Published papers

Amino acid selective cross-saturation (ASCS) method not only provides information about the interface of a protein assembly by the spin relaxation experiment, but also identifies the amino acid residues in the acceptor protein, which are located close to the selectively labeled amino acid residues in the donor protein. Here, a new method was developed to build a precise structural model of a protein assembly, which satisfies the experimental ASCS values, using simulated annealing computation. This method was applied to the ubiquitin-yeast ubiquitin hydrolase 1 (Ub-YUH1) complex to build a precise complex structure compatible with that determined by X-ray crystallography.

The membrane-proximal C-terminal region (Pro-C) is important for the regulation of G-protein-coupled receptors (GPCRs), but the binding of the Pro-C region to a cytosolic regulator has not been structurally analyzed. The chemokine receptor CCR2 is a member of the GPCR superfamily, and the Pro-C region of CCR2 binds to the cytosolic regulator FROUNT. Studying the interaction between CCR2 Pro-C and FROUNT at an atomic level provides a basis for understanding the signal transduction mechanism via GPCRs. NOE-based NMR experiments showed that, when bound to FROUNT, CCR2 Pro-C adopted a helical conformation, as well as when embedded in dodecylphosphocholine micelles. A comparison of two types of cross-saturation-based NMR experiments, applied to a three-component mixture of Pro-C, FROUNT and micelles or a two-component mixture of Pro-C and micelles, revealed that the hydrophobic binding surface on Pro-C for FROUNT mostly overlapped with the binding site for micelles, suggesting competitive binding of Pro-C between FROUNT and micelles. Leu316 was important for both FROUNT and micelle binding. Phe319 was newly identified to be crucial for FROUNT binding, by NMR and mutational analyses. The association and dissociation rates of CCR2 Pro-C for lipid bilayer biomembranes were faster than those for FROUNT. We previously reported that FROUNT binding to CCR2 is detectable even in unstimulated cells and increases in response to chemokine stimulation. Taken together, these results support a model of CCR2 equilibrium: chemokine binding changes the conformational equilibrium of CCR2 toward the active state, and Pro-C switches its binding partner from the membrane to FROUNT.

MgtE is a prokaryotic Mg(2+) transporter that controls cellular Mg(2+) concentrations. We previously reported crystal structures of the cytoplasmic region of MgtE, consisting of 2 domains, that is, N and CBS, in the Mg(2+)-free and Mg(2+)-bound forms. The Mg(2+)-binding sites lay at the interface of the 2 domains, making the Mg(2+)-bound form compact and globular. In the Mg(2+)-free structure, however, the domains are far apart, and the Mg(2+)-binding sites are destroyed. Therefore, it is unclear how Mg(2+)-free MgtE changes its conformation to accommodate Mg(2+) ions. Here, we used paramagnetic relaxation enhancement (PRE) to characterize the relative orientation of the N and CBS domains in the absence of Mg(2+) in solution. When the residues on the surface of the CBS domain were labeled with nitroxide tags, significant PRE effects were observed for the residues in the N domain. No single structure satisfied the PRE profiles, suggesting that the N and CBS domains are not fixed in a particular orientation in solution. We then conducted ensemble simulated annealing calculations in order to obtain the atomic probability density and visualize the spatial distribution of the N domain in solution. The results indicate that the N domain tends to occupy the space near its position in the Mg(2+)-bound crystal structure, facilitating efficient capture of Mg(2+) with increased intracellular Mg(2+) concentration, which is necessary to close the gate.

G protein-gated inwardly rectifying potassium channel (GIRK) plays a crucial role in regulating heart rate and neuronal excitability. The gating of GIRK is regulated by the association and dissociation of G protein betagamma subunits (Gbetagamma), which are released from pertussis toxin-sensitive G protein alpha subunit (Galpha(i/o)) upon GPCR activation in vivo. Several lines of evidence indicate that Galpha(i/o) also interacts directly with GIRK, playing functional roles in the signaling efficiency and the modulation of the channel activity. However, the underlying mechanism for GIRK regulation by Galpha(i/o) remains to be elucidated. Here, we performed NMR analyses of the interaction between the cytoplasmic region of GIRK1 and Galpha(i3) in the GTP-bound state. The NMR spectral changes of Galpha upon the addition of GIRK as well as the transferred cross-saturation (TCS) results indicated their direct binding mode, where the K(d) value was estimated as approximately 1 mm. The TCS experiments identified the direct binding sites on Galpha and GIRK as the alpha2/alpha3 helices on the GTPase domain of Galpha and the alphaA helix of GIRK. In addition, the TCS and paramagnetic relaxation enhancement results suggested that the helical domain of Galpha transiently interacts with the alphaA helix of GIRK. Based on these results, we built a docking model of Galpha and GIRK, suggesting the molecular basis for efficient GIRK deactivation by Galpha(i/o).

