Distinct activation mechanisms of β-arrestin-1 revealed by F NMR spectroscopy.

β-Arrestins (βarrs) are functionally versatile proteins that play critical roles in the G-protein-coupled receptor (GPCR) signaling pathways. While it is well established that the phosphorylated receptor tail plays a central role in βarr activation, emerging evidence highlights the contribution from membrane lipids. However, detailed molecular mechanisms of βarr activation by different binding partners remain elusive.

Function and dynamics of the intrinsically disordered carboxyl terminus of β2 adrenergic receptor.

Advances in structural biology have provided important mechanistic insights into signaling by the transmembrane core of G-protein coupled receptors (GPCRs); however, much less is known about intrinsically disordered regions such as the carboxyl terminus (CT), which is highly flexible and not visible in GPCR structures. The β adrenergic receptor's (βAR) 71 amino acid CT is a substrate for GPCR kinases and binds β-arrestins to regulate signaling. Here we show that the βAR CT directly inhibits basal and agonist-stimulated signaling in cell lines lacking β-arrestins.

Structural and dynamic insights into supra-physiological activation and allosteric modulation of a muscarinic acetylcholine receptor.

The M2 muscarinic receptor (M2R) is a prototypical G-protein-coupled receptor (GPCR) that serves as a model system for understanding GPCR regulation by both orthosteric and allosteric ligands. Here, we investigate the mechanisms governing M2R signaling versatility using cryo-electron microscopy (cryo-EM) and NMR spectroscopy, focusing on the physiological agonist acetylcholine and a supra-physiological agonist iperoxo, as well as a positive allosteric modulator LY2119620. These studies reveal that acetylcholine stabilizes a more heterogeneous M2R-G-protein complex than iperoxo, where two conformers with distinctive G-protein orientations were determined.

Construction of nano receptors for ubiquitin and ubiquitinated proteins based on the region-specific interactions between ubiquitin and polydopamine.

Ubiquitination is a prevalent post-translational modification that controls a multitude of important biological processes. Due to the low abundance of ubiquitinated proteins, highly efficient separation and enrichment approaches are required for ubiquitinome analysis. In this work, we disclose the region-specific interactions between the hydrophobic patch of ubiquitin and polydopamine.

Computational strategy for intrinsically disordered protein ligand design leads to the discovery of p53 transactivation domain I binding compounds that activate the p53 pathway.

Intrinsically disordered proteins or intrinsically disordered regions (IDPs) have gained much attention in recent years due to their vital roles in biology and prevalence in various human diseases. Although IDPs are perceived as attractive therapeutic targets, rational drug design targeting IDPs remains challenging because of their conformational heterogeneity. Here, we propose a hierarchical computational strategy for IDP drug virtual screening (IDPDVS) and applied it in the discovery of p53 transactivation domain I (TAD1) binding compounds.

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

Arrestins 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.

DeSiphering receptor core-induced and ligand-dependent conformational changes in arrestin via genetic encoded trimethylsilyl H-NMR probe.

Characterization of the dynamic conformational changes in membrane protein signaling complexes by nuclear magnetic resonance (NMR) spectroscopy remains challenging. Here we report the site-specific incorporation of 4-trimethylsilyl phenylalanine (TMSiPhe) into proteins, through genetic code expansion. Crystallographic analysis revealed structural changes that reshaped the TMSiPhe-specific amino-acyl tRNA synthetase active site to selectively accommodate the trimethylsilyl (TMSi) group.

Analysis of βAR-G and βAR-G complex formation by NMR spectroscopy.

The β-adrenergic receptor (βAR) is a prototypical G protein-coupled receptor (GPCR) that preferentially couples to the stimulatory G protein G and stimulates cAMP formation. Functional studies have shown that the βAR also couples to inhibitory G protein G, activation of which inhibits cAMP formation [R. P.

Conformational Dynamics of the Periplasmic Chaperone SurA.

The periplasmic protein SurA is the primary chaperone involved in the biogenesis of bacterial outer membrane proteins and is a potential antibacterial drug target. The three-dimensional structure of SurA can be divided into three parts, a core module formed by the N- and C-terminal regions and two peptidyl-prolyl isomerase (PPIase) domains, P1 and P2. Despite the determination of the structures of several SurA-peptide complexes, the functional mechanism of this chaperone remains elusive and the roles of the two PPIase domains are yet unclear.

Backbone H, C and N resonance assignments of the proteasome lid subunit Rpn12 from Saccharomyces cerevisiae.

The 26S proteasome degrades selected polyubiquitinated proteins in the ubiquitin-proteasome system, which is the major pathway for programmed protein degradation in eukaryotic cells. The Saccharomyces cerevisiae Rpn12 locates in the lid of the 19S regulatory particle within the 26S proteasome and plays a role in recruiting the extrinsic ubiquitin receptor Rpn10. Rpn12 contains a N-terminal TPR (tetratrico peptide repeat)-like domain and a C-terminal WH (winged helix) domain.

H, C and N resonance assignments of the second peptidyl-prolyl isomerase domain of chaperone SurA from Escherichia coli.

The periplasmic chaperone SurA in Gram-negative bacteria plays a central role in the biogenesis of integral outer membrane proteins and is critical to the maintenance of bacterial membrane integrity. SurA contains a core chaperone module comprising the N- and C-terminal domains, along with two peptidyl-prolyl isomerase (PPIase) domains. The chaperone activity of SurA has been demonstrated to rely on the core module, whereas recent works suggested that the PPIase domains may regulate the chaperone activity through large conformational rearrangements.

Structural basis and mechanism of the unfolding-induced activation of HdeA, a bacterial acid response chaperone.

