Membrane proteins play vital roles in living organisms, serving as targets for most currently prescribed drugs. Membrane protein structural biology aims to provide accurate structural information to understand their mechanisms of action. The advance of membrane protein structural biology has primarily relied on detergent-based methods over the past several decades. However, detergent-based approaches have significant drawbacks because detergents often damage the native protein–lipid interactions, which are often crucial for maintaining the natural structure and function of membrane proteins. Detergent-free methods recently have emerged as alternatives with a great promise, e.g. for high-resolution structure determinations of membrane proteins in their native cell membrane lipid environments. This minireview critically examines the current status of detergent-free methods by a comparative analysis of five groups of membrane protein structures determined using detergent-free and detergent-based methods. This analysis reveals that current detergent-free systems, such as the styrene-maleic acid lipid particles (SMALP), the diisobutyl maleic acid lipid particles (DIBMALP), and the cycloalkane-modified amphiphile polymer (CyclAPol) technologies are not better than detergent-based approaches in terms of maintenance of native cell membrane lipids on the transmembrane domain and high-resolution structure determination. However, another detergent-free technology, the native cell membrane nanoparticles (NCMN) system, demonstrated improved maintenance of native cell membrane lipids with the studied membrane proteins, and produced particles that were suitable for high-resolution structural analysis. The ongoing development of new membrane-active polymers and their optimization will facilitate the maturation of these new detergent-free systems.

In the twelve years since styrene maleic acid (SMA) was first used to extract and purify a membrane protein within a native lipid bilayer, this technological breakthrough has provided insight into the structural and functional details of protein–lipid interactions. Most recently, advances in cryo-EM have demonstrated that SMA-extracted membrane proteins are a rich-source of structural data. For example, it has been possible to resolve the details of annular lipids and protein–protein interactions within complexes, the nature of lipids within central cavities and binding pockets, regions involved in stabilising multimers, details of terminal residues that would otherwise remain unresolved and the identification of physiologically relevant states. Functionally, SMA extraction has allowed the analysis of membrane proteins that are unstable in detergents, the characterization of an ultrafast component in the kinetics of electron transfer that was not possible in detergent-solubilised samples and quantitative, real-time measurement of binding assays with low concentrations of purified protein. While the use of SMA comes with limitations such as its sensitivity to low pH and divalent cations, its major advantage is maintenance of a protein's lipid bilayer. This has enabled researchers to view and assay proteins in an environment close to their native ones, leading to new structural and mechanistic insights.

Phosphoglycosyl transferases (PGTs) play a pivotal role at the inception of complex glycoconjugate biosynthesis pathways across all domains of life. PGTs promote the first membrane-committed step in the en bloc biosynthetic strategy by catalyzing the transfer of a phospho-sugar from a nucleoside diphospho-sugar to a membrane-resident polyprenol phosphate. Studies on the PGTs have been hampered because they are integral membrane proteins, and often prove to be recalcitrant to expression, purification and analysis. However, in recent years exciting new information has been derived on the structures and the mechanisms of PGTs, revealing the existence of two unique superfamilies of PGT enzymes that enact catalysis at the membrane interface. Genome neighborhood analysis shows that these superfamilies, the polytopic PGT (polyPGT) and monotopic PGT (monoPGT), may initiate different pathways within the same organism. Moreover, the same fundamental two-substrate reaction is enacted through two different chemical mechanisms with distinct modes of catalysis. This review highlights the structural and mechanistic divergence between the PGT enzyme superfamilies and how this is reflected in differences in regulation in their varied glycoconjugate biosynthesis pathways.

Cdc42 is a member of the Rho family of small GTPases and a key regulator of the actin cytoskeleton, controlling cell motility, polarity and cell cycle progression. It signals downstream of the master regulator Ras and is essential for cell transformation by this potent oncogene. Overexpression of Cdc42 is observed in several cancers, where it is linked to poor prognosis. As a regulator of both cell architecture and motility, deregulation of Cdc42 is also linked to tumour metastasis. Like Ras, Cdc42 and other components of the signalling pathways it controls represent important potential targets for cancer therapeutics. In this review, we consider the progress that has been made targeting Cdc42, its regulators and effectors, including new modalities and new approaches to inhibition. Strategies under consideration include inhibition of lipid modification, modulation of Cdc42–GEF, Cdc42–GDI and Cdc42-effector interactions, and direct inhibition of downstream effectors.

PRC2 deposits the H3K27me3 repressive mark, which facilitates transcription repression of developmental genes. The decision of whether a particular gene is silenced at a given point during development is heavily dependent on the chromatin context. More than just a simple epigenetic writer, PRC2 employs several distinct chromatin reading capabilities to sense the local chromatin environment and modulate the H3K27me3 writer activity in a context-dependent manner. Here we discuss the complex interplay of PRC2 with the hallmarks of active and repressive chromatin, how it affects H3K27me3 deposition and how it guides transcriptional activity.

Systemic amyloidosis is defined as a protein misfolding disease in which the amyloid is not necessarily deposited within the same organ that produces the fibril precursor protein. There are different types of systemic amyloidosis, depending on the protein constructing the fibrils. This review will focus on recent advances made in the understanding of the structural basis of three major forms of systemic amyloidosis: systemic AA, AL and ATTR amyloidosis. The three diseases arise from the misfolding of serum amyloid A protein, immunoglobulin light chains or transthyretin. The presented advances in understanding were enabled by recent progress in the methodology available to study amyloid structures and protein misfolding, in particular concerning cryo-electron microscopy (cryo-EM) and nuclear magnetic resonance (NMR) spectroscopy. An important observation made with these techniques is that the structures of previously described in vitro formed amyloid fibrils did not correlate with the structures of amyloid fibrils extracted from diseased tissue, and that in vitro fibrils were typically more protease sensitive. It is thus possible that ex vivo fibrils were selected in vivo by their proteolytic stability.

Driven by transmembrane electrochemical ion gradients, F-type ATP synthases are the primary source of the universal energy currency, adenosine triphosphate (ATP), throughout all domains of life. The ATP synthase found in the thylakoid membranes of photosynthetic organisms has some unique features not present in other bacterial or mitochondrial systems. Among these is a larger-than-average transmembrane rotor ring and a redox-regulated switch capable of inhibiting ATP hydrolysis activity in the dark by uniquely adapted rotor subunit modifications. Here, we review recent insights into the structure and mechanism of ATP synthases specifically involved in photosynthesis and explore the cellular physiological consequences of these adaptations at short and long time scales.

