Identification of Gα12-vs-Gα13-coupling determinants and development of a Gα12/13-coupled designer GPCR.

G-protein-coupled receptors (GPCRs) transduce diverse signals into the cell by coupling to one or several Gα subtypes. Of the 16 Gα subtypes in human cells, Gα12 and Gα13 belong to the G12 subfamily and are reported to be functionally different. Notably, certain GPCRs display selective coupling to either Gα12 or Gα13, highlighting their significance in various cellular contexts. However, the structural basis underlying this selectivity remains unclear. Here, using a Gα12-coupled designer receptor exclusively activated by designer drugs (DREADD; G12D) as a model system, we identified residues in the α5 helix and the receptor that collaboratively determine Gα12-vs-Gα13 selectivity. Residue-swapping experiments showed that G12D distinguishes differences between Gα12 and Gα13 in the positions G.H5.09 and G.H5.23 in the α5 helix. Molecular dynamics simulations observed that I378G.H5.23 in Gα12 interacts with N1032.39, S1693.53 and Y17634.53 in G12D, while H364G.H5.09 in Gα12 interact with Q2645.71 in G12D. Screening of mutations at these positions in G12D identified G12D mutants that enhanced coupling with Gα12 and to an even greater extent with Gα13. Combined mutations, most notably the dual Y17634.53H and Q2645.71R mutant, further enhanced Gα12/13 coupling, thereby serving as a potential Gα12/13-DREADD. Such novel Gα12/13-DREADD may be useful in future efforts to develop drugs that target Gα12/13 signaling as well as to identify their therapeutic indications.

Towards a structural and functional analysis of the immunoglobulin-fold proteome.

The immunoglobulin fold (Ig fold) domain is a super-secondary structural motif consisting of a sandwich with two layers of ß-sheets that is present in many proteins with very diverse biological functions covering a wide range of physiological processes. This domain presents a modular architecture built with ß strands connected by variable length loops that has a highly conserved structural core of four ß-strands and quite variable ß-sheet extensions in the two sandwich layers that enable both divergent and convergent evolutionary mechanisms in the known Ig fold proteome. The central role of this Ig fold's structural plasticity in the evolutionary success of antibodies in our immune system is well established. Nature has also utilized this Ig fold in all domains of life in many different physiological contexts that go way beyond the immune system. Here we will present a structural and functional overview of the utilization of the Ig fold in different biological processes and in different cellular contexts to highlight some of the innumerable ways that this structural motif can interact in multidomain proteins to enable their diversity of functions. This includes shareable specific protein structure visualizations behind those functions that serve as starting points for further explorations of the biomolecular interactions spanning the Ig fold proteome. This overview also highlights how this Ig fold is being utilized through natural adaptation, engineering, and even building from scratch for a range of biotechnological applications.

Three-stage model of helical membrane protein folding: Role of membrane-water interface as the intermediate stage vestibule for TM helices during their in membrano assembly.

Integral membrane proteins (MPs) are dominated by transmembrane α-helical (TMH) proteins playing critical roles in cellular signaling processes. These proteins display a wide range of sizes from one TMH domain to at least 26 TMH domains and diverse structural folds. A common feature of most of these folds is the TM orientation of the helical domains and the approximately parallel packing of these domains into helical bundles of varying stability, however, it has been challenging to study the folding of these proteins experimentally. The contribution of helix stabilization in membrane and interface to the folding energy landscape are investigated here for the full range of TMH protein sizes containing 1 TM domain (1-TMH protein) to 24 TM domains (24-TMH protein) for all TMH proteins with available structures using structural bioinformatics based hydropathy analysis. The TM helix insertion stabilization energies from Water to membrane-water Interface (WAT→INT energies) are on average half of those insertion energies from water to transmembrane orientation (WAT→TM energies) for the whole polytopic helical membrane proteome (1-TMH to 24-TMH proteins). This suggests a potentially dominant role of the membrane-water interface as a viable holding vestibule for the TM helices during their release from the translocon. This provides proteome-level evidence for the broadly applicable four-step thermodynamic framework by White and co-workers as well as a natural extension of Popot and Engelman's original two-stage model of helical MP folding to a three-stage model, where, in the new intermediate stage, the membrane-water interface acts as a holding vestibule for the translated TM helices, reconciling the interface's critical role in MP folding seen in many previous studies. Support for this model is provided by showing the stability of hydrophobic TM helices at the membrane-water interface through several microsecond long molecular dynamics simulations of five hydrophobic helical domains and a helical hairpin pre-folded from the ribosomal exit vestibule.

