Kuntal Pal

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin–arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.

Structure and mechanism for recognition of peptide hormones by Class B G-protein-coupled receptors

Class B G-protein-coupled receptors (GPCRs) are receptors for peptide hormones that include glucagon, parathyroid hormone, and calcitonin. These receptors are involved in a wide spectrum of physiological activities, from metabolic regulation and stress control to development and maintenance of the skeletal system. As such, they are important drug targets for the treatment of diabetes, osteoporosis, and stress related disorders. Class B GPCRs are organized into two modular domains: an extracellular domain (ECD) and a helical bundle that contains seven transmembrane helices (TM domain). The ECD is responsible for the high affinity and specificity of hormone binding, and the TM domain is required for receptor activation and signal coupling to downstream G-proteins. Although the structure of the full-length receptor remains unknown, the ECD structures have been well characterized for a number of Class B GPCRs, revealing a common fold for ligand recognition. This review summarizes the general structural principles that guide hormone binding by Class B ECDs and their implications in the design of peptide hormone analogs for therapeutic purposes.

Crystal structure of full-length Mycobacterium tuberculosis H37Rv glycogen branching enzyme: insights of N-terminal beta-sandwich in substrate specificity and enzymatic activity.

The open reading frame Rv1326c of Mycobacterium tuberculosis (Mtb) H37Rv encodes for an α-1,4-glucan branching enzyme (MtbGlgB, EC 2.4.1.18, Uniprot entry Q10625). This enzyme belongs to glycoside hydrolase (GH) family 13 and catalyzes the branching of a linear glucose chain during glycogenesis by cleaving a 1→4 bond and making a new 1→6 bond. Here, we show the crystal structure of full-length MtbGlgB (MtbGlgBWT) at 2.33-Å resolution. MtbGlgBWT contains four domains: N1 β-sandwich, N2 β-sandwich, a central (β/α)8 domain that houses the catalytic site, and a C-terminal β-sandwich. We have assayed the amylase activity with amylose and starch as substrates and the glycogen branching activity using amylose as a substrate for MtbGlgBWT and the N1 domain-deleted (the first 108 residues deleted) MtbΔ108GlgB protein. The N1 β-sandwich, which is formed by the first 105 amino acids and superimposes well with the N2 β-sandwich, is shown to have an influence in substrate binding in the amylase assay. Also, we have checked and shown that several GH13 family inhibitors are ineffective against MtbGlgBWT and MtbΔ108GlgB. We propose a two-step reaction mechanism, for the amylase activity (1→4 bond breakage) and isomerization (1→6 bond formation), which occurs in the same catalytic pocket. The structural and functional properties of MtbGlgB and MtbΔ108GlgB are compared with those of the N-terminal 112-amino acid-deleted Escherichia coli GlgB (ECΔ112GlgB).

Glucocorticoids as prophylaxis against acute mountain sickness

Objective Acute mountain sickness (AMS) characterized by presence of symptoms including headache, nausea, excessive fatigue, loss of appetite, irritability and insomnia is a major impediment to work performance in human subjects who are rapidly inducted to high altitude (HA) during the initial phase of induction. The present study aims at to evaluate the efficacy of prophylactic administration of low dose glucocorticoids in prevention of AMS in normal healthy men who are inducted to HA by air.

Differential Requirement of the Extracellular Domain in Activation of Class B G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) from the secretin-like (class B) family are key players in hormonal homeostasis and are important drug targets for the treatment of metabolic disorders and neuronal diseases. They consist of a large N-terminal extracellular domain (ECD) and a transmembrane domain (TMD) with the GPCR signature of seven transmembrane helices. Class B GPCRs are activated by peptide hormones with their C termini bound to the receptor ECD and their N termini bound to the TMD. It is thought that the ECD functions as an affinity trap to bind and localize the hormone to the receptor. This in turn would allow the hormone N terminus to insert into the TMD and induce conformational changes of the TMD to activate downstream signaling. In contrast to this prevailing model, we demonstrate that human class B GPCRs vary widely in their requirement of the ECD for activation. In one group, represented by corticotrophin-releasing factor receptor 1 (CRF1R), parathyroid hormone receptor (PTH1R), and pituitary adenylate cyclase activating polypeptide type 1 receptor (PAC1R), the ECD requirement for high affinity hormone binding can be bypassed by induced proximity and mass action effects, whereas in the other group, represented by glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R), the ECD is required for signaling even when the hormone is covalently linked to the TMD. Furthermore, the activation of GLP-1R by small molecules that interact with the intracellular side of the receptor is dependent on the presence of its ECD, suggesting a direct role of the ECD in GLP-1R activation.