The MET receptor tyrosine kinase (RTK) and its cognate ligand hepatocyte growth factor (HGF) comprise a signaling axis essential for development, wound healing and tissue homeostasis. Aberrant HGF/MET signaling is a driver of many cancers and contributes to drug resistance to several approved therapeutics targeting other RTKs, making MET itself an important drug target. In RTKs, homeostatic receptor signaling is dependent on autoinhibition in the absence of ligand binding and orchestrated set of conformational changes induced by ligand-mediated receptor dimerization that result in activation of the intracellular kinase domains. A fundamental understanding of these mechanisms in the MET receptor remains incomplete, despite decades of research. This is due in part to the complex structure of the HGF ligand, which remains unknown in its full-length form, and a lack of high-resolution structures of the complete MET extracellular portion in an apo or ligand-bound state. A current view of HGF-dependent MET activation has evolved from biochemical and structural studies of HGF and MET fragments and here we review what these findings have thus far revealed.

Serpins ( ser ine p roteinase in hibitor s ) are an ancient superfamily of structurally similar proteins, the majority of which use an elegant suicide inhibition mechanism to target serine proteinases. Despite likely evolving from a single common ancestor, the 36 human serpins have established roles regulating diverse biological processes, such as blood coagulation, embryonic development and extracellular matrix (ECM) turnover. Genetic mutations in serpin genes underpin a host of monogenic disorders — collectively termed the ‘serpinopathies’ — but serpin dysregulation has also been shown to drive pathological mechanisms in many common diseases. Osteoarthritis is a degenerative joint disorder, characterised by the progressive destruction of articular cartilage. This breakdown of the cartilage is driven by the metalloproteinases, and it has long been established that an imbalance of metalloproteinases to their inhibitors is of critical importance. More recently, a role for serine proteinases in cartilage destruction is emerging; including the activation of latent matrix metalloproteinases and cell-surface receptors, or direct proteolysis of the ECM. Serpins likely regulate these processes, as well as having roles beyond serine proteinase inhibition. Indeed, serpins are routinely observed to be highly modulated in osteoarthritic tissues and fluids by ‘omic analysis, but despite this, they are largely ignored. Confusing nomenclature and an underappreciation for the role of serine proteinases in osteoarthritis (OA) being the likely causes. In this narrative review, serpin structure, biochemistry and nomenclature are introduced, and for the first time, their putative importance in maintaining joint tissues — as well as their dysregulation in OA — are explored.

Histone lysine methyltransferases (HKMTs) are key regulators of many cellular processes. By definition, HKMTs catalyse the methylation of lysine residues in histone proteins. The enzymatic activities of HKMTs are under precise control, with their allosteric regulation emerging as a prevalent paradigm. We review the molecular mechanisms of allosteric regulation of HKMTs using well-studied histone H3 (K4, K9, K27 and K36) methyltransferases as examples. We discuss the current advances and future potential in targeting allosteric sites of HKMTs for drug development.

Ubiquitination is the major criteria for the recognition of a substrate-protein by the 26S proteasome. Additionally, a disordered segment on the substrate — either intrinsic or induced — is critical for proteasome engagement. The proteasome is geared to interact with both of these substrate features and prepare it for degradation. To facilitate substrate accessibility, resting proteasomes are characterised by a peripheral distribution of ubiquitin receptors on the 19S regulatory particle (RP) and a wide-open lateral surface on the ATPase ring. In this substrate accepting state, the internal channel through the ATPase ring is discontinuous, thereby obstructing translocation of potential substrates. The binding of the conjugated ubiquitin to the ubiquitin receptors leads to contraction of the 19S RP. Next, the ATPases engage the substrate at a disordered segment, energetically unravel the polypeptide and translocate it towards the 20S catalytic core (CP). In this substrate engaged state, Rpn11 is repositioned at the pore of the ATPase channel to remove remaining ubiquitin modifications and accelerate translocation. C-termini of five of the six ATPases insert into corresponding lysine-pockets on the 20S α-ring to complete 20S CP gate opening. In the resulting substrate processing state, the ATPase channel is fully contiguous with the translocation channel into the 20S CP, where the substrate is proteolyzed. Complete degradation of a typical ubiquitin-conjugate takes place over a few tens of seconds while hydrolysing tens of ATP molecules in the process (50 kDa/∼50 s/∼80ATP). This article reviews recent insight into biochemical and structural features that underlie substrate recognition and processing by the 26S proteasome.

Bifidobacteria have attracted significant attention because they provide health-promoting effects in the human gut. In this review, we present a current overview of the three-dimensional structures of bifidobacterial proteins involved in carbohydrate uptake, degradation, and metabolism. As predominant early colonizers of the infant's gut, distinct bifidobacterial species are equipped with a panel of transporters and enzymes specific for human milk oligosaccharides (HMOs). Interestingly, Bifidobacterium bifidum and Bifidobacterium longum possess lacto- N -biosidases with unrelated structural folds to release the disaccharide lacto- N -biose from HMOs, suggesting the convergent evolution of this activity from different ancestral proteins. The crystal structures of enzymes that confer the degradation of glycans from the mucin glycoprotein layer provide a structural basis for the utilization of this sustainable nutrient in the gastrointestinal tract. The utilization of several plant dietary oligosaccharides has been studied in detail, and the prime importance of oligosaccharide-specific ATP-binding cassette (ABC) transporters in glycan utilisations by bifidobacteria has been revealed. The structural elements underpinning the high selectivity and roles of ABC transporter binding proteins in establishing competitive growth on preferred oligosaccharides are discussed. Distinct ABC transporters are conserved across several bifidobacterial species, e.g. those targeting arabinoxylooligosaccharide and α-1,6-galactosides/glucosides. Less prevalent transporters, e.g. targeting β-mannooligosaccharides, may contribute to the metabolic specialisation within Bifidobacterium . Some bifidobacterial species have established symbiotic relationships with humans. Structural studies of carbohydrate-utilizing systems in Bifidobacterium have revealed the interesting history of molecular coevolution with the host, as highlighted by the early selection of bifidobacteria by mucin and breast milk glycans.

