The molecular sociology of NHERF1 PDZ proteins controlling renal hormone-regulated phosphate transport.

Parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF23) control extracellular phosphate levels by regulating renal NPT2A-mediated phosphate transport by a process requiring the PDZ scaffold protein NHERF1. NHERF1 possesses two PDZ domains, PDZ1 and PDZ2, with identical core-binding GYGF motifs explicitly recognizing distinct binding partners that play different and specific roles in hormone-regulated phosphate transport. The interaction of PDZ1 and the carboxy-terminal PDZ-binding motif of NPT2A (C-TRL) is required for basal phosphate transport.

Scribble scrambles parathyroid hormone receptor interactions to regulate phosphate and vitamin D homeostasis.

G protein-coupled receptors, including PTHR, are pivotal for controlling metabolic processes ranging from serum phosphate and vitamin D levels to glucose uptake, and cytoplasmic interactors may modulate their signaling, trafficking, and function. We now show that direct interaction with Scribble, a cell polarity-regulating adaptor protein, modulates PTHR activity. Scribble is a crucial regulator for establishing and developing tissue architecture, and its dysregulation is involved in various disease conditions, including tumor expansion and viral infections.

Mutations in an unrecognized internal NPT2A PDZ motif disrupt phosphate transport and cause congenital hypophosphatemia.

The Na+-dependent phosphate cotransporter-2A (NPT2A, SLC34A1) is a primary regulator of extracellular phosphate homeostasis. Its most prominent structural element is a carboxy-terminal PDZ ligand that binds Na+/H+ Exchanger Regulatory Factor-1 (NHERF1, SLC9A3R1). NHERF1, a multidomain PDZ protein, establishes NPT2A membrane localization and is required for hormone-inhibitable phosphate transport.

Mutations in an unrecognized internal NPT2A PDZ motif disrupt phosphate transport causing congenital hypophosphatemia.

The Na -dependent phosphate cotransporter-2A (NPT2A, SLC34A1) is a primary regulator of extracellular phosphate homeostasis. Its most prominent structural element is a carboxy-terminal PDZ ligand that binds Na /H Exchanger Regulatory Factor-1 (NHERF1, SLC9A3R1). NHERF1, a multidomain PDZ protein,establishes NPT2A membrane localization and is required for hormone-sensitive phosphate transport.

RGS14 regulates PTH- and FGF23-sensitive NPT2A-mediated renal phosphate uptake via binding to the NHERF1 scaffolding protein.

Phosphate homeostasis, mediated by dietary intake, renal absorption, and bone deposition, is incompletely understood because of the uncharacterized roles of numerous implicated protein factors. Here, we identified a novel role for one such element, regulator of G protein signaling 14 (RGS14), suggested by genome-wide association studies to associate with dysregulated Pi levels. We show that human RGS14 possesses a carboxy-terminal PDZ ligand required for sodium phosphate cotransporter 2a (NPT2A) and sodium hydrogen exchanger regulatory factor-1 (NHERF1)-mediated renal Pi transport.

ACE2 interaction with cytoplasmic PDZ protein enhances SARS-CoV-2 invasion.

SARS-CoV-2 is responsible for the global COVID-19 pandemic. Angiotensin converting enzyme 2 (ACE2) is the membrane-delimited receptor for SARS-CoV-2. Lung, intestine, and kidney, major sites of viral infection, express ACE2 that harbors an intracellular, carboxy-terminal PDZ-recognition motif.

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.

Rearrangement of 7-Aryloxazolo[5,4-]pyridines to Benzo[][1,7]naphthyridine-4(3)-ones and Thieno[3,2-][1,7]naphthyridine-6(7)-ones.

In this work, we describe the development of the rearrangement for 7-aryl-substituted oxazolo[5,4-]pyridines treated with aluminum chloride into synthetically hard-to-reach benzo[][1,7]naphthyridinones. The discovered rearrangement is applied to a variety of electron-rich (hetero)arene substrates. It offers the advantages of mild conditions (90 °C temperature), fast reaction rates (<4 h), compatibility with air moisture, and the use of inexpensive commercial reagents.

Parallel Post-Translational Modification Scanning Enhancing Hydrogen-Deuterium Exchange-Mass Spectrometry Coverage of Key Structural Regions.

