Biology subjects

Skiniotis, G.

Publications and source records attributed to Skiniotis, G..

26 records · Page 2Linked to original sources

bioRxiv2021

Plasticity in Ligand Recognition at Somatostatin Receptors

Somatostatin is a signaling peptide that plays a pivotal and wide-ranging role in physiologic processes relating to metabolism and growth through its actions at somatostatin receptors (SSTRs). Members of the somatostatin receptor subfamily, particularly SSTR2, are key drug targets for neuroendocrine neoplasms, with several synthetic peptide agonists currently in clinical use. Here, we show the cryogenic electron microscopy (cryo-EM) structures of active-state SSTR2 in complex with heterotrimeric Gi protein and either the endogenous ligand SST14 or the FDA-approved drug octreotide. Complemented by biochemical assays and molecular dynamics simulations, these structures reveal key details of ligand recognition, receptor activation, and subtype-selectivity at somatostatin receptors. We find that SSTR ligand recognition is highly diverse, as demonstrated by ligand-induced conformational changes in ECL2, substantial sequence divergence across subtypes in extracellular regions, and loss of ligand binding upon several structurally homologous substitutions between subtypes. Despite this complexity, we rationalize several previously described sources of SSTR subtype selectivity and identify an additional key interaction for SSTR2/3/5 specific binding. These results shed light on the basis of ligand recognition by somatostatin receptors and provide valuable insights for structure-based drug discovery at these targets.

biophysics↗

bioRxiv2020

Structural basis of GIRK2 channel modulation by cholesterol and PIP2

G protein-gated inwardly rectifying potassium (GIRK) channels play important roles in controlling cellular excitability in the heart and brain. While structural data begin to unravel the molecular basis for G protein and alcohol dependent activation of GIRK channels, little is known about the modulation by cholesterol. Here, we present cryo-electron microscopy (cryoEM) structures of GIRK2 in the presence and absence of the cholesterol analog cholesteryl hemisuccinate (CHS), and PIP2. The structures and their comparison reveal that CHS binds near PIP2 in lipid-facing hydrophobic pockets of the transmembrane domain (TMD). CHS potentiates the effects of PIP2, which stabilizes the inter-domain region and promotes the engagement of the cytoplasmic domain (CTD) onto the transmembrane region. The results suggest that CHS acts as a positive allosteric modulator and identify novel therapeutic sites for modulating GIRK channels in the brain.

neuroscience↗

bioRxiv2020

Structures of metabotropic GABAB receptor

GABA ({gamma}-aminobutyric acid) stimulation of the metabotropic GABAB receptor results in prolonged inhibition of neurotransmission that is central to brain physiology. GABAB belongs to the Family C of G protein-coupled receptors (GPCRs), which operate as dimers to relay synaptic neurotransmitter signals into a cellular response through the binding and activation of heterotrimeric G proteins. GABAB, however, is unique in its function as an obligate heterodimer in which agonist binding and G protein activation take place on distinct subunits. Here we show structures of heterodimeric and homodimeric full-length GABAB receptors. Complemented by cellular signaling assays and atomistic simulations, the structures reveal an essential role for the GABAB extracellular loop 2 (ECL2) in relaying structural transitions by ordering the linker connecting the extracellular ligand-binding domain to the transmembrane region. Furthermore, the ECL2 of both GABAB subunits caps and interacts with the hydrophilic head of a phospholipid occupying the extracellular half of the transmembrane domain, thereby providing a potentially crucial link between ligand binding and the receptor core that engages G protein. These results provide a starting framework to decipher mechanistic modes of signal transduction mediated by GABAB dimers and have important implications for rational drug design targeting these receptors.

neuroscience↗

bioRxiv2020

Structure of the Visual Signaling Complex between Transducin and Phosphodiesterase 6

Heterotrimeric G proteins communicate signals from activated G protein-coupled receptors to downstream effector proteins. In the phototransduction pathway responsible for vertebrate vision, the G protein-effector complex is comprised of the GTP-bound transducin subunit (GT{middle dot}GTP) and the cyclic GMP (cGMP) phosphodiesterase 6 (PDE6), which stimulates cGMP hydrolysis to transmit signals to the optic nerve. Here we report a cryo-electron microscopy (cryoEM) structure of PDE6 complexed to GTP-bound GT. The structure reveals two GT{middle dot}GTP subunits engaging the PDE6 hetero-tetramer at both the PDE6 catalytic core and the PDE{gamma} subunits, driving extensive rearrangements to relieve all inhibitory constraints on enzyme catalysis. Analysis of the conformational ensemble in the cryoEM data highlights the dynamic nature of the contacts between the two GT{middle dot}GTP subunits and PDE6 that support an alternating-site catalytic mechanism.