The biological activities of living organisms involve various inputs and outputs. The ATP-driven substances (biomolecules) responsible for these kinds of activities through membrane (i.e. uptake and efflux of substrates) include ATP-binding cassette (ABC) transporters, some of which play important roles in multidrug resistance. The basic architecture of ABC transporters comprises transmembrane domains (TMDs) and nucleotide-binding domains (NBDs). The functional dynamics (substrate transport) of ABC transporters are realized by concerted motions, such as NBD dimerization, mechanical transmission via coupling helices (CHs), and the translocation of substrates through TMDs, which are induced by the binding and/or hydrolysis of ATP molecules and substrates. In this mini-review, we briefly discuss recent progresses in the structural dynamics as revealed by molecular simulation studies at all-atom (AA), coarse-grained (CG), and quantum mechanics/molecular mechanics (QM/MM) levels.

The material properties of cellulose are heavily influenced by the organisation of β-1,4-glucan chains into a microfibril. It is likely that the structure of this microfibril is determined by the spatial arrangement of catalytic cellulose synthase (CESA) proteins within the cellulose synthase complex (CSC). In land plants, CESA proteins form a large complex composed of a hexamer of trimeric lobes termed the rosette. Each rosette synthesises a single microfibril likely composed of 18 glucan chains. In this review, the biochemical events leading to plant CESA protein assembly into the rosette are explored. The protein interfaces responsible for CESA trimerization are formed by regions that define rosette-forming CESA proteins. As a consequence, these regions are absent from the ancestral bacterial cellulose synthases (BcsAs) that do not form rosettes. CSC assembly occurs within the context of the endomembrane system, however the site of CESA assembly into trimers and rosettes is not determined. Both the N-Terminal Domain and Class Specific Region of CESA proteins are intrinsically disordered and contain all of the identified phosphorylation sites, making both regions candidates as sites for protein–protein interactions and inter–lobe interface formation. We propose a sequential assembly model, whereby CESA proteins form stable trimers shortly after native folding, followed by sequential recruitment of lobes into a rosette, possibly assisted by Golgi-localised STELLO proteins. A comprehensive understanding of CESA assembly into the CSC will enable directed engineering of CESA protein spatial arrangements, allowing changes in cellulose crystal packing that alter its material properties.

Super-resolution microscopy has revolutionised the way we observe biological systems. These methods are now a staple of fluorescence microscopy. Researchers have used super-resolution methods in myriad systems to extract nanoscale spatial information on multiple interacting parts. These methods are continually being extended and reimagined to further push their resolving power and achieve truly single protein resolution. Here, we explore the most recent advances at the frontier of the ‘super-resolution’ limit and what opportunities remain for further improvements in the near future.

Allostery, in which binding of ligands to remote sites causes a functional change in the active sites, is a fascinating phenomenon observed in enzymes. Allostery can occur either with or without significant conformational changes in the enzymes, and the molecular basis of its mechanism can be difficult to decipher using only experimental techniques. Computational tools for analyzing enzyme sequences, structures, and dynamics can provide insights into the allosteric mechanism at the atomic level. Combining computational and experimental methods offers a powerful strategy for the study of enzyme allostery. The aromatic amino acid biosynthesis pathway is essential in microorganisms and plants. Multiple enzymes involved in this pathway are sensitive to feedback regulation by pathway end products and are known to use allostery to control their activities. To date, four enzymes in the aromatic amino acid biosynthesis pathway have been computationally investigated for their allosteric mechanisms, including 3-deoxy- d - arabino -heptulosonate 7-phosphate synthase, anthranilate synthase, chorismate mutase, and tryptophan synthase. Here we review the computational studies and findings on the allosteric mechanisms of these four enzymes. Results from these studies demonstrate the capability of computational tools and encourage future computational investigations of allostery in other enzymes of this pathway.

Lytic polysaccharide monooxygenases (LPMOs) are mononuclear copper enzymes that catalyse the oxidative cleavage of glycosidic bonds. They are characterised by two histidine residues that coordinate copper in a configuration termed the Cu-histidine brace. Although first identified in bacteria and fungi, LPMOs have since been found in all biological kingdoms. LPMOs are now included in commercial enzyme cocktails used in industrial biorefineries. This has led to increased process yield due to the synergistic action of LPMOs with glycoside hydrolases. However, the introduction of LPMOs makes control of the enzymatic step in industrial stirred-tank reactors more challenging, and the operational stability of the enzymes is reduced. It is clear that much is still to be learned about the interaction between LPMOs and their complex natural and industrial environments, and fundamental scientific studies are required towards this end. Several atomic-resolution structures have been solved providing detailed information on the Cu-coordination sphere and the interaction with the polysaccharide substrate. However, the molecular mechanisms of LPMOs are still the subject of intense investigation; the key question being how the proteinaceous environment controls the copper cofactor towards the activation of the O-O bond in O 2 and cleavage of the glycosidic bonds in polysaccharides. The need for biochemical characterisation of each putative LPMO is discussed based on recent reports showing that not all proteins with a Cu-histidine brace are enzymes.

Enzyme catalysis is omnipresent in the cell. The mechanisms by which highly evolved protein folds enable rapid and specific chemical transformation of substrates belong to the marvels of structural biology. Targeting of enzymes with inhibitors has immediate application in drug discovery, from chemotherapeutics over antibiotics to antivirals. NMR spectroscopy combines multiple assets for the investigation of enzyme function. The non-invasive technique can probe enzyme structure and dynamics and map interactions with substrates, cofactors and inhibitors at the atomic level. With experiments performed at close to native conditions, catalytic transformations can be monitored in real time, giving access to kinetic parameters. The power of NMR in the solid state, in contrast with solution, lies in the absence of fundamental size limitations, which is crucial for enzymes that are either membrane-embedded or assemble into large soluble complexes exceeding hundreds of kilodaltons in molecular weight. Here we review recent progress in solid-state NMR methodology, which has taken big leaps in the past years due to steady improvements in hardware design, notably magic angle spinning, and connect it to parallel biochemical advances that enable isotope labelling of increasingly complex enzymes. We first discuss general concepts and requirements of the method and then highlight the state-of-the-art in sample preparation, structure determination, dynamics and interaction studies. We focus on examples where solid-state NMR has been instrumental in elucidating enzyme mechanism, alone or in integrative studies.