Development of enhanced conformational sampling methods to probe the activation landscape of GPCRs.

G protein-coupled receptors (GPCRs) make up the largest superfamily of integral membrane proteins and play critical signal transduction roles in many physiological processes. Developments in molecular biology, biophysical, biochemical, pharmacological, and computational techniques aimed at these important therapeutic targets are beginning to provide unprecedented details on the structural as well as functional basis of their pleiotropic signaling mediated by G proteins, ß arrestins, and other transducers. This pleiotropy presents a pharmacological challenge as the same ligand-receptor interaction can cause a therapeutic effect as well as an undesirable on-target side-effect through different downstream pathways. GPCRs don't function as simple binary on-off switches but as finely tuned shape-shifting machines described by conformational ensembles, where unique subsets of conformations may be responsible for specific signaling cascades. X-ray crystallography and more recently cryo-electron microscopy are providing snapshots of some of these functionally-important receptor conformations bound to ligands and/or transducers, which are being utilized by computational methods to describe the dynamic conformational energy landscape of GPCRs. In this chapter, we review the progress in computational conformational sampling methods based on molecular dynamics and discrete sampling approaches that have been successful in complementing biophysical and biochemical studies on these receptors in terms of their activation mechanisms, allosteric effects, actions of biased ligands, and effects of pathological mutations. Some of the sampled simulation time scales are beginning to approach receptor activation time scales. The list of conformational sampling methods and example uses discussed is not exhaustive but includes representative examples that have pushed the limits of classical molecular dynamics and discrete sampling methods to describe the activation energy landscape of GPCRs.

The complex role of the N-terminus and acidic residues of HdeA as pH-dependent switches in its chaperone function.

HdeA is a small acid-stress chaperone protein found in the periplasm of several pathogenic gram-negative bacteria. In neutral pH environments HdeA is an inactive folded homodimer but when exposed to strong acidic environments it partially unfolds and, once activated, binds to other periplasmic proteins, protecting them from irreversible aggregation. Here we use a combination of hydrogen/deuterium exchange NMR experiments and constant pH molecular dynamics simulations to elucidate the role of HdeA's N-terminus in its activation mechanism. Previous work indicates that the N-terminus is flexible and unprotected at high pH while exhibiting interactions with some HdeA client binding site residues. It, however, becomes partially solvent-protected at pH 2.6 - 2.8 and then loses protection again at pH 2.0. This protection is not due to the appearance of new secondary structure, but rather increased contacts between N-terminal residues and the C-terminus of the other protomer in the dimer, as well as concurrent loosening of its hold on the client binding site residues, priming HdeA for interactions with periplasmic client proteins. This work also uncovers unusual protonation profiles of some titratable residues and suggests their complex role in chaperone function.

Pseudo-Symmetric Assembly of Protodomains as a Common Denominator in the Evolution of Polytopic Helical Membrane Proteins.

