Search

G protein–coupled receptors (GPCRs) are the largest family of cell surface receptors. Class B1 GPCRs constitute a subfamily of 15 receptors that characteristically contain large extracellular domains (ECDs) and respond to long polypeptide hormones. Class B1 GPCRs are critical regulators of homeostasis, and, as such, many are important drug targets. While most transmembrane proteins, including GPCRs, are recalcitrant to crystallization, recent advances in cryo-electron microscopy (cryo-EM) have facilitated a rapid expansion of the structural understanding of membrane proteins. As a testament to this success, structures for all the class B1 receptors bound to G proteins have been determined by cryo-EM in the past 5 years. Further advances in cryo-EM have uncovered dynamics of these receptors, ligands, and signaling partners. Here, we examine the recent structural underpinnings of the class B1 GPCRs with an emphasis on structure–function relationships.

Membrane proteins are highly diverse in both structure and function and can, therefore, present different challenges for structure determination. They are biologically important for cells and organisms as gatekeepers for information and molecule transfer across membranes, but each class of membrane proteins can present unique obstacles to structure determination. Historically, many membrane protein structures have been investigated using highly engineered constructs or using larger fusion proteins to improve solubility and/or increase particle size. Other strategies included the deconstruction of the full-length protein to target smaller soluble domains. These manipulations were often required for crystal formation to support X-ray crystallography or to circumvent lower resolution due to high noise and dynamic motions of protein subdomains. However, recent revolutions in membrane protein biochemistry and cryo-electron microscopy now provide an opportunity to solve high resolution structures of both large,1 megadalton (MDa), and small,100 kDa (kDa), drug targets in near-native conditions, routinely reaching resolutions around or below 3 Å. This review provides insights into how the recent advances in membrane biology and biochemistry, as well as technical advances in cryo-electron microscopy, help us to solve structures of a large variety of membrane protein groups, from small receptors to large transporters and more complex machineries.

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen that presents great health concerns. Treatment requires the use of last-line antibiotics, such as members of the oxazolidinone family, of which linezolid is the first member to see regular use in the clinic. Here, we report a short time scale selection experiment in which strains of MRSA were subjected to linezolid treatment. Clonal isolates which had evolved a linezolid-resistant phenotype were characterized by whole-genome sequencing. Linezolid-resistant mutants were identified which had accumulated mutations in the ribosomal protein uL3. Multiple clones which had two mutations in uL3 exhibited resistance to linezolid, 2-fold higher than the clinical breakpoint. Ribosomes from this strain were isolated and subjected to single-particle cryo-electron microscopic analysis and compared to the ribosomes from the parent strain. We found that the mutations in uL3 lead to a rearrangement of a loop that makes contact with Helix 90, propagating a structural change over 15 Å away. This distal change swings nucleotide U2504 into the binding site of the antibiotic, causing linezolid resistance.

Summary The complex etiology of neurodegeneration continues to stifle efforts to develop effective therapeutics. New agents elucidating key pathways causing neurodegeneration might serve to increase our understanding and potentially lead to improved treatments. Here, we demonstrate that a water-soluble manganese(II) texaphyrin (MMn) is a suitable magnetic resonance imaging (MRI) contrast agent for detecting larger amyloid beta constructs. The imaging potential of MMn was inferred on the basis of in vitro studies and in vivo detection in Alzheimer’s disease C. elegans models via MRI and ICP-MS. In vitro antioxidant- and cellular-based assays provide support for the notion that this porphyrin analog shows promise as a therapeutic agent able to mitigate the oxidative and nitrative toxic effects considered causal in neurodegeneration. The present report marks the first elaboration of an MRI-active metalloantioxidant that confers diagnostic and therapeutic benefit in Alzheimer’s disease models without conjugation of a radioisotope, targeting moiety, or therapeutic payload., Graphical Abstract, Highlights • Manganese texaphyrin binds to Aβ constructs (i.e., monomer, fibrils, and aggregates) • MRI studies demonstrate the utility of metallotexaphyrins for detecting Aβ aggregates • Antioxidant assays show potential utility of MMn for treating CNS neurodegeneration, The Bigger Picture Neurodegenerative disease is correlated with aberrant oxidation and nitration of small-molecule neurotransmitters, proteins, DNA, and cellular membrane components. Cellular damage enhanced by these modifications propagates disease progression, leading to death. Effective tools that enable insight into the complex etiology of CNS diseases could allow researchers to find a cure. Part of this effort is focused on developing imaging modalities that can assist in understanding these diseases. Here, we demonstrate a new theranostic strategy that shows promise in addressing the oxidative and nitrative malfunctions considered causal in disease progression. The approach detailed in this report also allows the MRI of aggregated Aβ, which could lead to improvements in disease diagnosis. The present work is thus expected to set the stage for the mechanistic-based design of theranostics, useful in understanding and treating neurodegeneration., Sessler et al. have studied manganese(II) texaphyrin as a catalytic metalloantioxidant theranostic agent for the detection of Aβ aggregates. A series of in vitro and in vivo analyses allowed for the inference that metallotexaphyrins could be used for MRI-based detection of Aβ biomarkers. In vitro and cellular-based assays coupled with mechanistic analysis demonstrated an efficacious protective effect against oxidative and nitrative damage. The present results could thus guide future efforts in developing new theranostic modalities for use in neurodegenerative disease.

