Noncanonical Sequences Involving NHERF1 Interaction with NPT2A Govern Hormone-Regulated Phosphate Transport: Binding Outside the Box

Na+/H+ exchange factor-1 (NHERF1), a multidomain PDZ scaffolding phosphoprotein, is required for the type II sodium-dependent phosphate cotransporter (NPT2A)-mediated renal phosphate absorption. Both PDZ1 and PDZ2 domains are involved in NPT2A-dependent phosphate uptake. Though harboring identical core-binding motifs, PDZ1 and PDZ2 play entirely different roles in hormone-regulated phosphate transport. PDZ1 is required for the interaction with the C-terminal PDZ-binding sequence of NPT2A (-TRL). Remarkably, phosphocycling at Ser290 distant from PDZ1, the penultimate step for both parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF23) regulation, controls the association between NHERF1 and NPT2A. PDZ2 interacts with the C-terminal PDZ-recognition motif (-TRL) of G Protein-coupled Receptor Kinase 6A (GRK6A), and that promotes phosphorylation of Ser290. The compelling biological puzzle is how PDZ1 and PDZ2 with identical GYGF core-binding motifs specifically recognize distinct binding partners. Binding determinants distinct from the canonical PDZ-ligand interactions and located “outside the box” explain PDZ domain specificity. Phosphorylation of NHERF1 by diverse kinases and associated conformational changes in NHERF1 add more complexity to PDZ-binding diversity.

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NMR investigation of protein–ligand interactions for G-protein coupled receptors

Future Medicinal Chemistry ◽

10.4155/fmc-2018-0312 ◽

2019 ◽

Vol 11 (14) ◽

pp. 1811-1825 ◽

Cited By ~ 1

Author(s):

Claire Raingeval ◽

Isabelle Krimm

Keyword(s):

Conformational Changes ◽

Functional Characterization ◽

Allosteric Modulation ◽

G Protein Coupled Receptors ◽

Nmr Studies ◽

Protein Ligand Interactions ◽

Ligand Interactions ◽

Therapeutic Compounds ◽

G Protein Coupled

In this review, we report NMR studies of ligand–GPCR interactions, including both ligand-observed and protein-observed NMR experiments. Published studies exemplify how NMR can be used as a powerful tool to design novel GPCR ligands and investigate the ligand-induced conformational changes of GPCRs. The strength of NMR also lies in its capability to explore the diverse signaling pathways and probe the allosteric modulation of these highly dynamic receptors. By offering unique opportunities for the identification, structural and functional characterization of GPCR ligands, NMR will likely play a major role for the generation of novel molecules both as new tools for the understanding of the GPCR function and as therapeutic compounds for a large diversity of pathologies.

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Emerging roles of adhesion G protein-coupled receptors

Biochemical Society Transactions ◽

10.1042/bst20201144 ◽

2021 ◽

Author(s):

Matthew Rosa ◽

Timothy Noel ◽

Matthew Harris ◽

Graham Ladds

Keyword(s):

G Protein ◽

Conformational Changes ◽

Current Knowledge ◽

Receptor Activation ◽

G Protein Coupled Receptors ◽

Extracellular Region ◽

Extracellular Matrix Components ◽

Ligand Interactions ◽

Tethered Ligand ◽

G Protein Coupled

Adhesion G protein-coupled receptors (aGPCRs) form a sub-group within the GPCR superfamily. Their distinctive structure contains an abnormally large N-terminal, extracellular region with a GPCR autoproteolysis-inducing (GAIN) domain. In most aGPCRs, the GAIN domain constitutively cleaves the receptor into two fragments. This process is often required for aGPCR signalling. Over the last two decades, much research has focussed on aGPCR-ligand interactions, in an attempt to deorphanize the family. Most ligands have been found to bind to regions N-terminal to the GAIN domain. These receptors may bind a variety of ligands, ranging across membrane-bound proteins and extracellular matrix components. Recent advancements have revealed a conserved method of aGPCR activation involving a tethered ligand within the GAIN domain. Evidence for this comes from increased activity in receptor mutants exposing the tethered ligand. As a result, G protein-coupling partners of aGPCRs have been more extensively characterised, making use of their tethered ligand to create constitutively active mutants. This has led to demonstrations of aGPCR function in, for example, neurodevelopment and tumour growth. However, questions remain around the ligands that may bind many aGPCRs, how this binding is translated into changes in the GAIN domain, and the exact mechanism of aGPCR activation following GAIN domain conformational changes. This review aims to examine the current knowledge around aGPCR activation, including ligand binding sites, the mechanism of GAIN domain-mediated receptor activation and how aGPCR transmembrane domains may relate to activation. Other aspects of aGPCR signalling will be touched upon, such as downstream effectors and physiological roles.

