Function and dynamics of the intrinsically disordered carboxyl terminus of ß2 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 ß2 adrenergic receptor's (ß2AR) 71 amino acid CT is a substrate for GPCR kinases and binds ß-arrestins to regulate signaling. Here we show that the ß2AR CT directly inhibits basal and agonist-stimulated signaling in cell lines lacking ß-arrestins. Combining single-molecule fluorescence resonance energy transfer (FRET), NMR spectroscopy, and molecular dynamics simulations, we reveal that the negatively charged ß2AR-CT serves as an autoinhibitory factor via interacting with the positively charged cytoplasmic surface of the receptor to limit access to G-proteins. The stability of this interaction is influenced by agonists and allosteric modulators, emphasizing that the CT plays important role in allosterically regulating GPCR activation.

Conformational flexibility of adenine riboswitch aptamer in apo and bound states using NMR and an X-ray free electron laser.

Riboswitches are structured cis-regulators mainly found in the untranslated regions of messenger RNA. The aptamer domain of a riboswitch serves as a sensor for its ligand, the binding of which triggers conformational changes that regulate the behavior of its expression platform. As a model system for understanding riboswitch structures and functions, the add adenine riboswitch has been studied extensively. However, there is a need for further investigation of the conformational dynamics of the aptamer in light of the recent real-time crystallographic study at room temperature (RT) using an X-ray free electron laser (XFEL) and femtosecond X-ray crystallography (SFX). Herein, we investigate the conformational motions of the add adenine riboswitch aptamer domain, in the presence or absence of adenine, using nuclear magnetic resonance relaxation measurements and analysis of RT atomic displacement factors (B-factors). In the absence of ligand, the P1 duplex undergoes a fast exchange where the overall molecule exhibits a motion at kex ~ 319 s-1, based on imino signals. In the presence of ligand, the P1 duplex adopts a highly ordered conformation, with kex~ 83 s-1, similar to the global motion of the molecule, excluding the loops and binding pocket, at 84 s-1. The µs-ms motions in both the apo and bound states are consistent with RT B-factors. Reduced spatial atomic fluctuation, ~ 50%, in P1 upon ligand binding coincides with significantly attenuated temporal dynamic exchanges. The binding pocket is structured in the absence or presence of ligand, as evidenced by relatively low and similar RT B-factors. Therefore, despite the dramatic rearrangement of the binding pocket, those residues exhibit similar spatial thermal fluctuation before and after binding.

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. Herein, we used advanced solution NMR methods to characterize the activated-state conformation of HdeA under acidic conditions and identify its client-binding sites. We observed that the structure of activated HdeA becomes largely disordered and exposes two hydrophobic patches essential for client interactions. Furthermore, using the pH-dependent chemical exchange saturation transfer (CEST) NMR method, we identified three acid-sensitive regions that act as structural locks in regulating the exposure of the two client-binding sites during the activation process, revealing a multistep activation mechanism of HdeA's chaperone function at the atomic level. Our results highlight the role of intrinsic protein disorder in chaperone function and the self-inhibitory role of ordered structures under nonstress conditions, offering new insights for improving our understanding of protein structure-function paradigms.

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. Herein, we use two state-of-the-art NMR methods, the chemical exchange saturation transfer (CEST) and the CPMG-based relaxation dispersion (CPMG RD) experiments, to probe the conformational dynamics of B. subtilis ArsC in all reaction stages, namely the enzymatic active reduced state, the intra-molecular C10-C82 disulfide-bonded intermediate state, the inactive oxidized state, and the inter-molecular disulfide-bonded protein complex with Trx. Our results reveal highly rugged energy landscapes in the active reduced state, and suggest global collective motions in both the C10-C82 disulfide-bonded intermediate and the mixed-disulfide Trx-ArsC complex.