Small GTPases, in association with their GEFs, GAPs and effectors, control major intracellular processes such as signal transduction, cytoskeletal dynamics and membrane trafficking. Accordingly, dysfunctions in their biochemical properties are associated with many diseases, including cancers, diabetes, infections, mental disorders and cardiac diseases, which makes them attractive targets for therapies. However, small GTPases signalling modules are not well-suited for classical inhibition strategies due to their mode of action that combines protein–protein and protein–membrane interactions. As a consequence, there is still no validated drug available on the market that target small GTPases, whether directly or through their regulators. Alternative inhibitory strategies are thus highly needed. Here we review recent studies that highlight the unique modalities of the interaction of small GTPases and their GEFs at the periphery of membranes, and discuss how they can be harnessed in drug discovery.

Ion Mobility (IM) coupled to mass spectrometry (MS) is a useful tool for separating species of interest out of small quantities of heterogenous mixtures via a combination of m/z and molecular shape. While tandem MS instruments are common, instruments which employ tandem IM are less so with the first commercial IM–MS instrument capable of multiple IM selection rounds being released in 2019. Here we explore the history of tandem IM instruments, recent developments, the applications to biological systems and expected future directions.

The transport of histones from the cytoplasm to the nucleus of the cell, through the nuclear membrane, is a cellular process that regulates the supply of new histones in the nucleus and is key for DNA replication and transcription. Nuclear import of histones is mediated by proteins of the karyopherin family of nuclear transport receptors. Karyopherins recognize their cargos through linear motifs known as nuclear localization/export sequences or through folded domains in the cargos. Karyopherins interact with nucleoporins, proteins that form the nuclear pore complex, to promote the translocation of their cargos into the nucleus. When binding to histones, karyopherins not only function as nuclear import receptors but also as chaperones, protecting histones from non-specific interactions in the cytoplasm, in the nuclear pore and possibly in the nucleus. Studies have also suggested that karyopherins might participate in histones deposition into nucleosomes. In this review we describe structural and biochemical studies from the last two decades on how karyopherins recognize and transport the core histone proteins H3, H4, H2A and H2B and the linker histone H1 from the cytoplasm to the nucleus, which karyopherin is the major nuclear import receptor for each of these histones, the oligomeric state of histones during nuclear import and the roles of post-translational modifications, histone-chaperones and RanGTP in regulating these nuclear import pathways.

The bacterial SbcC/SbcD DNA repair proteins were identified over a quarter of a century ago. Following the subsequent identification of the homologous Mre11/Rad50 complex in the eukaryotes and archaea, it has become clear that this conserved chromosomal processing machinery is central to DNA repair pathways and the maintenance of genomic stability in all forms of life. A number of experimental studies have explored this intriguing genome surveillance machinery, yielding significant insights and providing conceptual advances towards our understanding of how this complex operates to mediate DNA repair. However, the inherent complexity and dynamic nature of this chromosome-manipulating machinery continue to obfuscate experimental interrogations, and details regarding the precise mechanisms that underpin the critical repair events remain unanswered. This review will summarize our current understanding of the dramatic structural changes that occur in Mre11/Rad50 complex to mediate chromosomal tethering and accomplish the associated DNA processing events. In addition, undetermined mechanistic aspects of the DNA enzymatic pathways driven by this vital yet enigmatic chromosomal surveillance and repair apparatus will be discussed. In particular, novel and putative models of DNA damage recognition will be considered and comparisons will be made between the modes of action of the Rad50 protein and other related ATPases of the overarching SMC superfamily.

The race to identify a successful treatment for COVID19 will be defined by fundamental research into the replication cycle of the SARS-CoV-2 virus. This has identified five distinct stages from which numerous vaccination and clinical trials have emerged alongside an innumerable number of drug discovery studies currently in development for disease intervention. Informing every step of the viral replication cycle has been an unprecedented ‘call-to-arms' by the global structural biology community. Of the 20 main SARS-CoV-2 proteins, 13 have been resolved structurally for SARS-CoV-2 with most having a related SARS-CoV and MERS-CoV structural homologue totalling some 300 structures currently available in public repositories. Herein, we review the contribution of structural studies to our understanding of the virus and their role in structure-based development of therapeutics.

Varicella–zoster virus (VZV) is the causative agent of chicken pox (varicella) and shingles (zoster). Although considered benign diseases, both varicella and zoster can cause complications. Zoster is painful and can lead to post herpetic neuralgia. VZV has also been linked to stroke, related to giant cell arteritis in some cases. Vaccines are available but the attenuated vaccine is not recommended in immunocompromised individuals and the efficacy of the glycoprotein E (gE) based subunit vaccine has not been evaluated for the prevention of varicella. A hallmark of VZV pathology is the formation of multinucleated cells termed polykaryocytes in skin lesions. This cell–cell fusion (abbreviated as cell fusion) is mediated by the VZV glycoproteins gB, gH and gL, which constitute the fusion complex of VZV, also needed for virion entry. Expression of gB, gH and gL during VZV infection and trafficking to the cell surface enables cell fusion. Recent evidence supports the concept that cellular processes are required for regulating cell fusion induced by gB/gH–gL. Mutations within the carboxyl domains of either gB or gH have profound effects on fusion regulation and dramatically restrict the ability of VZV to replicate in human skin. This loss of regulation modifies the transcriptome of VZV infected cells. Furthermore, cellular proteins have significant effects on the regulation of gB/gH–gL-mediated cell fusion and the replication of VZV, exemplified by the cellular phosphatase, calcineurin. This review provides the current state-of-the-art knowledge about the molecular controls of cell fusion-dependent pathogenesis caused by VZV.