The polytopic helical membrane proteome is dominated by proteins containing seven transmembrane helices (7TMHs). They cannot be grouped under a monolithic fold or superfold. However, a parallel structural analysis of folds around that magic number of seven in distinct protein superfamilies (SWEET, PnuC, TRIC, FocA, Aquaporin, GPCRs) reveals a common homology, not in their structural fold, but in their systematic pseudo-symmetric construction during their evolution. Our analysis leads to guiding principles of intragenic duplication and pseudo-symmetric assembly of ancestral transmembrane helical protodomains, consisting of 3 (or 4) helices. A parallel deconstruction and reconstruction of these domains provides a structural and mechanistic framework for their evolutionary paths. It highlights the conformational plasticity inherent to fold formation itself, the role of structural as well as functional constraints in shaping that fold, and the usefulness of protodomains as a tool to probe convergent vs divergent evolution. In the case of FocA vs. Aquaporin, this protodomain analysis sheds new light on their potential divergent evolution at the protodomain level followed by duplication and parallel evolution of the two folds. GPCR domains, whose function does not seem to require symmetry, nevertheless exhibit structural pseudo-symmetry. Their construction follows the same protodomain assembly as any other pseudo-symmetric protein suggesting their potential evolutionary origins. Interestingly, all the 6/7/8TMH pseudo-symmetric folds in this study also assemble as oligomeric forms in the membrane, emphasizing the role of symmetry in evolution, revealing self-assembly and co-evolution not only at the protodomain level but also at the domain level.

Understanding G Protein Selectivity of Muscarinic Acetylcholine Receptors Using Computational Methods.

The neurotransmitter molecule acetylcholine is capable of activating five muscarinic acetylcholine receptors, M1 through M5, which belong to the superfamily of G-protein-coupled receptors (GPCRs). These five receptors share high sequence and structure homology; however, the M1, M3, and M5 receptor subtypes signal preferentially through the Gαq/11 subset of G proteins, whereas the M2 and M4 receptor subtypes signal through the Gαi/o subset of G proteins, resulting in very different intracellular signaling cascades and physiological effects. The structural basis for this innate ability of the M1/M3/M5 set of receptors and the highly homologous M2/M4 set of receptors to couple to different G proteins is poorly understood. In this study, we used molecular dynamics (MD) simulations coupled with thermodynamic analyses of M1 and M2 receptors coupled to both Gαi and Gαq to understand the structural basis of the M1 receptor's preference for the Gαq protein and the M2 receptor's preference for the Gαi protein. The MD studies showed that the M1 and M2 receptors can couple to both Gα proteins such that the M1 receptor engages with the two Gα proteins in slightly different orientations and the M2 receptor engages with the two Gα proteins in the same orientation. Thermodynamic studies of the free energy of binding of the receptors to the Gα proteins showed that the M1 and M2 receptors bind more strongly to their cognate Gα proteins compared to their non-cognate ones, which is in line with previous experimental studies on the M3 receptor. A detailed analysis of receptor-G protein interactions showed some cognate-complex-specific interactions for the M2:Gαi complex; however, G protein selectivity determinants are spread over a large overlapping subset of residues. Conserved interaction between transmembrane helices 5 and 6 far away from the G-protein-binding receptor interface was found only in the two cognate complexes and not in the non-cognate complexes. An analysis of residues implicated previously in G protein selectivity, in light of the cognate and non-cognate structures, shaded a more nuanced role of those residues in affecting G protein selectivity. The simulation of both cognate and non-cognate receptor-G protein complexes fills a structural gap due to difficulties in determining non-cognate complex structures and provides an enhanced framework to probe the mechanisms of G protein selectivity exhibited by most GPCRs.

HER3-targeted protein chimera forms endosomolytic capsomeres and self-assembles into stealth nucleocapsids for systemic tumor homing of RNA interference in vivo.

