The three-dimensional solution structure of the recombinant B domain (FB) of staphylococcal protein A, which specifically binds to the Fc portion of immunoglobulin G, was determined by NMR spectroscopy and hybrid distance geometry-dynamical simulated annealing calculations. On the basis of 692 experimental constraints including 587 distance constraints obtained from the nuclear Overhauser effect (NOE), 57 torsion angle (phi, chi 1) constraints, and 48 constraints associated with 24 hydrogen bonds, a total of 10 converged structures of FB were obtained. The atomic root mean square difference among the 10 converged structures is 0.52 +/- 0.10 A for the backbone atoms and 0.98 +/- 0.08 A for all heavy atoms (excluding the N-terminal segment from Thr1 to Glu9 and the C-terminal segment from Gln56 to Ala60, which are partially disordered). FB is composed of a bundle of three alpha-helices, i.e., helix I (Gln10-His19), helix II (Glu25-Asp37), and helix III (Ser42-Ala55). Helix II and helix III are antiparallel to each other, whereas the long axis of helix I is tilted at an angle of about 30 degrees with respect to those of helix II and helix III. Most of the hydrophobic residues of FB are buried in the interior of the bundle of the three helices. It is suggested that the buried hydrophobic residues form a hydrophobic core, contributing to the stability of FB.(ABSTRACT TRUNCATED AT 250 WORDS)

In eukaryotes, shortening of the 3-poly(A) tail is the rate-limiting step in the degradation of most mRNAs, and two major mRNA deadenylase complexes-Caf1-Ccr4 and Pan2-Pan3-play central roles in this process, referred to as deadenylation. However, the molecular mechanism triggering deadenylation remains elusive. Previously, we demonstrated that eukaryotic releasing factor eRF3 mediates deadenylation and decay of mRNA in a manner coupled to translation termination. Here, we report the mechanism of mRNA deadenylation. The eRF3-mediated deadenylation is catalyzed by both Caf1-Ccr4 and Pan2-Pan3. Interestingly, translation termination complexes eRF1-eRF3, Pan2-Pan3, and Caf1-Ccr4 competitively interact with polyadenylate-binding protein PABPC1. In each complex, eRF3, Pan3, and Tob, respectively, mediate PABPC1 binding, and a combination of a PAM2 motif and a PABC domain is commonly utilized for their contacts. A translation-dependent exchange of eRF1-eRF3 for the deadenylase occurs on PABPC1. Consequently, PABPC1 binding leads to the activation of Pan2-Pan3 and Caf1-Ccr4. From these results, we suggest a mechanism of mRNA deadenylation by Pan2-Pan3 and Caf1-Ccr4 in cooperation with eRF3 and PABPC1.[Keywords: Translation termination; deadenylation; eRF3; PABPC1] Supplemental material is available at http://www.genesdev.org.

Many drugs that target G-protein-coupled receptors (GPCRs) induce or inhibit their signal transduction with different strengths, which affect their therapeutic properties. However, the mechanism underlying the differences in the signalling levels is still not clear, although several structures of GPCRs complexed with ligands determined by X-ray crystallography are available. Here we utilized NMR to monitor the signals from the methionine residue at position 82 in neutral antagonist- and partial agonist-bound states of β2-adrenergic receptor (β2AR), which are correlated with the conformational changes of the transmembrane regions upon activation. We show that this residue exists in a conformational equilibrium between the inverse agonist-bound states and the full agonist-bound state, and the population of the latter reflects the signal transduction level in each ligand-bound state. These findings provide insights into the multi-level signalling of β2AR and other GPCRs, including the basal activity, and the mechanism of signal transduction mediated by GPCRs.

Abstract. We present a complete list of extremal elliptic K3 surfaces (Theorem 1

We make a complete list of all possible ADE-types of singular fibers of complex elliptic K3 surfaces and the torsion parts of their Mordell-Weil groups.