Interaction of PCNA with DNA polymerase is vital to efficient and processive DNA synthesis. PCNA being a homotrimeric ring possesses three hydrophobic pockets mostly involved in an interaction with its binding partners. PCNA interacting proteins contain a short sequence of eight amino acids, popularly coined as PIP motif, which snuggly fits into the hydrophobic pocket of PCNA to stabilize the interaction. In the last two decades, several PIP motifs have been mapped or predicted in eukaryotic DNA polymerases. In this review, we summarize our understandings of DNA polymerase-PCNA interaction, the function of such interaction during DNA synthesis, and emphasize the lacunae that persist. Because of the presence of multiple ligands in the replisome complex and due to many interaction sites in DNA polymerases, we also propose two modes of DNA polymerase positioning on PCNA required for DNA synthesis to rationalize the tool-belt model of DNA replication.

TRIM proteins form a protein family that is characterized by a conserved tripartite motif domain comprising a RING domain, one or two B-box domains and a coiled-coil region. Members of this large protein family are important regulators of numerous cellular functions including innate immune responses, transcriptional regulation and apoptosis. Key to their cellular role is their E3 ligase activity which is conferred by the RING domain. Self-association is an important characteristic of TRIM protein activity and is mediated by homodimerization via the coiled-coil region, and in some cases higher order association via additional domains of the tripartite motif. In many of the TRIM family proteins studied thus far, RING dimerization is an important prerequisite for E3 ligase enzymatic activity though the propensity of RING domains to dimerize differs significantly between different TRIMs and can be influenced by other regions of the protein.

Why proteins are fuzzy? Constant adaptation to the cellular environment requires a wide range of changes in protein structure and interactions. Conformational ensembles of disordered proteins in particular exhibit large shifts to activate or inhibit alternative pathways. Fuzziness is critical for liquid–liquid phase separation and conversion of biomolecular condensates into fibrils. Interpretation of these phenomena presents a challenge for the classical structure-function paradigm. Here I discuss a multi-valued formalism, based on fuzzy logic, which can be applied to describe complex cellular behavior of proteins.

The aim of structural biology has been always the study of biological macromolecules structures and their mechanistic behaviour at molecular level. To achieve its goal, multiple biophysical methods and approaches have become part of the structural biology toolbox. Considered as one of the pillars of structural biology, X-ray crystallography has been the most successful method for solving three-dimensional protein structures at atomic level to date. It is however limited by the success in obtaining well-ordered protein crystals that diffract at high resolution. This is especially true for challenging targets such as membrane proteins (MPs). Understanding structure-function relationships of MPs at the biochemical level is vital for medicine and drug discovery as they play critical roles in many cellular processes. Though difficult, structure determination of MPs by X-ray crystallography has significantly improved in the last two decades, mainly due to many relevant technological and methodological developments. Today, numerous MP crystal structures have been solved, revealing many of their mechanisms of action. Yet the field of structural biology has also been through significant technological breakthroughs in recent years, particularly in the fields of single particle electron microscopy (cryo-EM) and X-ray free electron lasers (XFELs). Here we summarise the most important advancements in the field of MP crystallography and the significance of these developments in the present era of modern structural biology.

The structure-function paradigm has guided investigations into the molecules involved in cellular signalling for decades. The peripheries of this paradigm, however, start to unravel when considering the co-operation between proteins and the membrane in signalling processes. Intrinsically disordered regions hold distinct advantages over folded domains in terms of their binding promiscuity, sensitivity to their particular environment and their ease of modulation through post-translational modifications. Low sequence complexity and bias towards charged residues are also favourable for the multivalent electrostatic interactions that occur at the surfaces of lipid bilayers. This review looks at the principles behind the successful marriage between protein disorder and membranes in addition to the role of this partnership in modifying and regulating signalling in cellular processes. The HVR (hypervariable region) of small GTPases is highlighted as a well-studied example of the nuanced role a short intrinsically disordered region can play in the fine-tuning of signalling pathways.

Retromer (VPS26/VPS35/VPS29) is a highly conserved eukaryotic protein complex that localizes to endosomes to sort transmembrane protein cargoes into vesicles and elongated tubules. Retromer mediates retrieval pathways from endosomes to the trans -Golgi network in all eukaryotes and further facilitates recycling pathways to the plasma membrane in metazoans. In cells, retromer engages multiple partners to orchestrate the formation of tubulovesicular structures, including sorting nexin (SNX) proteins, cargo adaptors, GTPases, regulators, and actin remodeling proteins. Retromer-mediated pathways are especially important for sorting cargoes required for neuronal maintenance, which links retromer loss or mutations to multiple human brain diseases and disorders. Structural and biochemical studies have long contributed to the understanding of retromer biology, but recent advances in cryo-electron microscopy and cryo-electron tomography have further uncovered exciting new snapshots of reconstituted retromer structures. These new structures reveal retromer assembles into an arch-shaped scaffold and suggest the scaffold may be flexible and adaptable in cells. Interactions with cargo adaptors, particularly SNXs, likely orient the scaffold with respect to phosphatidylinositol-3-phosphate (PtdIns3 P )-enriched membranes. Pharmacological small molecule chaperones have further been shown to stabilize retromer in cultured cell and mouse models, but mechanisms by which these molecules bind remain unknown. This review will emphasize recent structural and biophysical advances in understanding retromer structure as the field moves towards a molecular view of retromer assembly and regulation on membranes.

RAS GTPases are fundamental regulators of development and drivers of an extraordinary number of human cancers. RAS oncoproteins constitutively signal through downstream effector proteins, triggering cancer initiation, progression and metastasis. In the absence of targeted therapeutics to mutant RAS itself, inhibitors of downstream pathways controlled by the effector kinases RAF and PI3K have become tools in the treatment of RAS-driven tumours. Unfortunately, the efficacy of this approach has been greatly minimized by the prevalence of acquired drug resistance. Decades of research have established that RAS signalling is highly complex, and in addition to RAF and PI3K these small GTPase proteins can interact with an array of alternative effectors that feature RAS binding domains. The consequence of RAS binding to these effectors remains relatively unexplored, but these pathways may provide targets for combinatorial therapeutics. We discuss here three candidate alternative effectors: RALGEFs, RASSF5 and AFDN, detailing their interaction with RAS GTPases and their biological significance. The metastatic nature of RAS-driven cancers suggests more attention should be granted to these alternate pathways, as they are highly implicated in the regulation of cell adhesion, polarity, cell size and cytoskeletal architecture.