We numerically study how the interactions between surface, subsurface, and rainfall create complex runoff behavior in a headwater-dominated high elevation, mountainous catchment in East River Watershed, Colorado, USA. In order to understand the effect of surface–subsurface interactions on the hydrological response, we compare model variations with different soil permeability. We compute hydrographs, hydrological indices, and spatio-temporal distributions of hydrological variables. The indices and distributions are then linked to the hydrograph at the outlet of the domain. Our results show that changing surface–subsurface interactions trigger different flow regimes, connectivity dynamics, and runoff generation mechanisms inside the catchment, and hence, affect the distributed hydrological response. Increased surface–subsurface interaction leads to a higher degree of connectivity in the catchment. Temporal dynamics of both distributed and aggregated hydrological response did not change significantly, which may be because the structural connectivity of the catchment remained untouched. Our results indicate that in higher order catchments, the effect of changes in distributed hydrological response may not always be observable through aggregated hydrological signatures.

G protein-coupled receptors (GPCRs) are the largest class of cell surface drug targets. Advances in biochemical approaches for the stabilisation of GPCR:transducer complexes together with improvements in the technology and application of cryo-EM has recently opened up new possibilities for structure-assisted drug design of GPCR agonists. Nonetheless, limitations in the commercial application of some of these approaches, including the use of nanobody 35 (Nb35) for stabilisation of GPCR:Gs complexes, and the high cost of 300kV imaging have restricted broad application of cryo-EM in drug discovery. Here, using the PF 06882961-bound GLP-1R as exemplar, we validated formation of stable complexes with a modified Gs protein in the absence of Nb35 that had equivalent resolution in the drug binding pocket to complexes solved in the presence of Nb35, while the G protein displayed increased conformational dynamics. In parallel, we assessed the performance of 200kV versus 300kV image acquisition using a Falcon 4 or K3 direct electron detector. We show that with 300kV Krios, both bottom mounted Falcon 4 and energy filtered (25eV slit) Bio-Quantum K3 produced similar resolution. Moreover, the 200kV Glacios with bottom mounted Falcon 4 yielded a 3.2 Å map with clear density for bound drug and multiple structurally ordered waters. Our work paves the way for broader commercial application of cryo-EM for GPCR drug discovery.

Salmonella is an important genus of Gram-negative pathogens, treatment of which has become problematic due to increases in antimicrobial resistance. This is partly attributable to the overexpression of tripartite efflux pumps, particularly the constitutively expressed AcrAB-TolC. Despite its clinical importance, the structure of the Salmonella AcrB transporter remained unknown to-date, with much of our structural understanding coming from the Escherichia coli orthologue. Here, by taking advantage of the styrene maleic acid (SMA) technology to isolate membrane proteins with closely associated lipids, we report the very first experimental structure of Salmonella AcrB transporter. Furthermore, this novel structure provides additional insight into mechanisms of drug efflux as it bears the mutation (G288D), originating from a clinical isolate of Salmonella Typhimurium presenting an increased resistance to fluoroquinolones. Experimental data are complemented by state-of-the-art molecular dynamics (MD) simulations on both the wild type and G288D variant of Salmonella AcrB. Together, these reveal several important differences with respect to the E. coli protein, providing insights into the role of the G288D mutation in increasing drug efflux and extending our understanding of the mechanisms underlying antibiotic resistance.