The 826 G protein-coupled receptors (GPCRs) in the human proteome regulate key physiological processes, and thus have long been attractive as drug targets. With crystal structure determinations of more than 50 different human GPCRs during the last decade, an initial platform for structure-based rational design has been established for drugs that target GPCRs, which is currently being augmented with cryo-EM structures of higher-order GPCR complexes. Nuclear magnetic resonance (NMR) spectroscopy in solution is one of the key approaches for expanding this platform with dynamic features, which can be accessed at physiological temperature and with minimal modification of the wild-type GPCR covalent structures. Here, we review strategies for the use of advanced biochemistry and NMR techniques with GPCRs, survey projects where crystal or cryo-EM structures have been complemented with NMR investigations, and discuss the impact of this integrative approach on GPCR biology and drug discovery. More than 30% of all drugs approved by the US Food and Drug Administration target G protein-coupled receptors (GPCRs)1–3, and these drugs are utilized in a wide range of therapeutic areas, including inflammation and diseases of the central nervous system as well as the cardiovascular, respiratory and gastrointestinal systems2,4. Currently more than 300 agents are in clinical trials, of which around 60 target novel GPCRs for which no drug has as yet been approved2. The novel GPCR targets also include orphan GPCRs, for which endogenous ligands have not yet been discovered2. Overall, the drugs approved so far target only 27% of the human non-olfactory GPCRs2, indicating that much excitement still lies ahead. Identifying new GPCR drugs will need additional detailed knowledge of GPCR biology, especially knowledge from structural biology, given the complex structure–function relationships involved in GPCR signaling. Along this line, recent reviews on GPCRs have covered studies with antibodies5 and nanobodies6, allosteric modulation7–10 biased signaling11–15, methods in GPCR structural biology16–19, GPCR crystal structures20–27 and drug development2,4,28,29, with some reviews addressing specific GPCR families30,31. Complementing the substantial number of GPCR crystal structures that have become available in the past decade, as well as the recent demonstrations of the potential for cryo-EM to provide information on higher-order GPCR complexes32–36, dynamic studies of GPCRs are important for providing new insights into GPCR biology that can assist drug discovery. In this respect, nuclear magnetic resonance (NMR) spectroscopy in solution is a key tool for analysing function-related conformational equilibria in GPCRs as they relate to allosteric coupling, variable efficacies and biased signaling of GPCR ligands, which are of particular interest for their potential as drugs. Furthermore, NMR spectroscopy is also a useful tool for fragment-based lead discovery with GPCR targets. In this article, we first overview the structural biology of GPCRs ba...

The chemokine stromal cell-derived factor-1 (SDF-1/ CXCL12) and its G-protein-coupled receptor (GPCR) CXCR4 play fundamental roles in many physiological processes, and CXCR4 is a drug target for various diseases such as cancer metastasis and human immunodeficiency virus, type 1, infection. However, almost no structural information about the SDF-1-CXCR4 interaction is available, mainly because of the difficulties in expression, purification, and crystallization of CXCR4. In this study, an extensive investigation of the preparation of CXCR4 and optimization of the experimental conditions enables NMR analyses of the interaction between the full-length CXCR4 and SDF-1. We demonstrated that the binding of an extended surface on the SDF-1 ␤-sheet, 50-s loop, and N-loop to the CXCR4 extracellular region and that of the SDF-1 N terminus to the CXCR4 transmembrane region, which is critical for G-protein signaling, take place independently by methyl-utilizing transferred cross-saturation experiments along with the usage of the CXCR4-selective antagonist AMD3100. Furthermore, based upon the data, we conclude that the highly dynamic SDF-1 N terminus in the 1st step bound state plays a crucial role in efficiently searching the deeply buried binding pocket in the CXCR4 transmembrane region by the "fly-casting" mechanism. This is the first structural analyses of the interaction between a full-length GPCR and its chemokine, and our methodology would be applicable to other GPCR-ligand systems, for which the structural studies are still challenging.Chemokines are a number of small (8 -10 kDa) secreted proteins that direct cell migration in immune systems by activating their receptors expressed on the cell surface (1, 2). The chemokine, stromal cell-derived factor-1 (SDF-1, 2 also known as CXCL12) (3, 4), and its receptor, CXCR4 (5-7), play many essential physiological roles, such as homeostatic regulation of leukocyte traffic, hematopoiesis, and embryonic development (8 -11). The interaction between SDF-1 and CXCR4 also controls cancer metastasis (12, 13), and CXCR4 is a co-receptor for T-tropic strains of human immunodeficiency virus, type 1 (5, 14).The most abundant splice variant of SDF-1 (SDF-1␣) is composed of 68 amino acids, and its NMR (15, 16) and crystal structures (17, 18) demonstrated that SDF-1␣ assumes a typical chemokine fold as follows: an unstructured N terminus (Lys 1 -Tyr 7 ) followed by a long flexible loop (N-loop), a three-stranded anti-parallel ␤-sheet, and an ␣-helix. The mutational analyses revealed that although the SDF-1␣ N terminus is critical for the CXCR4-mediated signaling (15), both the N terminus and the N-loop residues are implicated in the receptor binding (15,18,19). In addition, recent mutational analysis suggested that the residues on the SDF-1␣ ␤-sheet are also important for receptor binding (20).CXCR4, composed of 352 amino acids, belongs to the class A G-protein-coupled receptor (GPCR) family, with the seven transmembrane (TM) helices. Whereas GPCR activation is mediated by the conformation...

KcsA is a prokaryotic pH-dependent potassium (K) channel. Its activation, by a decrease in the intracellular pH, is coupled with its subsequent inactivation, but the underlying mechanisms remain elusive. Here, we have investigated the conformational changes and equilibrium of KcsA by using solution NMR spectroscopy. Controlling the temperature and pH of KcsA samples produced three distinct methyl-TROSY and NOESY spectra, corresponding to the resting, activated, and inactivated states. The pH-dependence of the signals from the extracellular side was affected by the mutation of H25 on the intracellular side, indicating the coupled conformational changes of the extracellular and intracellular gates. K þ titration and NOE experiments revealed that the inactivated state was obtained by the replacement of K þ with H 2 O, which may interfere with the K þ -permeation. This structural basis of the activation-coupled inactivation is closely related to the C-type inactivation of other K channels.potassium channel | solution NMR | gating | inactivation