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 betagamma subunits (Gbetagamma) upon stimulation of G protein-coupled receptors, such as M2 acetylcholine receptor. The binding of Gbetagamma 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 Gbetagamma 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 Gbetagamma, by ITC and NMR. The ITC analyses indicated that four Gbetagamma molecules bind to a tetramer of the CP region of GIRK with a dissociation constant of 250 muM. The NMR analyses revealed that the Gbetagamma 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.

Translation termination-coupled deadenylation is the first and often the rate-limiting step of eukaryotic mRNA decay in which two deadenylases, Ccr4-Caf1 and Pan2, play key roles. One of the deadenylases, Caf1, associates with Tob, which recruits Caf1 to the poly(A) tail through interactions with a cytoplasmic poly(A)-binding protein 1 (PABPC1). We previously proposed that the competition between Tob and eRF3 (a translation termination factor that interacts with PABPC1) is responsible for the regulation of deadenylase activity. However, the molecular mechanism of the regulation should be addressed by investigating the binding affinity and the cellular levels of these proteins. In this work, we characterized the human Tob interactions with Caf1 and a C-terminal domain of PABPC1 (PABC). Nuclear magnetic resonance (NMR) and Western blot analyses revealed that Tob consists of a structured N-terminal BTG-Tob domain and an unstructured C-terminal region with two conserved PAM2 (PABPC1-interacting motif 2) motifs. The BTG-TOB domain associates with Caf1, whereas the C-terminal PAM2 motif binds to PABC, with a K(d) value of 20 microM. Furthermore, we demonstrated that the levels of eRF3 and Tob in HeLa cells are 4-5 microM and less than 0.2 microM, respectively. On the basis of these results, we propose a thermodynamic mechanism for the translation termination-coupled deadenylation mediated by the Tob-Caf1 complex.

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.

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(+) titration and NOE experiments revealed that the inactivated state was obtained by the replacement of K(+) with H(2)O, which may interfere with the K(+)-permeation. This structural basis of the activation-coupled inactivation is closely related to the C-type inactivation of other K channels.

The gating of potassium ion (K+) channels is regulated by various kinds of protein-protein interactions (PPIs). Structural investigations of these PPIs provide useful information not only for understanding the gating mechanisms of K+ channels, but also for developing the pharmaceutical compounds targeting K+ channels. Here, we describe a nuclear magnetic resonance spectroscopic method, termed the cross saturation (CS) method, to accurately determine the binding surfaces of protein complexes, and its application to the investigation of the interaction between a G protein-coupled inwardly rectifying K+ channel and a G protein α subunit.

A number of peptide toxins isolated from animals target potassium ion (K+) channels. Many of them are particularly known to inhibit voltage-gated K+ (KV) channels and are mainly classified into pore-blocking toxins or gating-modifier toxins. Pore-blocking toxins directly bind to the ion permeation pores of KV channels, thereby physically occluding them. In contrast, gating-modifier toxins bind to the voltage-sensor domains of KV channels, modulating their voltage-dependent conformational changes. These peptide toxins are useful molecular tools in revealing the structure-function relationship of KV channels and have potential for novel treatments for diseases related to KV channels. This review focuses on the inhibition mechanism of pore-blocking and gating-modifier toxins that target KV channels.