Summary Drug-resistant epileptic encephalopathies of infancy have been associated with KCNT1 gain-of-function mutations, which increase the activity of KNa1.1 sodium-activated potassium channels. Pharmacological inhibition of hyperactive KNa1.1 channels by quinidine has been proposed as a stratified treatment, but mostly this has not been successful, being linked to the low potency and lack of specificity of the drug. Here we describe the use of a previously determined cryo-electron microscopy-derived KNa1.1 structure and mutational analysis to identify how quinidine binds to the channel pore and, using computational methods, screened for compounds predicated to bind to this site. We describe six compounds that inhibited KNa1.1 channels with low- and sub-micromolar potencies, likely also through binding in the intracellular pore vestibule. In hERG inhibition and cytotoxicity assays, two compounds were ineffective. These may provide starting points for the development of new pharmacophores and could become tool compounds to study this channel further., Graphical Abstract, Highlights • KNa1.1 potassium channel inhibitors work by blocking the channel pore • A chicken KNa1.1 cryo-EM structure was used for virtual compound screening • Six novel inhibitors of human KNa1.1 channels were identified • Inhibitors may help develop treatments for human KCNT1 gain-of-function disorders, Drugs; Neuroscience; Structural Biology

TRPC1/4/5 channels are non-specific cation channels implicated in a wide variety of diseases, and TRPC1/4/5 inhibitors have recently entered the first clinical trials. However, fundamental and translational studies require a better understanding of TRPC1/4/5 channel regulation by endogenous and exogenous factors. Although several potent and selective TRPC1/4/5 modulators have been reported, the paucity of mechanistic insights into their modes-of-action remains a barrier to the development of new chemical probes and drug candidates. The xanthine class of modulators includes the most potent and selective TRPC1/4/5 inhibitors described to date, as well as TRPC5 activators. Our previous studies suggest that xanthines interact with a, so far, elusive pocket of TRPC1/4/5 channels that is essential to channel gating. Targeting this pocket may be a promising strategy for TRPC1/4/5 drug discovery. Here we report the first structure of a small molecule-bound TRPC1/4/5 channel – human TRPC5 in complex with the xanthine Pico145 – to 3.0 Å. We found that Pico145 binds to a conserved lipid binding site of TRPC5, where it displaces a bound phospholipid. Our findings explain the mode-of-action of xanthine-based TRPC1/4/5 modulators, and suggest a structural basis for TRPC1/4/5 modulation by endogenous factors such as (phospho)lipids and Zn2+ ions. These studies lay the foundations for the structure-based design of new generations of TRPC1/4/5 modulators.

Structures of inhibitor-bound bovine cytochrome bc 1 were determined by cryo-EM and compared with X-ray crystallographic structures, demonstrating that cryo-EM could be a feasible tool for structure-based drug discovery., Cytochrome bc 1, a dimeric multi-subunit electron-transport protein embedded in the inner mitochondrial membrane, is a major drug target for the treatment and prevention of malaria and toxoplasmosis. Structural studies of cytochrome bc 1 from mammalian homologues co-crystallized with lead compounds have underpinned structure-based drug design to develop compounds with higher potency and selectivity. However, owing to the limited amount of cytochrome bc 1 that may be available from parasites, all efforts have been focused on homologous cytochrome bc 1 complexes from mammalian species, which has resulted in the failure of some drug candidates owing to toxicity in the host. Crystallographic studies of the native parasite proteins are not feasible owing to limited availability of the proteins. Here, it is demonstrated that cytochrome bc 1 is highly amenable to single-particle cryo-EM (which uses significantly less protein) by solving the apo and two inhibitor-bound structures to ∼4.1 Å resolution, revealing clear inhibitor density at the binding site. Therefore, cryo-EM is proposed as a viable alternative method for structure-based drug discovery using both host and parasite enzymes.