RNA interference represents a potent intervention for cancer treatment but requires a robust delivery agent for transporting gene-modulating molecules, such as small interfering RNAs (siRNAs). Although numerous molecular approaches for siRNA delivery are adequate in vitro, delivery to therapeutic targets in vivo is limited by payload integrity, cell targeting, efficient cell uptake, and membrane penetration. We constructed nonviral biomaterials to transport small nucleic acids to cell targets, including tumor cells, on the basis of the self-assembling and cell-penetrating activities of the adenovirus capsid penton base. Our recombinant penton base chimera contains polypeptide domains designed for noncovalent assembly with anionic molecules and tumor homing. Here, structural modeling, molecular dynamics simulations, and functional assays suggest that it forms pentameric units resembling viral capsomeres that assemble into larger capsid-like structures when combined with siRNA cargo. Pentamerization forms a barrel lined with charged residues mediating pH-responsive dissociation and exposing masked domains, providing insight on the endosomolytic mechanism. The therapeutic impact was examined on tumors expressing high levels of HER3/ErbB3 that are resistant to clinical inhibitors. Our findings suggest that our construct may utilize ligand mimicry to avoid host attack and target the siRNA to HER3+ tumors by forming multivalent capsid-like structures.

A beetle antifreeze protein protects lactate dehydrogenase under freeze-thawing.

Effects of a beetle antifreeze proteins (AFP) from Dendroides canadensis (DAFP-1) on a model freeze-labile enzyme, lactate dehydrogenase (LDH), were investigated under freezing and thawing conditions. The presence of DAFP-1 can effectively protect the enzymatic activity of LDH upon repeated freezing and thawing and the protective role of DAFP-1 is more significant than that of bovine serum albumin (BSA), a common protectant for freeze-labile proteins. The results of circular dichroism (CD) spectroscopy suggest that the presence of DAFP-1 provides protection to the denaturation of LDH under freezing and thawing. The molecular dynamics (MD) simulation of DAFP-1 and LDH suggests that DAFP-1 interacts with LDH using its ice-binding surface (IBS) and mainly through its arginine residues. A mutant of DAFP-1, where all the arginine residues were replaced by alanine residues, lost its effect in protecting LDH under freezing and thawing. The results demonstrated that DAFP-1 is an effective protectant for a freeze-labile protein under freezing and thawing and the arginine residues in DAFP-1 are important for its protective role. By correlating the protective effect of an AFP with its structure, new insights in the identification and development of effective protectants for freeze-labile proteins were provided.

Chiral Graphs: Reduced Representations of Ligand Scaffolds for Stereoselective Biomolecular Recognition, Drug Design, and Enhanced Exploration of Chemical Structure Space.

Rational structure-based drug design relies on a detailed, atomic-level understanding of protein-ligand interactions. The chiral nature of drug binding sites in proteins has led to the discovery of predominantly chiral drugs. A mechanistic understanding of stereoselectivity (which governs how one stereoisomer of a drug might bind stronger than the others to a protein) depends on the topology of stereocenters in the chiral molecule. Chiral graphs and reduced chiral graphs, introduced here, are new topological representations of chiral ligands using graph theory, to facilitate a detailed understanding of chiral recognition of ligands/drugs by proteins. These representations are demonstrated by application to all ≈14 000+ chiral ligands in the Protein Data Bank (PDB), which will facilitate an understanding of protein-ligand stereoselectivity mechanisms. Ligand modifications during drug development can be easily incorporated into these chiral graphs. In addition, these chiral graphs present an efficient tool for a deep dive into the enormous chemical structure space to enable sampling of unexplored structural scaffolds.

Ethanol Induced Disordering of Pancreatic Acinar Cell Endoplasmic Reticulum: An ER Stress/Defective Unfolded Protein Response Model.