The role of protein structural disorder in biological functions has gained increasing attention in the past decade. The bacterial acid-resistant chaperone HdeA belongs to a group of "conditionally disordered" proteins, because it is inactive in its well-structured state and becomes activated via an order-to-disorder transition under acid stress. However, the mechanism for unfolding-induced activation remains unclear because of a lack of experimental information on the unfolded state conformation and the chaperone-client interactions.

Intra- and inter-protein couplings of backbone motions underlie protein thiol-disulfide exchange cascade.

The thioredoxin (Trx)-coupled arsenate reductase (ArsC) is a family of enzymes that catalyzes the reduction of arsenate to arsenite in the arsenic detoxification pathway. The catalytic cycle involves a series of relayed intramolecular and intermolecular thiol-disulfide exchange reactions. Structures at different reaction stages have been determined, suggesting significant conformational fluctuations along the reaction pathway.

NMR H, C, N backbone and side chain resonance assignment of the N-terminal domain of yeast proteasome lid subunit Rpn5.

The 26S proteasome is responsible for the selective, ATP-dependent degradation of polyubiquitinated proteins in eukaryotic cells. It consists of a 20S barrel-shaped core particle capped by two 19S regulatory particle at both ends. The Rpn5 subunit is a non-ATPase subunit located in the lid subcomplex of the 19S regulatory particle and is identified to inhibit the Rpn11 deubiquitinase activity in the isolated lid.

Solution structure of the N-terminal domain of proteasome lid subunit Rpn5.

The 26S proteasome is the major protein degradation machinery in living cells. The Rpn5 protein is one scaffolding subunit in the lid subcomplex of the 19S regulatory particle in the proteasome holoenzyme. Herein we report the solution structure of the N-terminal domain (NTD) of yeast Rpn5 at high resolution by NMR spectroscopy.

Residue selective N CEST and CPMG experiments for studies of millisecond timescale protein dynamics.

Proteins are intrinsically dynamic molecules and undergo exchanges among multiple conformations to perform biological functions. The CPMG relaxation dispersion and CEST experiments are two important solution NMR techniques for characterizing the conformational exchange processes on the millisecond timescale. Traditional pseudo 3D N CEST and CPMG experiments have certain limitations in their applications.

Characterizations of the Interactions between Escherichia coli Periplasmic Chaperone HdeA and Its Native Substrates during Acid Stress.

The bacterial acid-resistant chaperone HdeA is a "conditionally disordered" protein that functions at low pH when it undergoes a transition from a well-folded dimer to an unfolded monomer. The dimer dissociation and unfolding processes result in exposure of hydrophobic surfaces that allows binding to a broad range of client proteins. To fully elucidate the chaperone mechanism of HdeA, it is crucial to understand how the activated HdeA interacts with its native substrates during acid stress.

Sap1 is a replication-initiation factor essential for the assembly of pre-replicative complex in the fission yeast .

A central step in the initiation of chromosomal DNA replication in eukaryotes is the assembly of pre-replicative complex (pre-RC) at late M and early G phase of the cell cycles. Since 1973, four proteins or protein complexes, including cell division control protein 6 (Cdc6)/Cdc18, minichromosome maintenance protein complex, origin recognition complex (ORC), and Cdt1, are known components of the pre-RC. Previously, we reported that a non-ORC protein binds to the essential element Δ9 of the DNA-replication origin ARS3001.

Structure-based Inhibitor Design for the Intrinsically Disordered Protein c-Myc.

Intrinsically disordered proteins (IDPs) are associated with various diseases and have been proposed as promising drug targets. However, conventional structure-based approaches cannot be applied directly to IDPs, due to their lack of ordered structures. Here, we describe a novel computational approach to virtually screen for compounds that can simultaneously bind to different IDP conformations.

HdeB chaperone activity is coupled to its intrinsic dynamic properties.

Enteric bacteria encounter extreme acidity when passing through hosts' stomach. Since the bacterial periplasmic space quickly equilibrates with outer environment, an efficient acid resistance mechanism is essential in preventing irreversible protein denaturation/aggregation and maintaining bacteria viability. HdeB, along with its homolog HdeA, was identified as a periplasmic acid-resistant chaperone.

A Hybrid Mechanism for the Synechocystis Arsenate Reductase Revealed by Structural Snapshots during Arsenate Reduction.

Evolution of enzymes plays a crucial role in obtaining new biological functions for all life forms. Arsenate reductases (ArsC) are several families of arsenic detoxification enzymes that reduce arsenate to arsenite, which can subsequently be extruded from cells by specific transporters. Among these, the Synechocystis ArsC (SynArsC) is structurally homologous to the well characterized thioredoxin (Trx)-coupled ArsC family but requires the glutaredoxin (Grx) system for its reactivation, therefore classified as a unique Trx/Grx-hybrid family.

Structural and functional characterization of tumor suppressors TIG3 and H-REV107.

H-REV107-like family proteins TIG3 and H-REV107 are class II tumor suppressors. Here we report that the C-terminal domains (CTDs) of TIG3 and H-REV107 can induce HeLa cell death independently. The N-terminal domain (NTD) of TIG3 enhances the cell death inducing ability of CTD, while NTD of H-REV107 plays an inhibitory role.

Solution structure of yeast Rpn9: insights into proteasome lid assembly.

The regulatory particle (RP) of the 26 S proteasome functions in preparing polyubiquitinated substrates for degradation. The lid complex of the RP contains an Rpn8-Rpn11 heterodimer surrounded by a horseshoe-shaped scaffold formed by six proteasome-COP9/CSN-initiation factor (PCI)-containing subunits. The PCI domains are essential for lid assembly, whereas the detailed molecular mechanisms remain elusive.