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.

Recognition and remodeling of endosomal zones by sorting nexins.

The proteolipid code determines how cytosolic proteins find and remodel membrane surfaces. Here, we investigate how this process works with sorting nexins Snx1 and Snx3. Both proteins form sorting machines by recognizing membrane zones enriched in phosphatidylinositol 3-phosphate (PI3P), phosphatidylserine (PS) and cholesterol. This co-localized combination forms a unique "lipid codon" or lipidon that we propose is responsible for endosomal targeting, as revealed by structures and interactions of their PX domain-based readers. We outline a membrane recognition and remodeling mechanism for Snx1 and Snx3 involving this code element alongside transmembrane pH gradients, dipole moment-guided docking and specific protein-protein interactions. This generates an initial membrane-protein assembly (memtein) that then recruits retromer and additional PX proteins to recruit cell surface receptors for sorting to the trans-Golgi network (TGN), lysosome and plasma membranes. Post-translational modification (PTM) networks appear to regulate how the sorting machines form and operate at each level. The commonalities and differences between these sorting nexins show how the proteolipid code orchestrates parallel flows of molecular information from ribosome emergence to organelle genesis, and illuminates a universally applicable model of the membrane.

A conserved epitope in VAR2CSA is targeted by a cross-reactive antibody originating from Plasmodium vivax Duffy binding protein.

During Plasmodium falciparum infection in pregnancy, VAR2CSA is expressed on the surface of infected erythrocytes (IEs) and mediates their sequestration in the placenta. As a result, antibodies to VAR2CSA are largely restricted to women who were infected during pregnancy. However, we discovered that VAR2CSA antibodies can also be elicited by P. vivax Duffy binding protein (PvDBP). We proposed that infection with P. vivax in non-pregnant individuals can generate antibodies that cross-react with VAR2CSA. To better understand the specificity of these antibodies, we took advantage of a mouse monoclonal antibody (3D10) raised against PvDBP that cross-reacts with VAR2CSA and identified the epitopes targeted by this antibody. We screened two peptide arrays that span the ectodomain of VAR2CSA from the FCR3 and NF54 alleles. Based on the top epitope recognized by 3D10, we designed a 34-amino acid synthetic peptide, which we call CRP1, that maps to a highly conserved region in DBL3X. Specific lysine residues are critical for 3D10 recognition, and these same amino acids are within a previously defined chondroitin sulfate A (CSA) binding site in DBL3X. We showed by isothermal titration calorimetry that the CRP1 peptide can bind directly to CSA, and antibodies to CRP1 raised in rats significantly blocked the binding of IEs to CSA in vitro. In our Colombian cohorts of pregnant and non-pregnant individuals, at least 45% were seroreactive to CRP1. Antibody reactivities to CRP1 and the 3D10 natural epitope in PvDBP region II, subdomain 1 (SD1), were strongly correlated in both cohorts. These findings suggest that antibodies arising from PvDBP may cross-react with VAR2CSA through the epitope in CRP1 and that CRP1 could be a potential vaccine candidate to target a distinct CSA binding site in VAR2CSA.

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.

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.

Discovery of allosteric SHP2 inhibitors through ensemble-based consensus molecular docking, endpoint and absolute binding free energy calculations.

SHP2 (Src homology-2 domain-containing protein tyrosine phosphatase-2) is a cytoplasmic protein -tyrosine phosphatase encoded by the gene PTPN11. It plays a crucial role in regulating cell growth and differentiation. Specifically, SHP2 is an oncoprotein associated with developmental pathologies and several different cancer types, including gastric, leukemia and breast cancer and is of great therapeutic interest. Given these roles, current research efforts have focused on developing SHP2 inhibitors. Allosteric SHP2 inhibitors have been shown to be more selective and pharmacologically appealing compared to competitive catalytic inhibitors targeting SHP2. Nevertheless, there remains a need for novel allosteric inhibitor scaffolds targeting SHP2 to develop compounds with improved selectivity, cell permeability, and bioavailability. Towards this goal, this study applied various computational tools to screen over 6 million compounds against the allosteric site within SHP2. The top-ranked hits from our in-silico screening were validated using protein thermal shift and biolayer interferometry assays, revealing three potent compounds. Kinetic binding assays were employed to measure the binding affinities of the top-ranked compounds and demonstrated that they all bind to SHP2 with a nanomolar affinity. Hence the compounds and the computational workflow described herein provide an effective approach for identifying and designing a generation of improved allosteric inhibitors of SHP2.