BACKGROUND & AIMS: Heavy alcohol drinking is associated with pancreatitis, whereas moderate intake lowers the risk. Mice fed ethanol long term show no pancreas damage unless adaptive/protective responses mediating proteostasis are disrupted. Pancreatic acini synthesize digestive enzymes (largely serine hydrolases) in the endoplasmic reticulum (ER), where perturbations (eg, alcohol consumption) activate adaptive unfolded protein responses orchestrated by spliced X-box binding protein 1 (XBP1). Here, we examined ethanol-induced early structural changes in pancreatic ER proteins. METHODS: Wild-type and Xbp1+/- mice were fed control and ethanol diets, then tissues were homogenized and fractionated. ER proteins were labeled with a cysteine-reactive probe, isotope-coded affinity tag to obtain a novel pancreatic redox ER proteome. Specific labeling of active serine hydrolases in ER with fluorophosphonate desthiobiotin also was characterized proteomically. Protein structural perturbation by redox changes was evaluated further in molecular dynamic simulations. RESULTS: Ethanol feeding and Xbp1 genetic inhibition altered ER redox balance and destabilized key proteins. Proteomic data and molecular dynamic simulations of Carboxyl ester lipase (Cel), a unique serine hydrolase active within ER, showed an uncoupled disulfide bond involving Cel Cys266, Cel dimerization, ER retention, and complex formation in ethanol-fed, XBP1-deficient mice. CONCLUSIONS: Results documented in ethanol-fed mice lacking sufficient spliced XBP1 illustrate consequences of ER stress extended by preventing unfolded protein response from fully restoring pancreatic acinar cell proteostasis during ethanol-induced redox challenge. In this model, orderly protein folding and transport to the secretory pathway were disrupted, and abundant molecules including Cel with perturbed structures were retained in ER, promoting ER stress-related pancreas pathology.

Resistance to receptor-blocking therapies primes tumors as targets for HER3-homing nanobiologics.

Resistance to anti-tumor therapeutics is an important clinical problem. Tumor-targeted therapies currently used in the clinic are derived from antibodies or small molecules that mitigate growth factor activity. These have improved therapeutic efficacy and safety compared to traditional treatment modalities but resistance arises in the majority of clinical cases. Targeting such resistance could improve tumor abatement and patient survival. A growing number of such tumors are characterized by prominent expression of the human epidermal growth factor receptor 3 (HER3) on the cell surface. This study presents a "Trojan-Horse" approach to combating these tumors by using a receptor-targeted biocarrier that exploits the HER3 cell surface protein as a portal to sneak therapeutics into tumor cells by mimicking an essential ligand. The biocarrier used here combines several functions within a single fusion protein for mediating targeted cell penetration and non-covalent self-assembly with therapeutic cargo, forming HER3-homing nanobiologics. Importantly, we demonstrate here that these nanobiologics are therapeutically effective in several scenarios of resistance to clinically approved targeted inhibitors of the human EGF receptor family. We also show that such inhibitors heighten efficacy of our nanobiologics on naïve tumors by augmenting HER3 expression. This approach takes advantage of a current clinical problem (i.e. resistance to growth factor inhibition) and uses it to make tumors more susceptible to HER3 nanobiologic treatment. Moreover, we demonstrate a novel approach in addressing drug resistance by taking inhibitors against which resistance arises and re-introducing these as adjuvants, sensitizing tumors to the HER3 nanobiologics described here.

The 3D Structure of Human DP Prostaglandin G-Protein-Coupled Receptor Bound to Cyclopentanoindole Antagonist, Predicted Using the DuplexBiHelix Modification of the GEnSeMBLE Method.

Prostaglandins play a critical physiological role in both cardiovascular and immune systems, acting through their interactions with 9 prostanoid G protein-coupled receptors (GPCRs). These receptors are important therapeutic targets for a variety of diseases including arthritis, allergies, type 2 diabetes, and cancer. The DP prostaglandin receptor is of interest because it has unique structural and physiological properties. Most notably, DP does not have the 3-6 ionic lock common to Class A GPCRs. However, the lack of X-ray structures for any of the 9 prostaglandin GPCRs hampers the application of structure-based drug design methods to develop more selective and active medications to specific receptors. We predict here 3D structures for the DP prostaglandin GPCR, based on the GEnSeMBLE complete sampling with hierarchical scoring (CS-HS) methodology. This involves evaluating the energy of 13 trillion packings to finally select the best 20 that are stable enough to be relevant for binding to antagonists, agonists, and modulators. To validate the predicted structures, we predict the binding site for the Merck cyclopentanoindole (CPI) selective antagonist docked to DP. We find that the CPI binds vertically in the 1-2-7 binding pocket, interacting favorably with residues R3107.40 and K762.54 with additional interactions with S3137.43, S3167.46, S191.35, etc. This binding site differs significantly from that of antagonists to known Class A GPCRs where the ligand binds in the 3-4-5-6 region. We find that the predicted binding site leads to reasonable agreement with experimental Structure-Activity Relationship (SAR). We suggest additional mutation experiments including K762.54, E1293.49, L1233.43, M2706.40, F2746.44 to further validate the structure, function, and activation mechanism of receptors in the prostaglandin family. Our structures and binding sites are largely consistent and improve upon the predictions by Li et al. ( J. Am. Chem. Soc. 2007 , 129 ( 35 ), 10720 ) that used our earlier MembStruk prediction methodology.

