Papers by ieva sutkeviciute
Journal of the Endocrine Society, 2019
Abstract Parathyroid hormone (PTH) receptor (PTHR) is a medically important family B G Protein Co... more Abstract Parathyroid hormone (PTH) receptor (PTHR) is a medically important family B G Protein Coupled Receptor (GPCR) that primarily couples to Gs/cAMP and Gq/Ca2+ signaling pathways and has a central role in regulating Ca2+ homeostasis and bone turnover. PTHR is activated by two endogenous hormones, endocrine PTH and paracrine PTH related protein (PTHrP), but only PTH mediates prolonged activation of the receptor and sustained cAMP responses after receptor internalization to the endosomes. To investigate the structural basis of prolonged activation of PTHR, we employed cryo-EM and solved the high resolution (3Å) structure of PTHR coupled to Gs and activated by long-acting PTH (LA-PTH), an analog of PTH that remarkably sustains endosomal PTHR signaling and induces significantly prolonged elevation in blood calcium in mice and monkeys. LA-PTH binds the receptor as a single extended α-helix with its N-terminus inserted deep into the transmembrane domain (TMD) of the receptor, while positioning the extracellular domain (ECD) of PTHR perpendicular to the cell membrane. The receptor activating N-terminal half of LA-PTH forms a dense network of hydrophobic and polar interactions with receptor’s TMD resulting in a tight binding and giving structural basis for remarkably sustained activation of PTHR by LA-PTH. Binding of LA-PTH activates the receptor by inducing partial unwinding of the C-terminus of transmembrane helix 6 (TM6) and a sharp kink at the middle of this helix, a hallmark of family B GPCR activation. This results in a pronounced outward movement of TM6, opening the cytosolic cavity of the receptor, which allows subsequent G protein coupling. Additionally, our cryo-EM data revealed structural dynamics of PTHR demonstrated by the ability of receptor ECD to adopt multiple conformational states. Together, these results reveal the structural determinants of PTHR signaling and provide a molecular framework for structure-based design of novel therapeutics for osteoporosis and hypocalcaemia treatment.
Journal of the Endocrine Society, 2019
Abstract The parathyroid hormone (PTH) type 1 receptor (PTHR) serves as the cognate receptor for ... more Abstract The parathyroid hormone (PTH) type 1 receptor (PTHR) serves as the cognate receptor for two endogenous ligands, PTH and its related peptide (PTHrP). Binding of PTHrP to the PTHR results in only transient cAMP production that is limited to the plasma membrane, while PTH induces sustained cAMP responses derived from endosomes following internalization of the ligand-receptor complex. The underlying mechanism and regulation of these kinetically-distinct signaling events are poorly understood. Here we show that extracellular Ca2+ acts as a positive allosteric modulator of the PTHR that promotes sustained cAMP production. Equilibrium and kinetic analysis of ligand binding and receptor activation revealed that extracellular Ca2+ increases the residence time of ligands on the receptor, consequently increasing the duration of receptor activation and cAMP production. Additionally, we found that this sensitivity to Ca2+ is lost for the PTH mutant R25C, identified as a cause of hypocalcemia in human patients. Using mass spectrometry (MS) approaches, we identified acidic clusters in the first extracellular loop of PTHR that serve a critical role in mediating Ca2+ allostery and sustained cAMP signaling. These findings provide insights toward understanding the determinants of PTHR-mediated signaling events and how these processes are regulated. Sources of Research Support: the National Institute of Diabetes and Digestive and Kidney Disease (NIDDK) and the National Institute of General Medical Sciences (NIGMS) of the US National Institutes of Health (NIH)
Http Www Theses Fr, Dec 14, 2012
Nature Chemical Biology
Class B G protein-coupled receptors (GPCRs) are notoriously difficult to target by small molecule... more Class B G protein-coupled receptors (GPCRs) are notoriously difficult to target by small molecules because their large orthosteric peptide-binding pocket embedded deep within the transmembrane domain limits the identification and development of nonpeptide small molecule ligands. Using the parathyroid hormone type 1 receptor (PTHR) as a prototypic class B GPCR target, and a combination of molecular dynamics simulations and elastic network model-based methods, we demonstrate that PTHR druggability can be effectively addressed. Here we found a key mechanical site that modulates the collective dynamics of the receptor and used this ensemble of PTHR conformers to identify selective small molecules with strong negative allosteric and biased properties for PTHR signaling in cell and PTH actions in vivo. This study provides a computational pipeline to detect precise druggable sites and identify allosteric modulators of PTHR signaling that could be extended to GPCRs to expedite discoveries of small molecules as novel therapeutic candidates. A computational pipeline linking molecular dynamics simulations and an elastic network model-based method identifies druggable sites in the parathyroid hormone class B GPCR and a nonpeptidic allosteric modulator of receptor signaling in cells.
Science Signaling
Physiological responses to parathyroid hormone depend on the subcellular location of receptor act... more Physiological responses to parathyroid hormone depend on the subcellular location of receptor activity.
Journal of Biological Chemistry
cAMP is the indispensable second messenger regulating cell metabolism and function in response to... more cAMP is the indispensable second messenger regulating cell metabolism and function in response to extracellular hormones and neurotransmitters. cAMP is produced via the activation of G protein–coupled receptors located at both the cell surface and inside the cell. Recently, Tsvetanova et al. explored cAMP generation in distinct locations and the impact on respective cell functions. Using a phospho-proteomic analysis, they provide insight into the unique role of localized cAMP production in cellular phospho-responses.