The use of neutrons as a scattering probe to investigate biological membranes has steadily grown in the past three decades, shedding light on the structure and behaviour of this ubiquitous and fundamental biological barrier. Meanwhile, the rise of antibiotic resistance has catalysed a renewed interest in understanding the mechanisms underlying the dynamics of antibiotics interaction with the bacterial cell envelope. It is widely recognised that the key reason behind the remarkable success of Gram-negative pathogens in developing antibiotic resistance lies in the effectiveness of their outer membrane (OM) in defending the cell from antibacterial compounds. Critical to its function, the highly asymmetric lipid distribution between the inner and outer bilayer leaflets of the OM, adds an extra level of complexity to the study of this crucial defence barrier. Here we review the opportunities offered by neutron scattering techniques, in particular reflectometry, to provide structural information on the interactions of antimicrobials with in vitro models of the OM. The differential sensitivity of neutrons towards hydrogen and deuterium makes them a unique probe to study the structure and behaviour of asymmetric membranes. Molecular-level understanding of the interactions between antimicrobials and the Gram-negative OM provides valuable insights that can aid drug development and broaden our knowledge of this critically important biological barrier.

Condensin and cohesin, both members of the structural maintenance of chromosome (SMC) family, contribute to the regulation and structure of chromatin. Recent work has shown both condensin and cohesin extrude DNA loops and most likely work via a conserved mechanism. This review focuses on condensin complexes, highlighting recent in vitro work characterising DNA loop formation and protein structure. We discuss similarities between condensin and cohesin complexes to derive a possible mechanistic model, as well as discuss differences that exist between the different condensin isoforms found in higher eukaryotes.

The efficacy of superoxide dismutase-1 (SOD1) folding impacts neuronal loss in motor system neurodegenerative diseases. Mutations can prevent SOD1 post-translational processing leading to misfolding and cytoplasmic aggregation in familial amyotrophic lateral sclerosis (ALS). Evidence of immature, wild-type SOD1 misfolding has also been observed in sporadic ALS, non-SOD1 familial ALS and Parkinson's disease. The copper chaperone for SOD1 (hCCS) is a dedicated and specific chaperone that assists SOD1 folding and maturation to produce the active enzyme. Misfolded or misfolding prone SOD1 also interacts with heat shock proteins and macrophage migration inhibitory factor to aid folding, refolding or degradation. Recognition of specific SOD1 structures by the molecular chaperone network and timely dissociation of SOD1-chaperone complexes are, therefore, important steps in SOD1 processing. Harnessing these interactions for therapeutic benefit is actively pursued as is the modulation of SOD1 behaviour with pharmacological and peptide chaperones. This review highlights the structural and mechanistic aspects of a selection of SOD1-chaperone interactions together with their impact on disease models.

The structural maintenance of chromosomes hinge domain containing protein 1 (SMCHD1) is a large multidomain protein involved in epigenetic gene silencing. Variations in the SMCHD1 gene are associated with two debilitating human disorders, facioscapulohumeral muscular dystrophy (FSHD) and Bosma arhinia microphthalmia syndrome (BAMS). Failure of SMCHD1 to silence the D4Z4 macro-repeat array causes FSHD, yet the consequences on gene silencing of SMCHD1 variations associated with BAMS are currently unknown. Despite the interest due to these roles, our understanding of the SMCHD1 protein is in its infancy. Most knowledge of SMCHD1 function is based on its similarity to the structural maintenance of chromosomes (SMC) proteins, such as cohesin and condensin. SMC proteins and SMCHD1 share similar domain organisation and affect chromatin conformation. However, there are important differences between the domain architectures of SMC proteins and SMCHD1, which distinguish SMCHD1 as a non-canonical member of the family. In the last year, the crystal structures of the two key domains crucial to SMCHD1 function, the ATPase and hinge domains, have emerged. These structures reveal new insights into how SMCHD1 may bind and regulate chromatin structure, and address how amino acid variations in SMCHD1 may contribute to BAMS and FSHD. Here, we contrast SMCHD1 with canonical SMC proteins, and relate the ATPase and hinge domain structures to their roles in SMCHD1-mediated epigenetic silencing and disease.

Recently, there have been significant advancements in dynamic-mode atomic force microscopy (AFM) for biological applications. With frequency modulation AFM (FM-AFM), subnanometer-scale surface structures of biomolecules such as secondary structures of proteins, phosphate groups of DNAs, and lipid-ion complexes have been directly visualized. In addition, three-dimensional AFM (3D-AFM) has been developed by combining a high-resolution AFM technique with a 3D tip scanning method. This method enabled visualization of 3D distributions of water (i.e. hydration structures) with subnanometer-scale resolution on various biological molecules such as lipids, proteins, and DNAs. Furthermore, 3D-AFM also allows visualization of subnanometer-scale 3D distributions of flexible surface structures such as thermally fluctuating lipid headgroups. Such a direct local information at nano-bio interfaces can play a critical role in determining the atomic- or molecular-scale model to explain interfacial structures and functions. Here, we present an overview of these recent advancements in the dynamic-mode AFM techniques and their biological applications.

Proteins are dynamic molecules that can transition between a potentially wide range of structures comprising their conformational ensemble. The nature of these conformations and their relative probabilities are described by a high-dimensional free energy landscape. While computer simulation techniques such as molecular dynamics simulations allow characterisation of the metastable conformational states and the transitions between them, and thus free energy landscapes, to be characterised, the barriers between states can be high, precluding efficient sampling without substantial computational resources. Over the past decades, a dizzying array of methods have emerged for enhancing conformational sampling, and for projecting the free energy landscape onto a reduced set of dimensions that allow conformational states to be distinguished, known as collective variables (CVs), along which sampling may be directed. Here, a brief description of what biomolecular simulation entails is followed by a more detailed exposition of the nature of CVs and methods for determining these, and, lastly, an overview of the myriad different approaches for enhancing conformational sampling, most of which rely upon CVs, including new advances in both CV determination and conformational sampling due to machine learning.