Predicting glycosaminoglycan surface protein interactions and implications for studying axonal growth.

Cell-surface carbohydrates play important roles in numerous biological processes through their interactions with various protein-binding partners. These interactions are made possible by the vast structural diversity of carbohydrates and the diverse array of carbohydrate presentations on the cell surface. Among the most complex and important carbohydrates are glycosaminoglycans (GAGs), which display varied stereochemistry, chain lengths, and patterns of sulfation. GAG-protein interactions participate in neuronal development, angiogenesis, spinal cord injury, viral invasion, and immune response. Unfortunately, little structural information is available for these complexes; indeed, for the highly sulfated chondroitin sulfate motifs, CS-E and CS-D, there are no structural data. We describe here the development and validation of the GAG-Dock computational method to predict accurately the binding poses of protein-bound GAGs. We validate that GAG-Dock reproduces accurately (<1-Å rmsd) the crystal structure poses for four known heparin-protein structures. Further, we predict the pose of heparin and chondroitin sulfate derivatives bound to the axon guidance proteins, protein tyrosine phosphatase σ (RPTPσ), and Nogo receptors 1-3 (NgR1-3). Such predictions should be useful in understanding and interpreting the role of GAGs in neural development and axonal regeneration after CNS injury.

Identifying multiple active conformations in the G protein-coupled receptor activation landscape using computational methods.

G protein-coupled receptors (GPCRs) are membrane proteins critical in cellular signaling, making them important targets for therapeutics. The activation of GPCRs is central to their function, requiring multiple conformations of the GPCRs in their activation landscape. To enable rational design of GPCR-targeting drugs, it is essential to obtain the ensemble of atomistic structures of GPCRs along their activation pathways. This is most challenging for structure determination experiments, making it valuable to develop reliable computational structure prediction methods. In particular, since the active-state conformations are higher in energy (less stable) than inactive-state conformations, they are difficult to stabilize. In addition, the computational methods are generally biased toward lowest energy structures by design and miss these high energy but functionally important conformations. To address this problem, we have developed a computationally efficient ActiveGEnSeMBLE method that systematically predicts multiple conformations that are likely in the GPCR activation landscape, including multiple active- and inactive-state conformations. ActiveGEnSeMBLE starts with a systematic coarse grid sampling of helix tilts/rotations (~13 trillion transmembrane domain conformations) and identifies multiple potential active-state energy wells, using the TM3-TM6 intracellular distance as a surrogate activation coordinate. These energy wells are then sampled locally using a finer grid in conformational space to find a locally minimized conformation in each energy well, which can be further relaxed using molecular dynamics (MD) simulations. This method, combining homology modeling, hierarchical complete conformational sampling, and nanosecond scale MD, provides one of the very few computational methods that predict multiple candidates for active-state conformations and is one of the most computationally affordable.

Activation mechanism of the G protein-coupled sweet receptor heterodimer with sweeteners and allosteric agonists.