How Choice of Model Membrane Affects Protein-Glycosphingolipid Interactions: Insights from Native Mass Spectrometry.

Interactions between glycan-binding proteins (GBPs) and glycosphingolipids (GSLs) are involved in numerous physiological and pathophysiological processes. Many model membrane systems are available for studying GBP-GSL interactions, but a systematic investigation has not been carried out on how the nature of the model membrane affects binding. In this work, we use electrospray ionization mass spectrometry (ESI-MS), both direct and competitive assays, to measure the binding of cholera toxin B subunit homopentamer (CTB5) to GM1 ganglioside in liposomes, bilayer islands [styrene maleic acid lipid particles (SMALPs), nanodiscs (NDs), and picodiscs (PDs)], and micelles. We find that direct ESI-MS analysis of CTB5 binding to GM1 is unreliable due to non-uniform response factors, incomplete extraction of bound GM1 in the gas phase, and nonspecific CTB5-GM1 interactions. Conversely, indirect proxy ligand ESI-MS measurements show that the intrinsic (per binding site) association constants of CTB5 for PDs, NDs, and SMALPs are similar and comparable to the affinity of soluble GM1 pentasaccharide (GM1os). The observed affinity decreases with increasing GM1 content due to molecular crowding stemming from GM1 clustering. Unlike the smaller model membranes, the observed affinity of CTB5 toward GM1 liposomes is ∼10-fold weaker than GM1os and relatively insensitive to the GM1 content. GM1 glycomicelles exhibit the lowest affinity, ∼35-fold weaker than GM1os. Together, the results highlight experimental design considerations for quantitative GBP-GSL binding studies involving multisubunit GBPs and factors to consider when comparing results obtained with different membrane systems. Notably, they suggest that bilayer islands with a low percentage of GSL, wherein clustering is minimized, are ideal for assessing intrinsic strength of GBP-GSL interactions in a membrane environment, while binding to liposomes, which is sub-optimal due to extensive clustering, may be more representative of authentic cellular environments.

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.

Effects of Specific Inhibitors for CaMK1D on a Primary Neuron Model for Alzheimer's Disease.

Alzheimer's disease (AD) is the most common cause of dementia worldwide. Despite extensive research and targeting of the main molecular components of the disease, beta-amyloid (Aß) and tau, there are currently no treatments that alter the progression of the disease. Here, we examine the effects of two specific kinase inhibitors for calcium/calmodulin-dependent protein kinase type 1D (CaMK1D) on Aß-mediated toxicity, using mouse primary cortical neurons. Tau hyperphosphorylation and cell death were used as AD indicators. These specific inhibitors were found to prevent Aß induced tau hyperphosphorylation in culture, but were not able to protect cells from Aß induced toxicity. While inhibitors were able to alter AD pathology in cell culture, they were insufficient to prevent cell death. With further research and development, these inhibitors could contribute to a multi-drug strategy to combat AD.

Tetraspanin 6 is a regulator of carcinogenesis in colorectal cancer.