Zinc ion dependent β-lactamases (MBLs) catalyze the hydrolysis of almost all β-lactam antibiotics and resist the action of clinically available β-lactamase inhibitors. We report how application of in silico fragment-based molecular design employing thiol-mediated metal anchorage leads to potent MBL inhibitors. The new inhibitors manifest potent inhibition of clinically important B1 subfamily MBLs, including the widespread NDM-1, IMP-1 and VIM-2 enzymes; with lower potency, some of them also inhibit clinically relevant Class A and D serine-β-lactamases. The inhibitors show selectivity for bacterial MBL enzymes compared to human MBL fold nucleases. Co-crystallization of one inhibitor, which shows potentiation of meropenem activity against MBL-expressing Enterobacteriaceae, with VIM-2 reveals an unexpected binding mode, involving interactions with residues from conserved active site bordering loops.

Recent developments in electron microscopy have provided new opportunities in the field of structure-based drug design. This review looks at the challenges that remain and the future prospects for the use of electron microscopy in this area., Recent developments in electron microscopy (EM) have led to a step change in our ability to solve the structures of previously intractable systems, especially membrane proteins and large protein complexes. This has provided new opportunities in the field of structure-based drug design, with a number of high-profile publications resolving the binding sites of small molecules and peptide inhibitors. There are a number of advantages of EM over the more traditional X-ray crystallographic approach, such as resolving different conformational states and permitting the dynamics of a system to be better resolved when not constrained by a crystal lattice. There are still significant challenges to be overcome using an EM approach, not least the speed of structure determination, difficulties with low-occupancy ligands and the modest resolution that is available. However, with the anticipated developments in the field of EM, the potential of EM to become a key tool for structure-based drug design, often complementing X-ray and NMR studies, seems promising.

Helping first-year students become involved in college via semester- or year-long first-year orientation programs is a major undertaking for college and university administrators. The effect of a weekend-long orientation program on students' involvement in college had yet to be determined until the current study. The authors describe such a program and evaluate its utility. Results revealed that incoming first-year students who attended a weekend-long orientation program were involved in school during their first year, than those who did not attend the program.

Several types of drug-resistant epileptic encephalopathies of infancy have been associated with mutations in the KCNT1 gene, which encodes the sodium-activated potassium channel subunit KNa1.1. These mutations are commonly gain-of-function, increasing channel activity, therefore inhibition by drugs is proposed as a stratified approach to treat disorders. To date, quinidine therapy has been trialled with several patients, but mostly with unsuccessful outcomes, which has been linked to its low potency and lack of specificity. Here we describe the use of a cryo-electron microscopy-derived KNa1.1 structure and mutational analysis to identify the quinidine biding site and identified novel inhibitors that target this site using computational methods. We describe six compounds that inhibit KNa1.1 channels with low- and sub-micromolar potencies, likely through binding in the intracellular pore vestibule. In preliminary hERG inhibition and cytotoxicity assays, two compounds showed little effect. These compounds may provide starting points for the development of novel pharmacophores for KNa1.1 inhibition, with the view to treating KCNT1-associated epilepsy and, with their potencies higher than quinidine, could become key tool compounds to further study this channel. Furthermore, this study illustrates the potential for utilising cryo-electron microscopy in ion channel drug discovery.

qNORs that catalyze the reduction of nitric oxide to nitrous oxide are dimeric and obtain their protons from cytoplasmic end., Quinol-dependent nitric oxide reductases (qNORs) are membrane-integrated, iron-containing enzymes of the denitrification pathway, which catalyze the reduction of nitric oxide (NO) to the major ozone destroying gas nitrous oxide (N2O). Cryo–electron microscopy structures of active qNOR from Alcaligenes xylosoxidans and an activity-enhancing mutant have been determined to be at local resolutions of 3.7 and 3.2 Å, respectively. They unexpectedly reveal a dimeric conformation (also confirmed for qNOR from Neisseria meningitidis) and define the active-site configuration, with a clear water channel from the cytoplasm. Structure-based mutagenesis has identified key residues involved in proton transport and substrate delivery to the active site of qNORs. The proton supply direction differs from cytochrome c–dependent NOR (cNOR), where water molecules from the cytoplasm serve as a proton source similar to those from cytochrome c oxidase.