The sweet taste in humans is mediated by the TAS1R2/TAS1R3 G protein-coupled receptor (GPCR), which belongs to the class C family that also includes the metabotropic glutamate and γ-aminobutyric acid receptors. We report here the predicted 3D structure of the full-length TAS1R2/TAS1R3 heterodimer, including the Venus Flytrap Domains (VFDs) [in the closed-open (co) active conformation], the cysteine-rich domains (CRDs), and the transmembrane domains (TMDs) at the TM56/TM56 interface. We observe that binding of agonists to VFD2 of TAS1R2 leads to major conformational changes to form a TM6/TM6 interface between TMDs of TAS1R2 and TAS1R3, which is consistent with the activation process observed biophysically on the metabotropic glutamate receptor 2 homodimer. We find that the initial effect of the agonist is to pull the bottom part of VFD3/TAS1R3 toward the bottom part of VFD2/TAS1R2 by ∼6 Šand that these changes get transmitted from VFD2 of TAS1R2 (where agonists bind) through the VFD3 and the CRD3 to the TMD3 of TAS1R3 (which couples to the G protein). These structural transformations provide a detailed atomistic mechanism for the activation process in GPCR, providing insights and structural details that can now be validated through mutation experiments.

Conformational and Thermodynamic Landscape of GPCR Activation from Theory and Computation.

We present a hybrid computational methodology to predict multiple energetically accessible conformations for G protein-coupled receptors (GPCRs) that might play a role in binding to ligands and different signaling partners. To our knowledge, this method, termed ActiveGEnSeMBLE, enables the first quantitative energy profile for GPCR activation that is consistent with the qualitative profile deduced from experiments. ActiveGEnSeMBLE starts with a systematic coarse grid sampling of helix tilts/rotations (∼13 trillion transmembrane-domain conformations) and selects the conformational landscape based on energy. This profile identifies multiple potential active-state energy wells, with the TM3-TM6 intracellular distance as an approximate activation coordinate. These energy wells are then sampled locally using a finer grid to find locally minimized conformation in each energy well. We validate this strategy using the inactive and active experimental structures of ß2 adrenergic receptor (hß2AR) and M2 muscarinic acetylcholine receptor. Structures of membrane-embedded hß2AR along its activation coordinate are subjected to molecular-dynamics simulations for relaxation and interaction energy analysis to generate a quantitative energy landscape for hß2AR activation. This landscape reveals several metastable states along this coordinate, indicating that for hß2AR, the agonist alone is not enough to stabilize the active state and that the G protein is necessary, consistent with experimental observations. The method's application to somatostatin receptor SSTR5 (no experimental structure available) shows that to predict an active conformation it is better to start from an inactive structure template based on a close homolog than to start from an active template based on a distant homolog. The energy landscape for hSSTR5 activation is consistent with hß2AR in the role of the G protein. These results demonstrate the utility of the ActiveGEnSeMBLE method for predicting multiple conformations along the pathways for activating GPCRs and the corresponding energy landscapes, thereby providing detailed structural insights into the initial molecular events of GPCR function that are not easily accessible by experiments.

A bitter pill for type 2 diabetes? The activation of bitter taste receptor TAS2R38 can stimulate GLP-1 release from enteroendocrine L-cells.

The bitter taste receptor TAS2R38 is a G protein coupled receptor (GPCR) that has been found in many extra-oral locations like the gastrointestinal (GI) system, respiratory system, and brain, though its function at these locations is only beginning to be understood. To probe the receptor's potential metabolic role, immunohistochemistry of human ileum tissues was performed, which showed that the receptor was co-localized with glucagon-like peptide 1 (GLP-1) in L-cells. In a previous study, we had modeled the structure of this receptor for its many taste-variant haplotypes (Tan et al. 2011), including the taster haplotype PAV. The structure of this haplotype was then used in a virtual ligand screening pipeline using a collection of ∼2.5 million purchasable molecules from the ZINC database. Three compounds (Z7, Z3, Z1) were purchased from the top hits and tested along with PTU (known TAS2R38 agonist) in in vitro and in vivo assays. The dose-response study of the effect of PTU and Z7 on GLP-1 release using wild-type and TAS2R38 knockout HuTu-80 cells showed that the receptor TAS2R38 plays a major role in GLP-1 release due to these molecules. In vivo studies of PTU and the three compounds showed that they each increase GLP-1 release. PTU was also chemical linked to cellulose to slow its absorption and when tested in vivo, it showed an enhanced and prolonged GLP-1 release. These results suggest that the GI lumen location of TAS2R38 on the L-cell makes it a relatively safe drug target as systemic absorption is not needed for a TAS2R38 agonist drug to effect GLP-1 release.