Trends in Endocrinology & Metabolism
The parathyroid hormone (PTH) type 1 receptor (PTHR) is the canonical G protein-coupled receptor ... more The parathyroid hormone (PTH) type 1 receptor (PTHR) is the canonical G protein-coupled receptor (GPCR) for PTH and PTH-related protein (PTHrP) and the key regulator of calcium homeostasis and bone turnover. PTHR function is critical for human health to maintain homeostatic control of ionized serum Ca2+ levels and has several unusual signaling features, such as endosomal cAMP signaling, that are well-studied but not structurally understood. In this review, we discuss how recently solved high resolution near-atomic structures of hormone-bound PTHR in its inactive and active signaling states and discovery of extracellular Ca2+ allosterism shed light on the structural basis for PTHR signaling and function.
Multivalent interactions between complex carbohydrates and oligomeric C-type lectins govern a wid... more Multivalent interactions between complex carbohydrates and oligomeric C-type lectins govern a wide range of immune responses. Up to date, standard SPR (surface plasmon resonance) competitive assays have largely been to evaluate binding properties from monosaccharide units (low affinity, mM) to multivalent elemental antagonists (moderate affinity, μM). Herein, we report typical case-studies of SPR competitive assays showing that they underestimate the potency of glycoclusters to inhibit the interaction between DC-SIGN and immobilized glycoconjugates. This paper describes the design and implementation of a SPR direct interaction over DC-SIGN oriented surfaces, extendable to other C-type lectin surfaces as such Langerin. This setup provides a microscopic overview of intrinsic avidity generation emanating simultaneously from multivalent glycoclusters and from DC-SIGN tetramers that are organized in nanoclusters on the cell membrane. For this purpose, covalent biospecific capture of DC-S...
Science
The parathyroid hormone receptor-1 (PTH1R) is a class B G protein–coupled receptor central to cal... more The parathyroid hormone receptor-1 (PTH1R) is a class B G protein–coupled receptor central to calcium homeostasis and a therapeutic target for osteoporosis and hypoparathyroidism. Here we report the cryo–electron microscopy structure of human PTH1R bound to a long-acting PTH analog and the stimulatory G protein. The bound peptide adopts an extended helix with its amino terminus inserted deeply into the receptor transmembrane domain (TMD), which leads to partial unwinding of the carboxyl terminus of transmembrane helix 6 and induces a sharp kink at the middle of this helix to allow the receptor to couple with G protein. In contrast to a single TMD structure state, the extracellular domain adopts multiple conformations. These results provide insights into the structural basis and dynamics of PTH binding and receptor activation.
Proceedings of the National Academy of Sciences
The parathyroid hormone (PTH) and its related peptide (PTHrP) activate PTH receptor (PTHR) signal... more The parathyroid hormone (PTH) and its related peptide (PTHrP) activate PTH receptor (PTHR) signaling, but only the PTH sustains GS-mediated adenosine 3′,5′-cyclic monophosphate (cAMP) production after PTHR internalization into early endosomes. The mechanism of this unexpected behavior for a G-protein–coupled receptor is not fully understood. Here, we show that extracellular Ca2+ acts as a positive allosteric modulator of PTHR signaling that regulates sustained cAMP production. Equilibrium and kinetic studies of ligand-binding and receptor activation reveal that Ca2+ prolongs the residence time of ligands on the receptor, thus, increasing both the duration of the receptor activation and the cAMP signaling. We further find that Ca2+ allostery in the PTHR is strongly affected by the point mutation recently identified in the PTH (PTHR25C) as a new cause of hypocalcemia in humans. Using high-resolution and mass accuracy mass spectrometry approaches, we identified acidic clusters in the r...
ACS chemical biology, Jan 22, 2017
At the surface of dendritic cells, C-type lectin receptors (CLRs) allow the recognition of carboh... more At the surface of dendritic cells, C-type lectin receptors (CLRs) allow the recognition of carbohydrate-based PAMPS or DAMPS (pathogen- or danger-associated molecular patterns respectively) and promote immune response regulation. However, some CLRs are hijacked by viral and bacterial pathogens. Thus, the design of ligands able to target specifically one CLR, to either modulate a immune response or to inhibit a given infection mechanism, has a great potential value in therapeutic design. A case study is the selective blocking of DC-SIGN, involved notably in HIV trans-infection of T lymphocytes, without interfering with Langerin-mediated HIV clearance. This is a challenging task due to their overlapping carbohydrate specificity. Towards the rational design of DC-SIGN selective ligands, we performed a comparative affinity study between DC-SIGN and Langerin with natural ligands. We found that GlcNAc is recognized by both CLRs, however, selective sulfatations are shown to increase the se...
Topics in Medicinal Chemistry
The parathyroid hormone (PTH) type 1 receptor (PTHR) is a medically important G protein-coupled r... more The parathyroid hormone (PTH) type 1 receptor (PTHR) is a medically important G protein-coupled receptor (GPCR) that triggers the cAMP/PKA signaling pathway in kidney and bone cells to regulate calcium ion homeostasis and bone turnover. It has been generally assumed that the production of cAMP mediated by GPCR and its termination take place exclusively at the plasma membrane. Recent studies reveal that the PTHR does not always follow this conventional paradigm. In the new model, PTH induces a prolonged cAMP response that is derived from the internalized ligand–PTHR complex located within endosomes. This model has been recognized as a new paradigm of GPCR signaling for peptide hormones, and the PTHR is a prototypical example. In this chapter we discuss molecular, structural, and cellular mechanisms responsible for this unexpected signaling process and its biological consequences.
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Papers by ieva sutkeviciute