Residue selective 15N 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 15N CEST and CPMG experiments have certain limitations in their applications. For example, both experiments have low sensitivity for broadened resonances, and the process of optimizing sample conditions and experimental parameters are often time consuming. To overcome these limitations, we herein present a new set of residue selective 15N CEST and CPMG pulse sequences by employing the Hartmann-Hahn cross-polarization transfer of magnetization in both 1D and 2D schemes. Combined with frequency labeling in the indirect dimension using only a small number of increments, the pulse sequences in the 2D scheme can be applied on resonances in overlapped regions of the 1H-15N HSQC spectrum. The pulse sequences were further applied on several proteins, demonstrating their advantages over the traditional CEST and CPMG experiments under specific circumstances.

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. Herein, we present a nuclear magnetic resonance study of the pH-dependent HdeA-substrate interactions. Our results show that the activation of HdeA is not only induced by acidification but also regulated by the presence of unfolded substrates. The variable extent of unfolding of substrates differentially regulates the HdeA-substrate interaction, and the binding further affects the HdeA conformation. Finally, we show that HdeA binds its substrates heterogeneously, and the "amphiphilic" model for HdeA-substrate interaction is discussed.

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

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 G1 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 Schizosaccharomyces pombe DNA-replication origin ARS3001. In this study, we identified that the non-ORC protein is Sap1. Like ORC, Sap1 binds to DNA origins during cell growth cycles. But unlike ORC, which binds to asymmetric AT-rich sequences through its nine AT-hook motifs, Sap1 preferentially binds to a DNA sequence of 5'-(A/T) n (C/G)(A/T)9-10(G/C)(A/T) n -3' (n ≥ 1). We also found that Sap1 and ORC physically interact. We further demonstrated that Sap1 is required for the assembly of the pre-RC because of its essential role in recruiting Cdc18 to DNA origins. Thus, we conclude that Sap1 is a replication-initiation factor that directly participates in the assembly of the pre-RC. DNA-replication origins in fission yeast are defined by possessing two essential elements with one bound by ORC and the other by Sap1.

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. Both proteins exist as homodimers and share similar monomeric structures under neutral pH, while showing different dimeric packing interfaces. Previous investigations show that HdeA functions through an acid-induced dimer-to-monomer transition and partial unfolding at low pH (pH 2-3), resulting in exposure of hydrophobic surfaces that bind substrate proteins. In contrast, HdeB appears to have a much higher optimal activation pH (pH 4-5), under which condition the protein maintains a well-folded dimer and the mechanism for its chaperone activity remains elusive. Herein, we present an NMR study of HdeB to investigate its dynamic properties. Our results reveal that HdeB undergoes significant micro- to milli-second timescale conformational exchanges at neutral to near-neutral pH, under the later condition it exhibits optimal activity. The current study indicates that HdeB activation is coupled to its intrinsic dynamics instead of structural changes, and therefore its functional mechanism is apparently different from HdeA.

Gene cloning, expression, and characterization of a nitrilase from Alcaligenes faecalis ZJUTB10.

Nitrilases are important industrial enzymes that convert nitriles directly into the corresponding carboxylic acids. In the current work, the fragment with a length of 1068 bp that encodes the A. faecalis ZJUTB10 nitrilase was obtained. Moreover, a catalytic triad was proposed and verified by site-directed mutagenesis, and the detailed mechanism of this nitrilase was clarified. The substrate specificity study demonstrated that the A. faecalis ZJUTB10 nitrilase belongs to the family of arylacetonitrilases. The optimum pH and temperature for the purified nitrilase was 7-8 and 40 °C, respectively. Mg(2+) stimulated hydrolytic activity, whereas Cu(2+), Co(2+), Ni(2+), Ag(+), and Hg(2+) showed a strong inhibitory effect. The K(m) and v(max) for mandelonitrile were 4.74 mM and 15.85 µmol min(-1) mg(-1) protein, respectively. After 30 min reaction using the nitrilase, mandelonitrile at the concentration of 20 mM was completely hydrolyzed and the enantiomeric excess against (R)-(-)-mandelic acid was >99%. Characteristics investigation indicates that this nitrilase is promising in catalysis applications.