We have determined the binding site on agitoxin2 (AgTx2) to the KcsA K(+) channel by a transferred cross-saturation (TCS) experiment. The residues significantly affected in the TCS experiments formed a contiguous surface on AgTx2, and substitutions of the surface residues decreased the binding affinity to the KcsA K(+) channel. Based on properties of the AgTx2 binding site with the KcsA K(+) channel, we present a surface motif that is observed in pore-blocking toxins affecting the K(+) channel. Furthermore, we also explain the structural basis of the specificity of the K(+) channel to the toxins. The TCS method utilized here is applicable not only for the channels, which are complexed with other inhibitors, but also with a variety of regulatory molecules, and provides important information about their interface in solution.

CC-chemokine receptor 5 (CCR5) belongs to the G protein-coupled receptor (GPCR) family and plays important roles in the inflammatory response. In addition, its ligands inhibit the HIV infection. Structural analyses of CCR5 have been hampered by its instability in the detergent-solubilized form. Here, CCR5 was reconstituted into high density lipoprotein (rHDL), which enabled CCR5 to maintain its functions for >24 h and to be suitable for structural analyses. By applying the methyl-directed transferred cross-saturation (TCS) method to the complex between rHDL-reconstituted CCR5 and its ligand MIP-1alpha, we demonstrated that valine 59 and valine 63 of MIP-1alpha are in close proximity to CCR5 in the complex. Furthermore, these results suggest that the protective influence on HIV-1 infection of a SNP of MIP-1alpha is due to its change of affinity for CCR5. This method will be useful for investigating the various and complex signaling mediated by GPCR, and will also provide structural information about the interactions of other GPCRs with lipids, ligands, G-proteins, and effector molecules.

Regnase-1 is an RNase that directly cleaves mRNAs of inflammatory genes such as IL-6 and IL-12p40, and negatively regulates cellular inflammatory responses. Here, we report the structures of four domains of Regnase-1 from Mus musculus—the N-terminal domain (NTD), PilT N-terminus like (PIN) domain, zinc finger (ZF) domain and C-terminal domain (CTD). The PIN domain harbors the RNase catalytic center; however, it is insufficient for enzymatic activity. We found that the NTD associates with the PIN domain and significantly enhances its RNase activity. The PIN domain forms a head-to-tail oligomer and the dimer interface overlaps with the NTD binding site. Interestingly, mutations blocking PIN oligomerization had no RNase activity, indicating that both oligomerization and NTD binding are crucial for RNase activity in vitro. These results suggest that Regnase-1 RNase activity is tightly controlled by both intramolecular (NTD-PIN) and intermolecular (PIN-PIN) interactions.

G protein-activated inwardly rectifying potassium channel (GIRK) plays crucial roles in regulating heart rate and neuronal excitability in eukaryotic cells. GIRK is activated by the direct binding of heterotrimeric G protein ␤␥ subunits (G␤␥) upon stimulation of G protein-coupled receptors, such as M2 acetylcholine receptor. The binding of G␤␥ to the cytoplasmic pore (CP) region of GIRK causes structural rearrangements, which are assumed to open the transmembrane ion gate. However, the crucial residues involved in the G␤␥ binding and the structural mechanism of GIRK gating have not been fully elucidated. Here, we have characterized the interaction between the CP region of GIRK and G␤␥, by ITC and NMR. The ITC analyses indicated that four G␤␥ molecules bind to a tetramer of the CP region of GIRK with a dissociation constant of 250 M. The NMR analyses revealed that the G␤␥ binding site spans two neighboring subunits of the GIRK tetramer, which causes conformational rearrangements between subunits. A possible binding mode and mechanism of GIRK gating are proposed.G protein-activated inwardly rectifying potassium channel (GIRK) 3 is a member of the inwardly rectifying potassium channel (Kir) family, which regulates heart rate and neuronal excitability (1, 2). The Kir proteins function as tetramers, consisting of a transmembrane (TM) region and a cytoplasmic pore (CP) region. The helix bundle at the cytoplasmic side of the TM region is assumed to be a K ϩ -ion gate. The opening and closing in the gate (gating) of Kirs are regulated by a variety of cytoplasmic factors. The gating of GIRK is triggered by the binding of its CP region with the heterotrimeric G-protein ␤␥ subunits (G␤␥), which are released from the pertussis toxin-sensitive G protein ␣ subunit (G␣ i/o ), subsequent to the stimulation of a G protein-coupled receptor, such as M2 acetylcholine receptor.Extensive mutational analyses to identify the GIRK residues that are critical for the G␤␥-induced activation revealed several critical residues such as His 57 , Leu 262 , Leu 333, and Gly 336 of GIRK1 (3-5). However, when these residues were mapped on the recently reported crystal structures of Kirs, they did not form a cluster on the protein surface (6 -9). Therefore, no clear consensus has been obtained regarding the region of GIRK that is essential for G␤␥ binding and/or GIRK activation. One of the reasons might be a structural alteration introduced by the mutagenesis (10), which could change the gating property of the channel. Although the various crystal structures have provided little information about the conformational change involved in the gating of the channel, FRET analyses have clearly demonstrated the conformational rearrangements in the CP region of GIRK upon G␤␥ binding (11). However, the resolution of the structural information obtained by the FRET analyses is low, primarily due to the large size of the fluorescent probe proteins.To reveal the structural mechanism by which G␤␥ binding activates GIRK, we have investigated the direct interacti...