Hydrogen-deuterium exchange-mass spectrometry (HDXMS) is a powerful technology to characterize conformations and conformational dynamics of proteins and protein complexes. HDXMS has been widely used in the field of therapeutics for the development of protein drugs. Although sufficient sequence coverage is critical to the success of HDXMS, it is sometimes difficult to achieve.

Parathyroid hormone initiates dynamic NHERF1 phosphorylation cycling and conformational changes that regulate NPT2A-dependent phosphate transport.

Na-H exchanger regulatory factor-1 (NHERF1) is a PDZ protein that scaffolds membrane proteins, including sodium-phosphate co-transport protein 2A (NPT2A) at the plasma membrane. NHERF1 is a phosphoprotein with 40 Ser and Thr residues. Here, using tandem MS analysis, we characterized the sites of parathyroid hormone (PTH)-induced NHERF1 phosphorylation and identified 10 high-confidence phosphorylation sites.

Origins of PDZ Binding Specificity. A Computational and Experimental Study Using NHERF1 and the Parathyroid Hormone Receptor.

Na/H exchanger regulatory factor-1 (NHERF1) is a scaffolding protein containing two PSD95/discs large protein/ZO1 (PDZ) domains that modifies the signaling, trafficking, and function of the parathyroid hormone receptor (PTHR), a family B G-protein-coupled receptor. PTHR and NHERF1 bind through a PDZ-ligand-recognition mechanism. We show that PTH elicits phosphorylation of Thr591 in the canonical -ETVM binding motif of PTHR.

Binding of EBP50 to Nox organizing subunit p47phox is pivotal to cellular reactive species generation and altered vascular phenotype.

Despite numerous reports implicating NADPH oxidases (Nox) in the pathogenesis of many diseases, precise regulation of this family of professional reactive oxygen species (ROS) producers remains unclear. A unique member of this family, Nox1 oxidase, functions as either a canonical or hybrid system using Nox organizing subunit 1 (NoxO1) or p47(phox), respectively, the latter of which is functional in vascular smooth muscle cells (VSMC). In this manuscript, we identify critical requirement of ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50; aka NHERF1) for Nox1 activation and downstream responses.

Actin-Sorting Nexin 27 (SNX27)-Retromer Complex Mediates Rapid Parathyroid Hormone Receptor Recycling.

The G protein-coupled parathyroid hormone receptor (PTHR) regulates mineral-ion homeostasis and bone remodeling. Upon parathyroid hormone (PTH) stimulation, the PTHR internalizes into early endosomes and subsequently traffics to the retromer complex, a sorting platform on early endosomes that promotes recycling of surface receptors. The C terminus of the PTHR contains a type I PDZ ligand that binds PDZ domain-containing proteins.

Canonical and Noncanonical Sites Determine NPT2A Binding Selectivity to NHERF1 PDZ1.

Na+/H+ Exchanger Regulatory Factor-1 (NHERF1) is a scaffolding protein containing 2 PDZ domains that coordinates the assembly and trafficking of transmembrane receptors and ion channels. Most target proteins harboring a C-terminus recognition motif bind more-or-less equivalently to the either PDZ domain, which contain identical core-binding motifs. However some substrates such as the type II sodium-dependent phosphate co-transporter (NPT2A), uniquely bind only one PDZ domain.

Phosphorylation of ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) by Akt promotes stability and mitogenic function of S-phase kinase-associated protein-2 (Skp2).

The regulation of the cell cycle by the ubiquitin-proteasome system is dependent on the activity of E3 ligases. Skp2 (S-phase kinase associated protein-2) is the substrate recognition subunit of the E3 ligase that ubiquitylates the cell cycle inhibitors p21(cip1) and p27(kip1) thus promoting cell cycle progression. Increased expression of Skp2 is frequently observed in diseases characterized by excessive cell proliferation, such as cancer and neointima hyperplasia.

Stability and rigidity/flexibility-two sides of the same coin?

Protein molecules require both flexibility and rigidity for functioning. The fast and accurate prediction of protein rigidity/flexibility is one of the important problems in protein science. We have determined flexible regions for four homologous pairs from thermophilic and mesophilic organisms by two methods: the fast FoldUnfold which uses amino acid sequence and the time consuming MDFirst which uses three-dimensional structures.