Translation of mRNAs is a fundamental process that occurs in all cell types of multicellular organisms. Conventionally, it has been considered a default step in gene expression, lacking specific regulation. However, recent studies have documented that certain mRNAs exhibit cell type–specific translation. Despite this, it remains unclear whether global translation is controlled in a cell type–specific manner. By using human cell lines and mouse models, we found that deletion of the ribosome-associated protein ribonuclease inhibitor 1 (RNH1) decreases global translation selectively in hematopoietic-origin cells but not in the non–hematopoietic-origin cells. RNH1-mediated cell type–specific translation is mechanistically linked to angiogenin-induced ribosomal biogenesis. Collectively, this study unravels the existence of cell type–specific global translation regulators and highlights the complex translation regulation in vertebrates.

Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules.

Eukaryotic releasing factor GSPT/eRF3 mediates translation termination-coupled mRNA decay via interaction with a cytosolic poly(A)-binding protein (PABPC1). A region of eRF3 containing two overlapping PAM2 (PABPC1-interacting motif 2) motifs is assumed to bind to the PABC domain of PABPC1, on the poly(A) tail of mRNA. PAM2 motifs are also found in the major deadenylases Caf1-Ccr4 and Pan2-Pan3, whose activities are enhanced upon PABPC1 binding to these motifs. Their deadenylase activities are regulated by eRF3, in which two overlapping PAM2 motifs competitively prevent interaction with PABPC1. However, it is unclear how these overlapping motifs recognize PABC and regulate deadenylase activity in a translation termination-coupled manner. We used a dominant-negative approach to demonstrate that the N-terminal PAM2 motif is critical for eRF3 binding to PABPC1 and that both motifs are required for function. Isothermal titration calorimetry (ITC) and NMR analyses revealed that the interaction is in equilibrium between the two PAM2-PABC complexes, where only one of the two overlapping PAM2 motifs is PABC-bound and the other is PABC-unbound and partially accessible to the other PABC. Based on these results, we proposed a biological role for the overlapping PAM2 motifs in the regulation of deadenylase accessibility to PABPC1 at the 3' end of poly(A).

KcsA is a tetrameric K(+) channel that is activated by acidic pH. Under open conditions of the helix bundle crossing, the selectivity filter undergoes an equilibrium between permeable and impermeable conformations. Here we report that the population of the permeable conformation (p(perm)) positively correlates with the tetrameric stability and that the population in reconstituted high density lipoprotein, where KcsA is surrounded by the lipid bilayer, is lower than that in detergent micelles, indicating that dynamic properties of KcsA are different in these two media. Perturbation of the membrane environment by the addition of 1-3% 2,2,2-trifluoroethanol increases p(perm) and the open probability, revealed by NMR and single-channel recording analyses. These results demonstrate that KcsA inactivation is determined not only by the protein itself but also by the surrounding membrane environments.

Solution NMR spectroscopy can analyze the dynamics of proteins on a wide range of timescales, from picoseconds to even days, in a site-specific manner, and thus its results are complementary to the detailed but largely static structural information obtained by X-ray crystallography. We review recent progresses in a variety of NMR techniques, including relaxation dispersion and paramagnetic relaxation enhancement (PRE), that permit the observation of the low-populated states, which had been 'invisible' with other techniques. In addition, we review how NMR spectroscopy can be used to elucidate functionally relevant protein dynamics.

The inwardly rectifying potassium channel (Kir) regulates resting membrane potential, K+ homeostasis, heart rate, and hormone secretion. The outward current is blocked in a voltage-dependent manner, upon the binding of intracellular polyamines or Mg2+ to the transmembrane pore domain. Meanwhile, electrophysiological studies have shown that mutations of several acidic residues in the intracellular regions affected the inward rectification. Although these acidic residues are assumed to bind polyamines, the functional role of the binding of polyamines and Mg2+ to the intracellular regions of Kirs remains unclear. Here, we report thermodynamic and structural studies of the interaction between polyamines and the cytoplasmic pore of mouse Kir3.1/GIRK1, which is gated by binding of G-protein betagamma-subunit (Gbetagamma). ITC analyses showed that two spermine molecules bind to a tetramer of Kir3.1/GIRK1 with a dissociation constant of 26 microM, which is lower than other blockers. NMR analyses revealed that the spermine binding site is Asp-260 and its surrounding area. Small but significant chemical shift perturbations upon spermine binding were observed in the subunit-subunit interface of the tetramer, suggesting that spermine binding alters the relative orientations of the four subunits. Our ITC and NMR results postulated a spermine binding mode, where one spermine molecule bridges two Asp-260 side chains from adjacent subunits, with rearrangement of the subunit orientations. This suggests the functional roles of spermine binding to the cytoplasmic pore: stabilization of the resting state conformation of the channel, and instant translocation to the transmembrane pore upon activation through the Gbetagamma-induced conformational rearrangement.