Insight is provided into the dynamic interactions of LAT1 and CD98hc and their role in the transport cycle of the complex., Solute carriers are a large class of transporters that play key roles in normal and disease physiology. Among the solute carriers, heteromeric amino-acid transporters (HATs) are unique in their quaternary structure. LAT1–CD98hc, a HAT, transports essential amino acids and drugs across the blood–brain barrier and into cancer cells. It is therefore an important target both biologically and therapeutically. During the course of this work, cryo-EM structures of LAT1–CD98hc in the inward-facing conformation and in either the substrate-bound or apo states were reported to 3.3–3.5 Å resolution [Yan et al. (2019), Nature (London), 568, 127–130]. Here, these structures are analyzed together with our lower resolution cryo-EM structure, and multibody 3D auto-refinement against single-particle cryo-EM data was used to characterize the dynamics of the interaction of CD98hc and LAT1. It is shown that the CD98hc ectodomain and the LAT1 extracellular surface share no substantial interface. This allows the CD98hc ectodomain to have a high degree of movement within the extracellular space. The functional implications of these aspects are discussed together with the structure determination.

Background: Parasite diseases are a huge burden on human health causing significant morbidity and mortality. However, parasite structure based drug discovery programmes have been hindered by a lack of high resolution structural information from parasite derived proteins and have often relied upon homology models from mammalian systems. The recent renaissance in electron microscopy (EM) has caused a dramatic rise in the number of structures being determined at high resolution and subsequently enabled it to be thought of as a tool in drug discovery. Results: In this review, we discuss the challenges associated with the structural determination of parasite proteins including the difficulties in obtaining sufficient quantities of protein. We then discuss the reasons behind the resurgence in EM, how it may overcome some of these challenges and provide examples of EM derived parasite protein structures. Finally, we discuss the challenges which EM needs to overcome before it is used as a mainstream technique in anti-parasite drug discovery. Conclusions: This review reports the progress that has been made in obtaining sufficient quantities of proteins for structural studies and the role EM may play in future structure based drug design programs. The outlook for future structure based drug design programs against some of the most devastating parasite diseases looks promising.

Lissencephaly is a malformation of cortical development typically caused by deficient neuronal migration resulting in cortical thickening and reduced gyration. Here we describe a "thin" lissencephaly (TLIS) variant characterized by megalencephaly, frontal predominant pachygyria, intellectual disability, and seizures. Trio-based whole-exome sequencing and targeted re-sequencing identified recessive mutations of CRADD in six individuals with TLIS from four unrelated families of diverse ethnic backgrounds. CRADD (also known as RAIDD) is a death-domain-containing adaptor protein that oligomerizes with PIDD and caspase-2 to initiate apoptosis. TLIS variants cluster in the CRADD death domain, a platform for interaction with other death-domain-containing proteins including PIDD. Although caspase-2 is expressed in the developing mammalian brain, little is known about its role in cortical development. CRADD/caspase-2 signaling is implicated in neurotrophic factor withdrawal- and amyloid-β-induced dendritic spine collapse and neuronal apoptosis, suggesting a role in cortical sculpting and plasticity. TLIS-associated CRADD variants do not disrupt interactions with caspase-2 or PIDD in co-immunoprecipitation assays, but still abolish CRADD's ability to activate caspase-2, resulting in reduced neuronal apoptosis in vitro. Homozygous Cradd knockout mice display megalencephaly and seizures without obvious defects in cortical lamination, supporting a role for CRADD/caspase-2 signaling in mammalian brain development. Megalencephaly and lissencephaly associated with defective programmed cell death from loss of CRADD function in humans implicate reduced apoptosis as an important pathophysiological mechanism of cortical malformation. Our data suggest that CRADD/caspase-2 signaling is critical for normal gyration of the developing human neocortex and for normal cognitive ability.