The Wnt signalling pathways are of great importance in embryonic development and oncogenesis. Canonical and non-canonical Wnt signalling pathways are known, with the canonical (or β-catenin dependent) pathway being perhaps the best studied of these. While structural knowledge of proteins and interactions involved in canonical Wnt signalling has accumulated over the past 20 years, the pace of discovery has increased in recent years, with the structures of several key proteins and assemblies in the pathway being released. In this review, we provide a brief overview of canonical Wnt signalling, followed by a comprehensive overview of currently available X-ray, NMR and cryoEM data elaborating the structures of proteins and interactions involved in canonical Wnt signalling. While the volume of structures available is considerable, numerous gaps in knowledge remain, particularly a comprehensive understanding of the assembly of large multiprotein complexes mediating key aspects of pathway, as well as understanding the structure and activation of membrane receptors in the pathway. Nonetheless, the presently available data affords considerable opportunities for structure-based drug design efforts targeting canonical Wnt signalling.

For more than 40 years, the oligomeric state of members of the mitochondrial carrier family (SLC25) has been the subject of debate. Initially, the consensus was that they were dimeric, based on the application of a large number of different techniques. However, the structures of the mitochondrial ADP/ATP carrier, a member of the family, clearly demonstrated that its structural fold is monomeric, lacking a conserved dimerisation interface. A re-evaluation of previously published data, with the advantage of hindsight, concluded that technical errors were at the basis of the earlier dimer claims. Here, we revisit this topic, as new claims for the existence of dimers of the bovine ADP/ATP carrier have emerged using native mass spectrometry of mitochondrial membrane vesicles. However, the measured mass does not agree with previously published values, and a large number of post-translational modifications are proposed to account for the difference. Contrarily, these modifications are not observed in electron density maps of the bovine carrier. If they were present, they would interfere with the structure and function of the carrier, including inhibitor and substrate binding. Furthermore, the reported mass does not account for three tightly bound cardiolipin molecules, which are consistently observed in other studies and are important stabilising factors for the transport mechanism. The monomeric carrier has all of the required properties for a functional transporter and undergoes large conformational changes that are incompatible with a stable dimerisation interface. Thus, our view that the native mitochondrial ADP/ATP carrier exists and functions as a monomer remains unaltered.

The phosphatidylinositol 3-kinase (PI3K) pathway is a critical regulator of many cellular processes including cell survival, growth, proliferation and motility. Not surprisingly therefore, the PI3K pathway is one of the most frequently mutated pathways in human cancers. In addition to their canonical role as part of the PI3K holoenzyme, the class IA PI3K regulatory subunits undertake critical functions independent of PI3K. The PI3K regulatory subunits exist in excess over the p110 catalytic subunits and therefore free in the cell. p110-independent p85 is unstable and exists in a monomer-dimer equilibrium. Two conformations of dimeric p85 have been reported that are mediated by N-terminal and C-terminal protein domain interactions, respectively. The role of p110-independent p85 is under investigation and it has been found to perform critical adaptor functions, sequestering or influencing compartmentalisation of key signalling proteins. Free p85 has roles in glucose homeostasis, cellular stress pathways, receptor trafficking and cell migration. As a regulator of fundamental pathways, the amount of p110-independent p85 in the cell is critical. Factors that influence the monomer-dimer equilibrium of p110-independent p85 offer additional control over this system, disruption to which likely results in disease. Here we review the current knowledge of the structure and functions of p110-independent class IA PI3K regulatory subunits.

Pygo is a nuclear protein containing two conserved domains, NHD and PHD, which play important roles in embryonic development and carcinogenesis. Pygo was first identified as a core component of the Wnt/β-catenin signalling pathway. However, it has also been reported that the function of Pygo is not always Wnt/β-catenin signalling dependent. In this review, we summarise the functions of both domains of Pygo and show that their functions are synergetic. The PHD domain mainly combines with transcription co-factors, including histone 3 and Bcl9/9l. The NHD domain mainly recruits histone methyltransferase/acetyltransferase (HMT/HAT) to modify lysine 4 of the histone 3 tail (H3K4) and interacts with Chip/LIM-domain DNA-binding proteins (ChiLS) to form enhanceosomes to regulate transcriptional activity. Furthermore, we summarised chromatin modification differences of Pygo in Drosophila (dPygo) and vertebrates, and found that Pygo displayes a chromatin silencing function in Drosophila , while in vertebates, Pygo has a chromatin-activating function due to the two substitution of two amino acid residues. Next, we confirmed the relationship between Pygo and Bcl9/9l and found that Pygo–Bcl/9l are specifically partnered both in the nucleus and in the cytoplasm. Finally, we discuss whether transcriptional activity of Pygo is Wnt/β-catenin dependent during embryonic development. Available information indications that the transcriptional activity of Pygo in embryonic development is either Wnt/β-catenin dependent or independent in both tissue-specific and cell-specific-modes.

Ubiquitination is a fundamental post-translational modification that regulates almost all aspects of cellular signalling and is ultimately catalysed by the action of E3 ubiquitin ligases. The RING-between-RING (RBR) family of E3 ligases encompasses 14 distinct human enzymes that are defined by a unique domain organisation and catalytic mechanism. Detailed characterisation of several RBR ligase family members in the last decade has revealed common structural and mechanistic features. At the same time these studies have highlighted critical differences with respect to autoinhibition, activation and catalysis. Importantly, the majority of RBR E3 ligases remain poorly studied, and thus the extent of diversity within the family remains unknown. In this mini-review we outline the current understanding of the RBR E3 mechanism, structure and regulation with a particular focus on recent findings and developments that will shape the field in coming years.

Crystallographic structural analysis is an essential method for the determination of protein structure. However, crystallization of a protein of interest is the most difficult process in the analysis. The process is often hampered during the sample preparation, including expression and purification. Even after a sample has been purified, not all candidate proteins crystallize. In this mini-review, the current methodologies used to overcome obstacles encountered during protein crystallization are sorted. Specifically, the strategy for an effective crystallization is compared with a pipeline where various expression hosts and constructs, purification and crystallization conditions, and crystallization chaperones as target-specific binder proteins are assessed by a precrystallization screening. These methodologies are also developed continuously to improve the process. The described methods are useful for sample preparation in crystallographic analysis and other structure determination techniques, such as cryo-electron microscopy.