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.

© Maruyama et al. Magnesium ions (Mg 2+ ) are divalent cations essential for various cellular functions. Mg 2+ homeostasis is maintained through Mg 2+ channels such as MgtE, a prokaryotic Mg 2+ channel whose gating is regulated by intracellular Mg 2+ levels. Our previous crystal structure of MgtE in the Mg 2+ -bound, closed state revealed the existence of seven crystallographically-independent Mg 2+ -binding sites, Mg1–Mg7. The role of Mg 2+ -binding to each site in channel closure remains unknown. Here, we investigated Mg 2+ -dependent changes in the structure and dynamics of MgtE using nuclear magnetic resonance spectroscopy. Mg 2+ -titration experiments, using wild-type and mutant forms of MgtE, revealed that the Mg 2+ binding sites Mg1, Mg2, Mg3, and Mg6, exhibited cooperativity and a higher affinity for Mg 2+ , enabling the remaining Mg 2+ binding sites, Mg4, Mg5, and Mg7, to play important roles in channel closure. This study revealed the role of each Mg 2+ -binding site in MgtE gating, underlying the mechanism of cellular Mg 2+ homeostasis.

Background: Human ether-à-go-go-related gene potassium channel 1 (hERG) is a voltage-gated potassium channel, the voltage-sensing domain (VSD) of which is targeted by a gating-modifier toxin, APETx1. APETx1 is a 42-residue peptide toxin of sea anemone Anthopleura elegantissima and inhibits hERG by stabilizing the resting state. A previous study that conducted cysteine-scanning analysis of hERG identified two residues in the S3-S4 region of the VSD that play important roles in hERG inhibition by APETx1. However, mutational analysis of APETx1 could not be conducted as only natural resources have been available until now. Therefore, it remains unclear where and how APETx1 interacts with the VSD in the resting state. Results: We established a method for preparing recombinant APETx1 and determined the NMR structure of the recombinant APETx1, which is structurally equivalent to the natural product. Electrophysiological analyses using wild type and mutants of APETx1 and hERG revealed that their hydrophobic residues, F15, Y32, F33, and L34, in APETx1, and F508 and I521 in hERG, in addition to a previously reported acidic hERG residue, E518, play key roles in the inhibition of hERG by APETx1. Our hypothetical docking models of the APETx1-VSD complex satisfied the results of mutational analysis. Conclusions: The present study identified the key residues of APETx1 and hERG that are involved in hERG inhibition by APETx1. These results would help advance understanding of the inhibitory mechanism of APETx1, which could provide a structural basis for designing novel ligands targeting the VSDs of KV channels.

Protein–protein interactions (PPIs) are prospective but challenging targets for drug discovery, because screening using traditional small-molecule libraries often fails to identify hits. Recently, we developed a PPI-oriented library comprising 12,593 small-to-medium-sized newly synthesized molecules. This study validates a promising combined method using PPI-oriented library and ligand-based virtual screening (LBVS) to discover novel PPI inhibitory compounds for Kelch-like ECH-associated protein 1 (Keap1) and nuclear factor erythroid 2-related factor 2 (Nrf2). We performed LBVS with two random forest models against our PPI library and the following time-resolved fluorescence resonance energy transfer (TR-FRET) assays of 620 compounds identified 15 specific hit compounds. The high hit rates for the entire PPI library (estimated 0.56–1.3%) and the LBVS (maximum 5.4%) compared to a conventional screening library showed the utility of the library and the efficiency of LBVS. All the hit compounds possessed novel structures with Tanimoto similarity ≤ 0.26 to known Keap1/Nrf2 inhibitors and aqueous solubility (AlogP < 5). Reasonable binding modes were predicted using 3D alignment of five hit compounds and a Keap1/Nrf2 peptide crystal structure. Our results represent a new, efficient method combining the PPI library and LBVS to identify novel PPI inhibitory ligands with expanded chemical space.