Heterotrimeric guanine-nucleotide-binding proteins (G proteins) serve as molecular switches in signalling pathways, by coupling the activation of cell surface receptors to intracellular responses. Mutations in the G protein α-subunit (Gα) that accelerate guanosine diphosphate (GDP) dissociation cause hyperactivation of the downstream effector proteins, leading to oncogenesis. However, the structural mechanism of the accelerated GDP dissociation has remained unclear. Here, we use magnetic field-dependent nuclear magnetic resonance relaxation analyses to investigate the structural and dynamic properties of GDP bound Gα on a microsecond timescale. We show that Gα rapidly exchanges between a ground-state conformation, which tightly binds to GDP and an excited conformation with reduced GDP affinity. The oncogenic D150N mutation accelerates GDP dissociation by shifting the equilibrium towards the excited conformation.

Background: Although G␤␥ is known to activate GIRK, G␣ i/o also modulates GIRK gating. Results: The ␣2/␣3 helices of G␣ i3 in the GTP-bound state directly bind to the ␣A helix of GIRK. Conclusion:The complex model explains how G␣ i/o sequesters G␤␥ efficiently from GIRK upon GTP hydrolysis. Significance: The structural basis for the rapid closure of GIRK by G␣ i/o is provided.

Eukaryotic mature mRNAs possess a poly adenylate tail (poly(A)), to which multiple molecules of poly(A)-binding protein C1 (PABPC1) bind. PABPC1 regulates translation and mRNA metabolism by binding to regulatory proteins. To understand functional mechanism of the regulatory proteins, it is necessary to reveal how multiple molecules of PABPC1 exist on poly(A). Here, we characterize the structure of the multiple molecules of PABPC1 on poly(A), by using transmission electron microscopy (TEM), chemical cross-linking, and NMR spectroscopy. The TEM images and chemical cross-linking results indicate that multiple PABPC1 molecules form a wormlike structure in the PABPC1-poly(A) complex, in which the PABPC1 molecules are linearly arrayed. NMR and cross-linking analyses indicate that PABPC1 forms a multimer by binding to the neighbouring PABPC1 molecules via interactions between the RNA recognition motif (RRM) 2 in one molecule and the middle portion of the linker region of another molecule. A PABPC1 mutant lacking the interaction site in the linker, which possesses an impaired ability to form the multimer, reduced the in vitro translation activity, suggesting the importance of PABPC1 multimer formation in the translation process. We therefore propose a model of the PABPC1 multimer that provides clues to comprehensively understand the regulation mechanism of mRNA translation.

G protein-gated inwardly rectifying potassium channel (GIRK) plays a key role in regulating neurotransmission. GIRK is opened by the direct binding of the G protein βγ subunit (Gβγ), which is released from the heterotrimeric G protein (Gαβγ) upon the activation of G protein-coupled receptors (GPCRs). GIRK contributes to precise cellular responses by specifically and efficiently responding to the Gi/o-coupled GPCRs. However, the detailed mechanisms underlying this family-specific and efficient activation are largely unknown. Here, we investigate the structural mechanism underlying the Gi/o family-specific activation of GIRK, by combining cell-based BRET experiments and NMR analyses in a reconstituted membrane environment. We show that the interaction formed by the αA helix of Gαi/o mediates the formation of the Gαi/oβγ-GIRK complex, which is responsible for the family-specific activation of GIRK. We also present a model structure of the Gαi/oβγ-GIRK complex, which provides the molecular basis underlying the specific and efficient regulation of GIRK.