid structures that surround cells and other biological compartments. We present a conceptual model wherein all membranes are organized into structural and functional zones. The assembly of zones such as receptor clusters, protein-coated pits, lamellipodia, cell junctions, and membrane fusion sites is explained to occur through a protein-lipid code. This challenges the theory that lipids sort proteins after forming stable membrane subregions independently of proteins.

Comprehensive classification of proteins based on structures that engage lipids by COMPOSEL.

Structures can now be predicted for any protein using programs like AlphaFold and Rosetta, which rely on a foundation of experimentally determined structures of architecturally diverse proteins. The accuracy of such artificial intelligence and machine learning (AI/ML) approaches benefits from the specification of restraints which assist in navigating the universe of folds to converge on models most representative of a given protein's physiological structure. This is especially pertinent for membrane proteins, with structures and functions that depend on their presence in lipid bilayers. Structures of proteins in their membrane environments could conceivably be predicted from AI/ML approaches with user-specificized parameters that describe each element of the architecture of a membrane protein accompanied by its lipid environment. We propose the Classification Of Membrane Proteins based On Structures Engaging Lipids (COMPOSEL), which builds on existing nomenclature types for monotopic, bitopic, polytopic and peripheral membrane proteins as well as lipids. Functional and regulatory elements are also defined in the scripts, as shown with membrane fusing synaptotagmins, multidomain PDZD8 and Protrudin proteins that recognize phosphoinositide (PI) lipids, the intrinsically disordered MARCKS protein, caveolins, the ß barrel assembly machine (BAM), an adhesion G-protein coupled receptor (aGPCR) and two lipid modifying enzymes - diacylglycerol kinase DGKε and fatty aldehyde dehydrogenase FALDH. This demonstrates how COMPOSEL communicates lipid interactivity as well as signaling mechanisms and binding of metabolites, drug molecules, polypeptides or nucleic acids to describe the operations of any protein. Moreover COMPOSEL can be scaled to express how genomes encode membrane structures and how our organs are infiltrated by pathogens such as SARS-CoV-2.

SARS-CoV-2 Omicron Subvariants Balance Host Cell Membrane, Receptor, and Antibody Docking via an Overlapping Target Site.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are emerging rapidly and offer surfaces that are optimized for recognition of host cell membranes while also evading antibodies arising from vaccinations and previous infections. Host cell infection is a multi-step process in which spike heads engage lipid bilayers and one or more angiotensin-converting enzyme 2 (ACE-2) receptors. Here, the membrane binding surfaces of Omicron subvariants are compared using cryo-electron microscopy (cEM) structures of spike trimers from BA.2, BA.2.12.1, BA.2.13, BA.2.75, BA.3, BA.4, and BA.5 viruses. Despite significant differences around mutated sites, they all maintain strong membrane binding propensities that first appeared in BA.1. Both their closed and open states retain elevated membrane docking capacities, although the presence of more closed than open states diminishes opportunities to bind receptors while enhancing membrane engagement. The electrostatic dipoles are generally conserved. However, the BA.2.75 spike dipole is compromised, and its ACE-2 affinity is increased, and BA.3 exhibits the opposite pattern. We propose that balancing the functional imperatives of a stable, readily cleavable spike that engages both lipid bilayers and receptors while avoiding host defenses underlies betacoronavirus evolution. This provides predictive criteria for rationalizing future pandemic waves and COVID-19 transmissibility while illuminating critical sites and strategies for simultaneously combating multiple variants.

Transmembrane Membrane Readers form a Novel Class of Proteins That Include Peripheral Phosphoinositide Recognition Domains and Viral Spikes.

Membrane proteins are broadly classified as transmembrane (TM) or peripheral, with functions that pertain to only a single bilayer at a given time. Here, we explicate a class of proteins that contain both transmembrane and peripheral domains, which we dub transmembrane membrane readers (TMMRs). Their transmembrane and peripheral elements anchor them to one bilayer and reversibly attach them to another section of bilayer, respectively, positioning them to tether and fuse membranes while recognizing signals such as phosphoinositides (PIs) and modifying lipid chemistries in proximity to their transmembrane domains. Here, we analyze full-length models from AlphaFold2 and Rosetta, as well as structures from nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography, using the Membrane Optimal Docking Area (MODA) program to map their membrane-binding surfaces. Eukaryotic TMMRs include phospholipid-binding C1, C2, CRAL-TRIO, FYVE, GRAM, GTPase, MATH, PDZ, PH, PX, SMP, StART and WD domains within proteins including protrudin, sorting nexins and synaptotagmins. The spike proteins of SARS-CoV-2 as well as other viruses are also TMMRs, seeing as they are anchored into the viral membrane while mediating fusion with host cell membranes. As such, TMMRs have key roles in cell biology and membrane trafficking, and include drug targets for diseases such as COVID-19.

Progressive membrane-binding mechanism of SARS-CoV-2 variant spike proteins.