Captive breeding and release programs, widely used to supplement populations of declining species, minimize juvenile mortality to achieve rapid population growth. However, raising animals in benign environments may promote traits that are adaptive in captivity but maladaptive in nature. In chinook salmon, hatchery rearing relaxes natural selection favoring large eggs, allowing fecundity selection to drive exceptionally rapid evolution of small eggs. Trends toward small eggs are also evident in natural populations heavily supplemented by hatcheries, but not in minimally supplemented populations. Unintentional selection in captivity can lead to rapid changes in critical life-history traits that may reduce the success of supplementation or reintroduction programs.

Transmembrane (TM) segments in proteins can be distinguished in amino acid sequences as continuous stretches of hydrophobic residues. However, examination of a data base of helical water-soluble (globular) proteins revealed that nearly one-third contained helices of sufficient length to span a bilayer (or =19 residues) that had mean hydrophobicityor =actual TM segments. We now report that synthetic peptides corresponding to these globular protein sequences, which we termed "delta-helices," behave like native TM sequences and readily insert into membrane mimetic environments in helical conformations. As well, certain delta-helix sequences can integrate into the membrane bilayer when placed into a membrane-targeted chimeric protein. We establish that delta-helices can be distinguished computationally from bona fide TM segments by the decreased frequency of occurrence of Ile/Val residues and by their relatively decreased solvent accessibilities (versus other globular helices) within tertiary structure. The further observations that (i) delta-helices generally contain three or more charged residues and (ii) delta-helices display relatively even distribution of these charged residues along their lengths, rather than concentration near their N and C termini as observed for TM segments, may constitute key recognition factors in diverting delta-helices from the membrane in vivo. Although a discrete biological role for delta-helices remains to be pinpointed, our overall results suggest that such segments may be required for globular protein folding and identify additional factors that may be important in the correct selection of TM segments by the cellular machinery.

Induced triploidy (3N) in salmon results from a blockage of maternal meiosis II, and hence provides a unique opportunity to study dosage effects on phenotypic variance. Chinook salmon families were bred using a paternal half-sib breeding design (62 females and 31 males) and half of each resulting family was treated to induce triploidy. The paired families were used to test for dosage effects (resulting from triploidy) on (1) the distribution and magnitude of phenotypic variation, (2) narrow-sense heritability and (3) maternal effects in fitness-related traits (i.e., survival, size-at-age, relative growth rate and serum lysozyme activity). Quantitative genetic analyses were performed separately for diploid and triploid family groups. Triploidization resulted in significantly higher levels of phenotypic variance and substantial differences in patterns of variance distribution for growth and survival-related traits, although the patterns were reversed for lysozyme activity. Triploids exhibited higher narrow sense heritability values relative to diploid Chinook salmon. However, maternal effects estimates were generally lower in triploids than in diploids. Thus, the dosage effects resulting from adding an extra set of chromosomes to the Chinook salmon genome are primarily additive. Somewhat counterintuitively, however, the relative magnitude of the combined effects of dominance, epistasis and maternal effects is not affected by dosage. Our results indicate that inheritance of fitness-related quantitative traits is profoundly affected by dosage effects associated with induced triploidy, and that triploidization can result in unpredictable performance and fitness outcomes.

Activin receptor-like kinase-1 (ALK-1), the gene mutated in HHT type 2 (HHT2), is a serine/threonine kinase receptor type I of the TGF-beta superfamily, specifically expressed on endothelial cells. We established an HHT2 genotype in 16 families and report nine novel mutations. These include insertions and deletions of single base pairs in exons 3, 8 and 9, as well as nonsense mutations in exons 4 and 8 of ALK-1, which would lead to premature truncation and unstable mRNA or protein. Three novel missense mutations were identified in exons 7 and 8 of the kinase domain. Five previously reported substitutions were also observed in the families analyzed. Our results bring to 36, the number of mutations associated with HHT2, and are mostly found in exons 8 and 3 followed by exons 4 and 7. To ascertain the potential functional implications of the missense mutations in the ALK-1 kinase domain, we generated a model based on the three-dimensional structure of the homologous ALK-5 kinase domain. Our data reveal that the 11 missense mutations modify residues conserved among type I receptors and alter the polarity, charge, hydrophobicity and/or size of the substituted amino-acid and likely lead to misfolded and nonfunctional proteins.