The unique architecture of the mycobacterial cell envelope plays an important role in Mycobacterium tuberculosis ( Mtb ) pathogenesis. A critical protein in cell envelope biogenesis in mycobacteria, required for transport of precursors, trehalose monomycolates (TMMs), is the Mycobacterial membrane protein large 3 (MmpL3). Due to its central role in TMM transport, MmpL3 has been an attractive therapeutic target and a key target for several preclinical agents. In 2019, the first crystal structures of the MmpL3 transporter and its complexes with lipids and inhibitors were reported. These structures revealed several unique structural features of MmpL3 and provided invaluable information on the mechanism of TMM transport. This review aims to highlight the recent advances made in the function of MmpL3 and summarises structural findings. The overall goal is to provide a mechanistic perspective of MmpL3-mediated lipid transport and inhibition, and to highlight the prospects for potential antituberculosis therapies.

A prominent attribute of chemical structure in microbial and plant natural products is aromatic C -glycosylation. In plants, various flavonoid natural products have a β- C - d -glucosyl moiety attached to their core structure. Natural product C -glycosides have attracted significant attention for their own unique bioactivity as well as for representing non-hydrolysable analogs of the canonical O -glycosides. The biosynthesis of natural product C -glycosides is accomplished by sugar nucleotide-dependent (Leloir) glycosyltransferases. Here, we provide an overview on the C -glycosyltransferases of microbial, plant and insect origin that have been biochemically characterized. Despite sharing basic evolutionary relationships, as evidenced by their common membership to glycosyltransferase family GT-1 and conserved GT-B structural fold, the known C -glycosyltransferases are diverse in the structural features that govern their reactivity, selectivity and specificity. Bifunctional glycosyltransferases can form C - and O -glycosides dependent on the structure of the aglycon acceptor. Recent crystal structures of plant C -glycosyltransferases and di- C -glycosyltransferases complement earlier structural studies of bacterial enzymes and provide important molecular insight into the enzymatic discrimination between C - and O -glycosylation. Studies of enzyme structure and mechanism converge on the view of a single displacement (S N 2)-like mechanism of enzymatic C -glycosyl transfer, largely analogous to O -glycosyl transfer. The distinction between reactions at the O - or C -acceptor atom is achieved through the precise positioning of the acceptor relative to the donor substrate in the binding pocket. Nonetheless, C -glycosyltransferases may differ in the catalytic strategy applied to induce nucleophilic reactivity at the acceptor carbon. Evidence from the mutagenesis of C -glycosyltransferases may become useful in engineering these enzymes for tailored reactivity.

Galectin-8 (Gal-8) is a tandem-repeat type galectin with affinity for β-galactosides, bearing two carbohydrate recognition domains (CRD) connected by a linker peptide. The N- and C-terminal domains (Gal-8N and Gal-8C) share 35% homology, and their glycan ligand specificity is notably dissimilar: while Gal-8N shows strong affinity for α(2-3)-sialylated oligosaccharides, Gal-8C has higher affinity for non-sialylated oligosaccharides, including poly- N -acetyllactosamine and/ or A and B blood group structures. Particularly relevant for understanding the biological role of this lectin, full-length Gal-8 can bind cell surface glycoconjugates with broader affinity than the isolated Gal-8N and Gal-8C domains, a trait also described for other tandem-repeat galectins. Herein, we aim to discuss the potential use of separate CRDs in modelling tandem-repeat galectin-8 and its biological functions. For this purpose, we will cover several aspects of the structure–function relationship of this protein including crystallographic structures, glycan specificity, cell function and biological roles, with the ultimate goal of understanding the potential role of each CRD in predicting full-length Gal-8 involvement in relevant biological processes.

HDX-MS has emerged as a powerful tool to interrogate the structure and dynamics of proteins and their complexes. Recent advances in the methodology and instrumentation have enabled the application of HDX-MS to membrane proteins. Such targets are challenging to investigate with conventional strategies. Developing new tools are therefore pertinent for improving our fundamental knowledge of how membrane proteins function in the cell. Importantly, investigating this central class of biomolecules within their native lipid environment remains a challenge but also a key goal ahead. In this short review, we outline recent progresses in dissecting the conformational mechanisms of membrane proteins using HDX-MS. We further describe how the use of computational strategies can aid the interpretation of experimental data and enable visualisation of otherwise intractable membrane protein states. This unique integration of experiments with computations holds significant potential for future applications.

Ribonucleic acids are driving a multitude of biological processes where they act alone or in complex with proteins (ribonucleoproteins, RNP). To understand these processes both structural and mechanistic information about RNA is necessary. Due to their conformational plasticity RNA pose a challenge for mainstream structural biology methods. Solid-state NMR (ssNMR) spectroscopy is an emerging technique that can be applied to biomolecular complexes of any size in close-to-native conditions. This review outlines recent methodological developments in ssNMR for structural characterization of RNA and protein–RNA complexes and provides relevant examples.

The activity of enzymes is subject to regulation at multiple levels. Cooperativity, the interconnected behavior of active sites within a protein complex, directly affects protein activity. Cooperativity is a mode of regulation that requires neither extrinsic factors nor protein modifications. Instead, it allows enzymes themselves to modulate reaction rates. Cooperativity is an important regulatory mechanism in soluble proteins, but also examples of cooperative membrane proteins have been described. In this review, we summarize the current knowledge on interprotomer cooperativity in elevator-type proteins, a class of membrane transporters characterized by large rigid-body movements perpendicular to the membrane, and highlight well-studied examples and experimental approaches.

Mass spectrometry (MS) has long been used to study proteins mainly via sequence identification and quantitation of expression abundance. In recent years, MS has emerged as a tool for structural biology. Intact protein structural analysis has been enabled by the development of methods such as native MS, top-down proteomics, and ion mobility MS. Other MS-based structural methods include affinity purification MS, chemical cross-linking, and protein footprinting. These methods have enabled the study of protein–protein and protein–ligand interactions and regions of conformational change. The coupling of MS with liquid chromatography has permitted the analysis of complex samples. This bottom-up proteomics workflow enables the study of protein structure in the native cellular environment and provides structural information across the proteome. It has been demonstrated that the crowded environment of the cell affects protein binding interactions and affinities. Performing studies in this complex environment is essential for understanding the functional roles of proteins. MS-based structural methods permit analysis of samples such as cell lysates, intact cells, and tissue to provide a more physiological view of protein structure. This mini-review discusses the various MS-based methods that can be used for proteome-wide structural studies and highlights some of their application.