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19F-NMR in Target-based Drug Discovery

Current Medicinal Chemistry ◽

10.2174/0929867326666190610160534 ◽

2019 ◽

Vol 26 (26) ◽

pp. 4964-4983 ◽

Cited By ~ 7

Author(s):

CongBao Kang

Keyword(s):

Drug Discovery ◽

Conformational Changes ◽

Protein Structures ◽

19F Nmr ◽

Binding Affinities ◽

Protein Ligand Interactions ◽

Compound Libraries ◽

Ligand Interactions ◽

Reference Ligand ◽

Hit Identification

Solution NMR spectroscopy plays important roles in understanding protein structures, dynamics and protein-protein/ligand interactions. In a target-based drug discovery project, NMR can serve an important function in hit identification and lead optimization. Fluorine is a valuable probe for evaluating protein conformational changes and protein-ligand interactions. Accumulated studies demonstrate that 19F-NMR can play important roles in fragment- based drug discovery (FBDD) and probing protein-ligand interactions. This review summarizes the application of 19F-NMR in understanding protein-ligand interactions and drug discovery. Several examples are included to show the roles of 19F-NMR in confirming identified hits/leads in the drug discovery process. In addition to identifying hits from fluorinecontaining compound libraries, 19F-NMR will play an important role in drug discovery by providing a fast and robust way in novel hit identification. This technique can be used for ranking compounds with different binding affinities and is particularly useful for screening competitive compounds when a reference ligand is available.

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Structural studies of phosphorylation-dependent interactions between the V2R receptor and arrestin-2

Nature Communications ◽

10.1038/s41467-021-22731-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Qing-Tao He ◽

Peng Xiao ◽

Shen-Ming Huang ◽

Ying-Li Jia ◽

Zhong-Liang Zhu ◽

...

Keyword(s):

Crystal Structures ◽

Conformational Changes ◽

Nmr Spectrum ◽

H Nmr ◽

Interaction Sites ◽

Receptor Interactions ◽

Remote Sites ◽

Genetic Code Expansion ◽

G Protein Coupled ◽

Interaction Change

AbstractArrestins recognize different receptor phosphorylation patterns and convert this information to selective arrestin functions to expand the functional diversity of the G protein-coupled receptor (GPCR) superfamilies. However, the principles governing arrestin-phospho-receptor interactions, as well as the contribution of each single phospho-interaction to selective arrestin structural and functional states, are undefined. Here, we determined the crystal structures of arrestin2 in complex with four different phosphopeptides derived from the vasopressin receptor-2 (V2R) C-tail. A comparison of these four crystal structures with previously solved Arrestin2 structures demonstrated that a single phospho-interaction change results in measurable conformational changes at remote sites in the complex. This conformational bias introduced by specific phosphorylation patterns was further inspected by FRET and 1H NMR spectrum analysis facilitated via genetic code expansion. Moreover, an interdependent phospho-binding mechanism of phospho-receptor-arrestin interactions between different phospho-interaction sites was unexpectedly revealed. Taken together, our results provide evidence showing that phospho-interaction changes at different arrestin sites can elicit changes in affinity and structural states at remote sites, which correlate with selective arrestin functions.

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Membrane Orientation and Binding Determinants of G Protein-Coupled Receptor Kinase 5 as Assessed by Combined Vibrational Spectroscopic Studies

PLoS ONE ◽

10.1371/journal.pone.0082072 ◽

2013 ◽

Vol 8 (11) ◽

pp. e82072 ◽

Cited By ~ 17

Author(s):

Pei Yang ◽

Alisa Glukhova ◽

John J. G. Tesmer ◽

Zhan Chen

Keyword(s):

G Protein ◽

Spectroscopic Studies ◽

Receptor Kinase ◽

G Protein Coupled Receptor ◽

Membrane Orientation ◽

Binding Determinants ◽

G Protein Coupled

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The Complexities of Organ Crosstalk in Phosphate Homeostasis: Time to Put Phosphate Sensing Back in the Limelight

International Journal of Molecular Sciences ◽

10.3390/ijms22115701 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5701

Author(s):

Lucile Figueres ◽

Sarah Beck-Cormier ◽

Laurent Beck ◽

Joanne Marks

Keyword(s):

Molecular Mechanisms ◽

Dominant Role ◽

Fibroblast Growth Factor 23 ◽

Hormone Secretion ◽

Phosphate Transport ◽

Phosphate Homeostasis ◽

Cellular Mechanisms ◽

Exact Function ◽

Phosphate Sensing ◽

Significant Research

Phosphate homeostasis is essential for health and is achieved via interaction between the bone, kidney, small intestine, and parathyroid glands and via intricate processes involving phosphate transporters, phosphate sensors, and circulating hormones. Numerous genetic and acquired disorders are associated with disruption in these processes and can lead to significant morbidity and mortality. The role of the kidney in phosphate homeostasis is well known, although it is recognized that the cellular mechanisms in murine models and humans are different. Intestinal phosphate transport also appears to differ in humans and rodents, with recent studies demonstrating a dominant role for the paracellular pathway. The existence of phosphate sensing has been acknowledged for decades; however, the underlying molecular mechanisms are poorly understood. At least three phosphate sensors have emerged. PiT2 and FGFR1c both act as phosphate sensors controlling Fibroblast Growth Factor 23 secretion in bone, whereas the calcium-sensing receptor controls parathyroid hormone secretion in response to extracellular phosphate. All three of the proposed sensors are expressed in the kidney and intestine but their exact function in these organs is unknown. Understanding organ interactions and the mechanisms involved in phosphate sensing requires significant research to develop novel approaches for the treatment of phosphate homeostasis disorders.