Molecular assembly of rhodopsin with G protein-coupled receptor kinases

G protein-coupled receptor kinases (GRKs) play pivotal roles in desensitizing GPCR signaling but little is known about how GRKs recognize and phosphorylate GPCRs due to the technical difficulties in detecting the highly dynamic GPCR/GRK interaction. By combining a genetic approach with multiple biochemical assays, we identified the key determinants for the assembly of the prototypical GPCR rhodopsin with its kinase GRK1. Our work reveals that the regulatory G-protein signaling homology (RH) domain of GRKs is the primary binding site to GPCRs and an active conformation of the GRK1 kinase domain is required for efficient interaction with rhodopsin. In addition, we provide a mechanistic solution for the longstanding puzzle about the gain-of-function Q41L mutation in GRK5. This mutation is in the RH domain and increases the capacity of the GRK mutant to interact with and to desensitize GPCRs. Finally we present the principal architecture of a rhodopsin/GRK complex through negative stain electron microscopy reconstruction. Together, these data define the key components for the rhodopsin/GRK1 interaction and provide a framework for understanding GRK-mediated desensitization of GPCRs.

Abstract B31: Development of fucose based pancreatic cancer biomarkers using modified lectins

Pancreatic cancer has poor prognosis because of ineffective and delayed diagnosis as well as treatment. Accurately identifying molecular changes could help with early detection and evaluation of treatment efficacy. Glycoprotein fucosylation is associated with various cancers. Interestingly, information at structural level such as diversity in fucose linkages can provide extensive details of disease state. But, lack of diagnostic reagents has impeded the research in identifying and interpreting these changes. This study is aimed to develop fucose based cancer biomarker reagents to identify important fucose linkages associated with cancer progression. We hypothesized that a panel of lectins, each specific for a particular type of fucose presentation, could identify differences between glycoproteins that are not discernable using any individual lectin or measurements of total fucose. Using a database of glycan array data, we identified lectins with high specificity for diverse linkages of fucose. We selected three for further testing: CGL2 for detecting fucose in a 2’ linkage, CCL2 for detecting fucose in a 3’ linkage, and RSL for detecting fucose in all linkages. To improve quality, production consistency, and test various modes of detection the lectins were recombinantly expressed with avitag (biotin) and poly-histidine fusion tags. The choice of terminus as well as the type of fusion tag could affect the molecular architecture and thereby lectin-glycan interaction. Therefore, two variants for each lectin (N terminus biotin and C terminus histidine and vice-versa) were expressed. The initial purification by size-exclusion chromatography showed similar results between the two tagging schemes for each protein. To test the accessibility of fusion tags and lectin multimerization with secondary detections (streptavidin or anti-poly-histindine), the two were pre-incubated prior to separation on an analytical scale size exclusion column resulting in protein elution that was entirely shifted to a higher molecular weight. Specificity for recombinant lectins to their respective linkages was performed using thermostability assay that resulted in a stability shift of lectins with respective glycan target but not with off-target glycan. To examine if the use of particular tag or secondary reagent was optimal for detection we performed competitive assay. Lectin binding to spotted glycoproteins in microarray assay was reduced upon pre-incubation with a competing sugar but not upon pre-incubation with a non-specific sugar. This suggests that recombinant lectins with fusion tags were functional. Lectins produced by recombinant expression showed specific glycan-binding using either a biotin or poly-histidine fusion tag at either terminus, but detection using the C-terminus tag was more reliable in each case. We probed the glycans on various glycoproteins with each lectin and predicted that the pattern of lectin binding would perform better than individual lectin measurements for identifying differences in fucose presentation. We tested this prediction using monoclonal antibodies against selected fucose-bearing glycans. Based on the known specificities of the antibodies, we could confirm differences in glycans predicted by the pattern of lectin binding but not by individual lectin measurements, thus confirming the ability to distinguish fucose presentations using a panel of lectins. This technique promises to help uncover details about the presentation of fucose on specific glycoproteins. The ability of these lectins to distinguish subtle changes in glycan presentation with enough precision at early stage and enable comparisons between tumor grades could prove valuable diagnostic reagents for pancreatic cancer. Citation Format: Sudhir Singh, Kuntal Pal, Elliot Ensink, Jessica Yadav, Doron Kletter, Marshall Bern, Anand Mehta, Karsten Melcher, Brian B. Haab. Development of fucose based pancreatic cancer biomarkers using modified lectins. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B31.

POTTALI KALPANA: A CONCISE AND POTENT PHARMACEUTICAL DOSAGE FORM OF INDIA N SYSTE M S OF MEDICINE

Indian system of medicine is a treasure of spectacular and peculiar pharmaceutical dosage forms. Ayurvedic herbo - mineral drugs contain several types of formulations with vivid variet ies. Maximum potency with minimum dosage is the philosophy of these drugs. Pottali kalpana is one of such, highly potent, concise and embossed dosage form in Ayurveda. The main ingredients of it, is the amalgam of mercury and sulphur, which is treated with different plant materials and ultimately given a compact form by the application of natural binder. This short review describe about the history, necessity, preparation and mode of application of the Pottali kalpana, an unique Ayurvedic pharmaceutical dos age form.

Structure Modeling Using Genetically Engineered Crosslinking

Class B G-protein-coupled receptors are exciting drug targets, yet the structure of a complete receptor bound to a peptide agonist has remained elusive. Coin et al. present a model of the receptor CRF1R bound to its native ligand based on partial structures and 44 spatial constraints revealed by new crosslinking approaches.