biophysics↗

bioRxiv2020

Limited Dishevelled/Axin oligomerization determines efficiency of Wnt/β-catenin signal transduction

In Wnt/{beta}-catenin signaling, the transcriptional coactivator {beta}-catenin is regulated by its phosphorylation in a complex that includes the scaffold protein Axin and associated kinases. Wnt binding to its coreceptors activates the cytosolic effector Dishevelled (Dvl), leading to the recruitment of Axin and the inhibition of {beta}-catenin phosphorylation. This process requires interaction of homologous DIX domains present in Dvl and Axin, but is mechanistically undefined. We show that Dvl DIX forms antiparallel, double-stranded oligomers in vitro, and that Dvl in cells forms oligomers typically <10 molecules at endogenous expression levels. Axin DIX (DAX) forms small single-stranded oligomers, but its self-association is stronger than that of DIX. DAX caps the ends of DIX oligomers, such that a DIX oligomer has at most four DAX binding sites. The relative affinities and stoichiometry of the DIX-DAX interaction provide a mechanism for efficient inhibition of {beta}-catenin phosphorylation upon Axin recruitment to the Wnt receptor complex.

biochemistry↗

bioRxiv2019

GemSpot: A Pipeline for Robust Modeling of Ligands into CryoEM Maps

Producing an accurate atomic model of biomolecule-ligand interactions from maps generated by cryo-electron microscopy often presents challenges inherent to the methodology and the dynamic nature of ligand binding. Here we have developed GemSpot, a pipeline of computational chemistry methods that take into account EM map potentials, quantum mechanics energy calculations, and water molecule site prediction to generate candidate poses and provide a measure of the degree of confidence. The pipeline is validated through several published cryoEM structures of complexes in different resolution ranges and various types of ligands. In all cases, at least one identified pose produced both excellent interactions with the target and agreement with the map. GemSpot will be valuable for the robust identification of ligand poses and drug discovery efforts through cryoEM.

biophysics↗

bioRxiv2019

Structural insights into ligand efficacy and activation of the glucagon receptor

The glucagon receptor family comprises Class B G protein-coupled receptors (GPCRs) that play a crucial role in regulating blood sugar levels. Receptors of this family represent important therapeutic targets for the treatment of diabetes and obesity. Despite intensive structural studies, we only have a poor understanding of the mechanism of peptide hormone-induced Class B receptor activation. This process involves the formation of a sharp kink in transmembrane helix 6 that moves out to allow formation of the nucleotide-free G protein complex. Here, we present the cryo-EM structure of the glucagon receptor (GCGR), a prototypical Class B GPCR, in complex with an engineered soluble glucagon derivative and the heterotrimeric G-protein, Gs. Comparison with the previously determined crystal structures of GCGR bound to a partial agonist reveals a structural framework to explain the molecular basis of ligand efficacy that is further supported by mutagenesis data.

biophysics↗

bioRxiv2018

De novo computational RNA modeling into cryoEM maps of large ribonucleoprotein complexes

RNA-protein assemblies carry out many critical biological functions including translation, RNA splicing, and telomere extension. Increasingly, cryo-electron microscopy (cryoEM) is used to determine the structures of these complexes, but nearly all maps determined with this method have regions in which the local resolution does not permit manual coordinate tracing. Because RNA coordinates typically cannot be determined by docking crystal structures of separate components and existing structure prediction algorithms cannot yet model RNA-protein complexes, RNA coordinates are frequently omitted from final models despite their biological importance. To address these omissions, we have developed a new framework for De novo Ribonucleoprotein modeling in Real-space through Assembly of Fragments Together with Electron density in Rosetta (DRRAFTER). We show that DRRAFTER recovers near-native models for a diverse benchmark set of small RNA-protein complexes, as well as for large RNA-protein machines, including the spliceosome, mitochondrial ribosome, and CRISPR-Cas9-sgRNA complexes where the availability of both high and low resolution maps enable rigorous tests. Blind tests on yeast U1 snRNP and spliceosomal P complex maps demonstrate that the method can successfully build RNA coordinates in real-world modeling scenarios. Additionally, to aid in final model interpretation, we present a method for reliable in situ estimation of DRRAFTER model accuracy. Finally, we apply this method to recently determined maps of telomerase, the HIV-1 reverse transcriptase initiation complex, and the packaged MS2 genome, demonstrating that DRRAFTER can be used to accelerate accurate model building in challenging cases.

biophysics↗