Membrane recognition by viral spike proteins is critical for infection. Here we show the host cell membrane-binding surfaces of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike variants Alpha, Beta, Gamma, Delta, Epsilon, Kappa, and Omicron as well as SARS-CoV-1 and pangolin and bat relatives. They show increases in membrane binding propensities over time, with all spike head mutations in variants, and particularly BA.1, impacting the protein's affinity to cell membranes. Comparison of hundreds of structures yields a progressive model of membrane docking in which spike protein trimers shift from initial perpendicular stances to increasingly tilted positions that draw viral particles alongside host cell membranes before optionally engaging angiotensin-converting enzyme 2 (ACE2) receptors. This culminates in the assembly of the symmetric fusion apparatus, with enhanced membrane interactions of variants explaining their unique cell fusion capacities and COVID-19 disease transmission rates.

Multifaceted membrane binding head of the SARS-CoV-2 spike protein.

The SARS-CoV-2 spike protein presents a surface with enormous membrane binding potential to host tissues and organelles of infected cells. Its exposed trimeric head binds not only the angiotensin-converting enzyme 2 (ACE2), but also host phospholipids which are missing from all existing structures. Hence, the membrane interaction surfaces that mediate viral fusion, entry, assembly and egress remain unclear. Here the spike:membrane docking sites are identified based on membrane optimal docking area (MODA) analysis of 3D structures of spike proteins in closed and open conformations at endocytic and neutral pH levels as well as ligand complexes. This reveals multiple membrane binding sites in the closed spike head that together prefer convex membranes and are modulated by pH, fatty acids and post-translational modifications including glycosylation. The exposure of the various membrane interaction sites adjusts upon domain repositioning within the trimer, allowing formation of intermediate bilayer complexes that lead to the prefusion state while also enabling ACE2 receptor recognition. In contrast, all antibodies that target the spike head would block the membrane docking process that precedes ACE2 recognition. Together this illuminates the engagements of the spike protein with plasma, endocytic, ER or exocytic vesicle membranes that help to drive the cycle of viral infection, and offers novel sites for intervention.

The phosphoinositide code is read by a plethora of protein domains.

INTRODUCTION: The proteins that decipher nucleic acid- and protein-based information are well known, however, those that read membrane-encoded information remain understudied. Here, we report 70 different human, microbial and viral protein folds that recognize phosphoinositides (PIs), comprising the readers of a vast membrane code. AREAS COVERED: Membrane recognition is best understood for FYVE, PH and PX domains, which exemplify hundreds of PI code readers. Comparable lipid interaction mechanisms may be mediated by kinases, adjacent C1 and C2 domains, trafficking arrestins, GAT and VHS modules, membrane-perturbing annexins, BAR, CHMP, ENTH, HEAT, syntaxin and Tubby helical bundles, multipurpose FERM, EH, MATH, PHD, PDZ, PROPPIN, PTB and SH2 domains, as well as systems that regulate receptors, GTPases and actin filaments, transfer lipids, and assemble bacterial and viral particles. EXPERT OPINION: The elucidation of how membranes are recognized has extended the genetic code to the PI code. Novel discoveries include PIP-stop and MET-stop residues to which phosphates and metabolites are attached to block phosphatidylinositol phosphate (PIP) recognition, memteins as functional membrane protein apparatuses and lipidons as lipid 'codons' recognized by membrane readers. At least 5% of the human proteome senses such membrane signals and allows eukaryotic organelles and pathogens to operate and replicate.

Phosphoinositide Recognition Sites Are Blocked by Metabolite Attachment.

Membrane readers take part in trafficking and signaling processes by localizing proteins to organelle surfaces and transducing molecular information. They accomplish this by engaging phosphoinositides (PIs), a class of lipid molecules which are found in different proportions in various cellular membranes. The prototypes are the PX domains, which exhibit a range of specificities for PIs. Our meta-analysis indicates that recognition of membranes by PX domains is specifically controlled by modification of lysine and arginine residues including acetylation, hydroxyisobutyrylation, glycation, malonylation, methylation and succinylation of sidechains that normally bind headgroups of phospholipids including organelle-specific PI signals. Such metabolite-modulated residues in lipid binding elements are named MET-stops here to highlight their roles as erasers of membrane reader functions. These modifications are concentrated in the membrane binding sites of half of all 49 PX domains in the human proteome and correlate with phosphoregulatory sites, as mapped using the Membrane Optimal Docking Area (MODA) algorithm. As these motifs are mutated and modified in various cancers and the responsible enzymes serve as potential drug targets, the discovery of MET-stops as a widespread inhibitory mechanism may aid in the development of diagnostics and therapeutics aimed at the readers, writers and erasers of the PI code.

Regulation of the Phosphoinositide Code by Phosphorylation of Membrane Readers.

The genetic code that dictates how nucleic acids are translated into proteins is well known, however, the code through which proteins recognize membranes remains mysterious. In eukaryotes, this code is mediated by hundreds of membrane readers that recognize unique phosphatidylinositol phosphates (PIPs), which demark organelles to initiate localized trafficking and signaling events. The only superfamily which specifically detects all seven PIPs are the Phox homology (PX) domains. Here, we reveal that throughout evolution, these readers are universally regulated by the phosphorylation of their PIP binding surfaces based on our analysis of existing and modelled protein structures and phosphoproteomic databases. These PIP-stops control the selective targeting of proteins to organelles and are shown to be key determinants of high-fidelity PIP recognition. The protein kinases responsible include prominent cancer targets, underscoring the critical role of regulated membrane readership.