Antifreeze proteins govern the precipitation of trehalose in a freezing-avoiding insect at low temperature.

The remarkable adaptive strategies of insects to extreme environments are linked to the biochemical compounds in their body fluids. Trehalose, a versatile sugar molecule, can accumulate to high levels in freeze-tolerant and freeze-avoiding insects, functioning as a cryoprotectant and a supercooling agent. Antifreeze proteins (AFPs), known to protect organisms from freezing by lowering the freezing temperature and deferring the growth of ice, are present at high levels in some freeze-avoiding insects in winter, and yet, paradoxically are found in some freeze-tolerant insects. Here, we report a previously unidentified role for AFPs in effectively inhibiting trehalose precipitation in the hemolymph (or blood) of overwintering beetle larvae. We determine the trehalose level (29.6 ± 0.6 mg/mL) in the larval hemolymph of a beetle, Dendroides canadensis, and demonstrate that the hemolymph AFPs are crucial for inhibiting trehalose crystallization, whereas the presence of trehalose also enhances the antifreeze activity of AFPs. To dissect the molecular mechanism, we examine the molecular recognition between AFP and trehalose crystal interfaces using molecular dynamics simulations. The theory corroborates the experiments and shows preferential strong binding of the AFP to the fast growing surfaces of the sugar crystal. This newly uncovered role for AFPs may help explain the long-speculated role of AFPs in freeze-tolerant species. We propose that the presence of high levels of molecules important for survival but prone to precipitation in poikilotherms (their body temperature can vary considerably) needs a companion mechanism to prevent the precipitation and here present, to our knowledge, the first example. Such a combination of trehalose and AFPs also provides a novel approach for cold protection and for trehalose crystallization inhibition in industrial applications.

Structure-Based Sequence Alignment of the Transmembrane Domains of All Human GPCRs: Phylogenetic, Structural and Functional Implications.

The understanding of G-protein coupled receptors (GPCRs) is undergoing a revolution due to increased information about their signaling and the experimental determination of structures for more than 25 receptors. The availability of at least one receptor structure for each of the GPCR classes, well separated in sequence space, enables an integrated superfamily-wide analysis to identify signatures involving the role of conserved residues, conserved contacts, and downstream signaling in the context of receptor structures. In this study, we align the transmembrane (TM) domains of all experimental GPCR structures to maximize the conserved inter-helical contacts. The resulting superfamily-wide GpcR Sequence-Structure (GRoSS) alignment of the TM domains for all human GPCR sequences is sufficient to generate a phylogenetic tree that correctly distinguishes all different GPCR classes, suggesting that the class-level differences in the GPCR superfamily are encoded at least partly in the TM domains. The inter-helical contacts conserved across all GPCR classes describe the evolutionarily conserved GPCR structural fold. The corresponding structural alignment of the inactive and active conformations, available for a few GPCRs, identifies activation hot-spot residues in the TM domains that get rewired upon activation. Many GPCR mutations, known to alter receptor signaling and cause disease, are located at these conserved contact and activation hot-spot residue positions. The GRoSS alignment places the chemosensory receptor subfamilies for bitter taste (TAS2R) and pheromones (Vomeronasal, VN1R) in the rhodopsin family, known to contain the chemosensory olfactory receptor subfamily. The GRoSS alignment also enables the quantification of the structural variability in the TM regions of experimental structures, useful for homology modeling and structure prediction of receptors. Furthermore, this alignment identifies structurally and functionally important residues in all human GPCRs. These residues can be used to make testable hypotheses about the structural basis of receptor function and about the molecular basis of disease-associated single nucleotide polymorphisms.