Early stages of colorectal cancer (CRC) development are characterized by a complex rewiring of transcriptional networks resulting in changes in the expression of multiple genes. Here, we demonstrate that the deletion of a poorly studied tetraspanin protein Tspan6 in Apcmin/+ mice, a well-established model for premalignant CRC, resulted in increased incidence of adenoma formation and tumor size. We demonstrate that the effect of Tspan6 deletion results in the activation of EGF-dependent signaling pathways through increased production of the transmembrane form of TGF-α (tmTGF-α) associated with extracellular vesicles. This pathway is modulated by an adaptor protein syntenin-1, which physically links Tspan6 and tmTGF-α. In support of this, the expression of Tspan6 is frequently decreased or lost in CRC, and this correlates with poor survival. Furthermore, the analysis of samples from the epidermal growth factor receptor (EGFR)-targeting clinical trial (COIN trial) has shown that the expression of Tspan6 in CRC correlated with better patient responses to EGFR-targeted therapy involving Cetuximab. Importantly, Tspan6-positive patients with tumors in the proximal colon (right-sided) and those with KRAS mutations had a better response to Cetuximab than the patients that expressed low Tspan6 levels. These results identify Tspan6 as a regulator of CRC development and a potential predictive marker for EGFR-targeted therapies in CRC beyond RAS pathway mutations.

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.

Structures and Dynamics of Native-State Transmembrane Protein Targets and Bound Lipids.

Membrane proteins work within asymmetric bilayers of lipid molecules that are critical for their biological structures, dynamics and interactions. These properties are lost when detergents dislodge lipids, ligands and subunits, but are maintained in native nanodiscs formed using styrene maleic acid (SMA) and diisobutylene maleic acid (DIBMA) copolymers. These amphipathic polymers allow extraction of multicomponent complexes of post-translationally modified membrane-bound proteins directly from organ homogenates or membranes from diverse types of cells and organelles. Here, we review the structures and mechanisms of transmembrane targets and their interactions with lipids including phosphoinositides (PIs), as resolved using nanodisc systems and methods including cryo-electron microscopy (cryo-EM) and X-ray diffraction (XRD). We focus on therapeutic targets including several G protein-coupled receptors (GPCRs), as well as ion channels and transporters that are driving the development of next-generation native nanodiscs. The design of new synthetic polymers and complementary biophysical tools bodes well for the future of drug discovery and structural biology of native membrane:protein assemblies (memteins).

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.

Structural biology of endogenous membrane protein assemblies in native nanodiscs.

The advent of amphiphilic copolymers enables integral membrane proteins to be solubilized into stable 10-30 nm native nanodiscs to resolve their multisubunit structures, post-translational modifications, endogenous lipid bilayers, and small molecule ligands. This breakthrough has positioned biological membrane:protein assemblies (memteins) as fundamental functional units of cellular membranes. Herein, we review copolymer design strategies and methods for the characterization of transmembrane proteins within native nanodiscs by cryo-electron microscopy (cryo-EM), transmission electron microscopy, nuclear magnetic resonance spectroscopy, electron paramagnetic resonance, X-ray diffraction, surface plasmon resonance, and mass spectrometry.

Multisite interactions of prions with membranes and native nanodiscs.

Although prions are known as protein-only infectious particles, they exhibit lipid specificities, cofactor dependencies and membrane-dependent activities. Such membrane interactions play key roles in how prions are processed, presented and regulated, and hence have significant functional consequences. The expansive literature related to prion protein interactions with lipids and native nanodiscs is discussed, and provides a unique opportunity to re-evaluate the molecular composition and mechanisms of its infectious and cellular states. A family of crystal and solution structures of prions are analyzed here for the first time using the membrane optimal docking area (MODA) program, revealling the presence of structured binding elements that could mediate specific lipid recognition. A set of motifs centerred around W99, L125, Y169 and Y226 are consistently predicted as being membrane interactive and form an exposed surface which includes α helical, ß strand and loop elements involving the prion protein (PrP) structural domain, while the scrapie form is radically different and doubles the size of the membrane interactive site into an extensible surface. These motifs are highly conserved throughout mammalian evolution, suggesting that prions have long been intrinsically attached to membranes at central and N- and C-terminal points, providing several opportunities for stable and specific bilayer interactions as well as multiple complexed orientations. Resistance or susceptibility to prion disease correlates with increased or decreased membrane binding propensity by mutant forms, respectively, indicating a protective role by lipids. The various prion states found in vivo are increasingly resolvable using native nanodiscs formed by styrene maleic acid (SMA) and stilbene maleic acid (STMA) copolymers rather than classical detergents, allowing the endogenous states to be tackled. These copolymers spontaneously fragment intact membranes into water-soluble discs holding a section of native bilayer, and can accommodate prion multimers and mini-fibrils. Such nanodiscs have also proven useful for understanding how ß amyloid and α synuclein proteins contribute to Alzheimer's and Parkinson's diseases, providing further biomedical applications. Structural and functional insights of such proteins in styrene maleic acid lipid particles (SMALPs) can be resolved at high resolution by methods including cryo-electron microscopy (cEM), motivating continued progress in polymer design to resolve biological and pathological mechanisms.