Ezrin-anchored protein kinase A coordinates phosphorylation-dependent disassembly of a NHERF1 ternary complex to regulate hormone-sensitive phosphate transport.

Congenital defects in the Na/H exchanger regulatory factor-1 (NHERF1) are linked to disordered phosphate homeostasis and skeletal abnormalities in humans. In the kidney, these mutations interrupt parathyroid hormone (PTH)-responsive sequestration of the renal phosphate transporter, Npt2a, with ensuing urinary phosphate wasting. We now report that NHERF1, a modular PDZ domain scaffolding protein, coordinates the assembly of an obligate ternary complex with Npt2a and the PKA-anchoring protein ezrin to facilitate PTH-responsive cAMP signaling events.

Structural basis for NHERF1 PDZ domain binding.

The Na(+)/H(+) exchange regulatory factor-1 (NHERF1) is a scaffolding protein that possesses two tandem PDZ domains and a carboxy-terminal ezrin-binding domain (EBD). The parathyroid hormone receptor (PTHR), type II sodium-dependent phosphate cotransporter (Npt2a), and β2-adrenergic receptor (β2-AR), through their respective carboxy-terminal PDZ-recognition motifs, individually interact with NHERF1 forming a complex with one of the PDZ domains. In the basal state, NHERF1 adopts a self-inhibited conformation, in which its carboxy-terminal PDZ ligand interacts with PDZ2.

Flexibility and mobility in mesophilic and thermophilic homologous proteins from molecular dynamics and FoldUnfold method.

To function properly protein molecules require both flexibility and rigidity, therefore fast and accurate prediction of protein rigidity/flexibility is one of the important problems in protein science. In this work we used two theoretical approaches to determine flexible regions in four homologous pairs of proteins from thermophilic and mesophilic organisms. Protein pairs chosen in this study were selected to represent four typical folding classes.

Energetics of the cleft closing transition and the role of electrostatic interactions in conformational rearrangements of the glutamate receptor ligand binding domain.

The ionotropic glutamate receptors are localized in the pre- and postsynaptic membrane of neurons in the brain. Activation by the principal excitatory neurotransmitter glutamate allows the ligand binding domain to change conformation, communicating opening of the channel for ion conduction. The free energy of the GluR2 S1S2 ligand binding domain (S1S2) closure transition was computed using a combination of thermodynamic integration and umbrella sampling modeling methods.

Interplay between structural rigidity and electrostatic interactions in the ligand binding domain of GluR2.

Using molecular dynamics (MD) simulations, computational protein modifications, and a novel theoretical methodology that determines structural rigidity/flexibility (the FIRST algorithm), we investigate how molecular structure and dynamics of the glutamate receptor ligand binding domain (GluR2 S1S2) facilitate its conformational transition. S1S2 is a two-lobe protein, which undergoes a cleft closure conformational transition upon binding an agonist in the cleft between the two lobes; hence it is expected that the mechanism of this conformational transition can be characterized as a hinge-type. However, in the rigidity analysis one lobe of the protein is identified as a single rigid cluster while the other one is structurally flexible, inconsistent with a presumed mechanical hinge mechanism.

Structure and energetics of channel-forming protein-polysaccharide complexes inferred via computational statistical thermodynamics.

The ion channel protein alpha-hemolysin (alphaHL) forms supramolecular complexes with the polysaccharide beta-cyclodextrin (betaCD). This system has potential uses in nanoscale device engineering. It has been found recently that betaCD formed longer- or shorter-lived complexes with some engineered alphaHL mutants then with a wild type protein (Gu et al.

Protein flexibility using constraints from molecular dynamics simulations.

Proteins are held together in the native state by hydrophobic interactions, hydrogen bonds and interactions with the surrounding water, whose strength as well as spatial and temporal distribution affects protein flexibility and hence function. We study these effects using 10 ns molecular dynamics simulations of pure water and of two proteins, the glutamate receptor ligand binding domain and barnase. We find that most of the noncovalent interactions flicker on and off over typically nanoseconds, and so we can obtain good statistics from the molecular dynamics simulations.