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Rationally designed chemokine-based toxin targeting the viral G protein-coupled receptor US28 potently inhibits cytomegalovirus infection in vivo

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1509392112 ◽

2015 ◽

Vol 112 (27) ◽

pp. 8427-8432 ◽

Cited By ~ 27

Author(s):

Katja Spiess ◽

Mads G. Jeppesen ◽

Mikkel Malmgaard-Clausen ◽

Karen Krzywkowski ◽

Kalpana Dulal ◽

...

Keyword(s):

G Protein ◽

Pseudomonas Exotoxin ◽

Viral Pathogen ◽

Virus Origin ◽

Ligand Interactions ◽

Toxin Protein ◽

Novel Strategy ◽

G Protein Coupled

The use of receptor–ligand interactions to direct toxins to kill diseased cells selectively has shown considerable promise for treatment of a number of cancers and, more recently, autoimmune disease. Here we move the fusion toxin protein (FTP) technology beyond cancer/autoimmune therapeutics to target the human viral pathogen, human cytomegalovirus (HCMV), on the basis of its expression of the 7TM G protein-coupled chemokine receptor US28. The virus origin of US28 provides an exceptional chemokine-binding profile with high selectivity and improved binding for the CX3C chemokine, CX3CL1. Moreover, US28 is constitutively internalizing by nature, providing highly effective FTP delivery. We designed a synthetic CX3CL1 variant engineered to have ultra-high affinity for US28 and greater specificity for US28 than the natural sole receptor for CX3CL1, CX3CR1, and we fused the synthetic variant with the cytotoxic domain of Pseudomonas Exotoxin A. This novel strategy of a rationally designed FTP provided unparalleled anti-HCMV efficacy and potency in vitro and in vivo.

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Structural insights into the activation of human calcium-sensing receptor

10.1101/2021.03.30.437720 ◽

2021 ◽

Author(s):

Xiaochen Chen ◽

Lu Wang ◽

Zhanyu Ding ◽

Qianqian Cui ◽

Li Han ◽

...

Keyword(s):

Conformational Changes ◽

Large Scale ◽

Transmembrane Domains ◽

Structural Basis ◽

Calcium Sensing Receptor ◽

Calcium Sensing ◽

Cryo Electron Microscopy ◽

Activation Mechanisms ◽

G Protein Coupled ◽

Structural Insights

AbstractHuman calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that maintains Ca2+ homeostasis in serum. Here, we present the cryo-electron microscopy structures of the CaSR in the inactive and active states. Complemented with previously reported crystal structures of CaSR extracellular domains, it suggests that there are three distinct conformations: inactive, intermediate and active state during the activation. We used a negative allosteric nanobody to stabilize the CaSR in the fully inactive state and found a new binding site for Ca2+ ion that acts as a composite agonist with L-amino acid to stabilize the closure of active Venus flytraps. Our data shows that the agonist binding leads to the compaction of the dimer, the proximity of the cysteine-rich domains, the large-scale transitions of 7-transmembrane domains, and the inter-and intrasubunit conformational changes of 7-transmembrane domains to accommodate the downstream transducers. Our results reveal the structural basis for activation mechanisms of the CaSR.

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Cryo-EM structure of phosphodiesterase 6 reveals insights into the allosteric regulation of type I phosphodiesterases

Science Advances ◽

10.1126/sciadv.aav4322 ◽

2019 ◽

Vol 5 (2) ◽

pp. eaav4322 ◽

Cited By ~ 13

Author(s):

Sahil Gulati ◽

Krzysztof Palczewski ◽

Andreas Engel ◽

Henning Stahlberg ◽

Lubomir Kovacik

Keyword(s):

Conformational Changes ◽

Second Messengers ◽

Structural Features ◽

Full Length ◽

Type I ◽

Regulatory Domains ◽

Catalytic Domains ◽

Cryo Electron Microscopy ◽

Density Map ◽

G Protein Coupled

Cyclic nucleotide phosphodiesterases (PDEs) work in conjunction with adenylate/guanylate cyclases to regulate the key second messengers of G protein–coupled receptor signaling. Previous attempts to determine the full-length structure of PDE family members at high-resolution have been hindered by structural flexibility, especially in their linker regions and N- and C-terminal ends. Therefore, most structure-activity relationship studies have so far focused on truncated and conserved catalytic domains rather than the regulatory domains that allosterically govern the activity of most PDEs. Here, we used single-particle cryo–electron microscopy to determine the structure of the full-length PDE6αβ2γ complex. The final density map resolved at 3.4 Å reveals several previously unseen structural features, including a coiled N-terminal domain and the interface of PDE6γ subunits with the PDE6αβ heterodimer. Comparison of the PDE6αβ2γ complex with the closed state of PDE2A sheds light on the conformational changes associated with the allosteric activation of type I PDEs.

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