Fgd5 is a Rac1-specific Rho GEF that is selectively inhibited by aurintricarboxylic acid.

Rho proteins are signalling molecules that control cellular dynamics, movement and morphological changes. They are activated by Rho guanine-nucleotide exchange factors (Rho GEFs) that transduce upstream signals into Rho-mediated activation of downstream processes. Fgd5 is a Rho GEF involved in angiogenesis and its target Rho protein for this process has been linked to Cdc42 activation. Here, we examined the function of purified Fgd5, specifically, which Rho proteins it activates and pinpoint the structural domains required for enzymatic activity. Using a GEF enzyme assay, we found that purified Fgd5 showed preferential activation of Rac1 and direct binding of Rac1 in pull-down and co-immunoprecipitation assays. Structural comparisons showed that the Fgd5 DH domain is highly similar to the Rac1 GEF, TrioN, supporting a role for Fgd5 as a Rac1 GEF. Compounds that bind to purified Fgd5 DH-PH protein were identified by screening a small molecule library via surface plasmon resonance. The effects of eleven ligands were further examined for their ability to inhibit the Fgd5 GEF enzymatic activity and Rac1 interaction. From these studies, we found that the compound aurintricarboxylic acid, and to a lesser extent mitoxantrone dihydrochloride, inhibited both Fgd5 GEF activation of Rac1 and their interaction. Aurintricarboxylic acid had no effect on the activity or binding of the Rac1 GEF, TrioN, thus demonstrating the feasibility of selectively disrupting Rho GEF activators. Abbreviations: a.a.: amino acid; ATA: aurintricarboxylic acid; DH: Dbl homology; DOCK: dictator of cytokinesis; Fgd: faciogenital dysplasia; GEF: guanine-nucleotide exchange factor; GST: glutathione S-transferase; LOPAC: library of pharmacologically active compounds; PH: pleckstrin homology; PDB: protein data bank; s.e.m.: standard error of the mean; SPR: surface plasmon resonance.

Structure and function of proteins in membranes and nanodiscs.

The field of membrane structural biology represents a fast-moving field with exciting developments including native nanodiscs that allow preparation of complexes of post-translationally modified proteins bound to biological lipids. This has led to conceptual advances including biological membrane:protein assemblies or "memteins" as the fundamental functional units of biological membranes. Tools including cryo-electron microscopy and X-ray crystallography are maturing such that it is becoming increasingly feasible to solve structures of large, multicomponent complexes, while complementary methods including nuclear magnetic resonance spectroscopy yield unique insights into interactions and dynamics. Challenges remain, including elucidating exactly how lipids and ligands are recognized at atomic resolution and transduce signals across asymmetric bilayers. In this special volume some of the latest thinking and methods are gathered through the analysis of a range of transmembrane targets. Ongoing work on areas including polymer design, protein labelling and microfluidic technologies will ensure continued progress on improving resolution and throughput, providing deeper understanding of this most important group of targets.