記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.bpj.2018.11.3122

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1007/s10858-018-0192-3

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担当区分：筆頭著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

DOI： 10.1111/febs.14397

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その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1111/febs.14397

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

The controlled activation of proteins in living cells is an important goal in protein-design research, but to introduce an artificial activation switch into membrane proteins through rational design is a significant challenge because of the structural and functional complexity of such proteins. Here we report the allosteric activation of two types of membrane-bound neurotransmitter receptors, the ion-channel type and the G-protein-coupled glutamate receptors, using coordination chemistry in living cells. The high programmability of coordination chemistry enabled two His mutations, which act as an artificial allosteric site, to be semirationally incorporated in the vicinity of the ligand-binding pockets. Binding of Pd(2,2'-bipyridine) at the allosteric site enabled the active conformations of the glutamate receptors to be stabilized. Using this approach, we were able to activate selectively a mutant glutamate receptor in live neurons, which initiated a subsequent signal-transduction pathway.

DOI： 10.1038/NCHEM.2554

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担当区分：筆頭著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.str.2011.05.009

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

There has been a renewal of interest in interactions of membrane proteins with detergents and lipids, sparked both by recent results that illuminate the structural details of these interactions and also by the realization that some experimental membrane protein structures are distorted by detergent-protein interactions. The integral membrane enzyme diacylglycerol kinase (DAGK) has long been thought to require the presence of lipid as an obligate "cofactor" in order to be catalytically viable in micelles. Here, we report that near-optimal catalytic properties are observed for DAGK in micelles composed of lysomyristoylphosphatidylcholine (LMPC), with significant activity also being observed in micelles composed of lysomyristoylphosphatidylglycerol and tetradecylphosphocholine. All three of these detergents were also sustained high stability of the enzyme. NMR measurements revealed significant differences in DAGK-detergent interactions involving LMPC micelles versus micelles composed of dodecylphosphocholine. These results highlight the fact that some integral membrane proteins can maintain native-like properties in lipid-free detergent micelles and also suggest that C(14)-based detergents may be worthy of more widespread use in studies of membrane proteins.

DOI： 10.1021/bi100575s

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DOI： 10.1016/j.bbalip.2010.03.008

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

Background: The determination of protein-protein interfaces is of crucial importance to understand protein function and to guide the design of compounds. To identify protein-protein interface by NMR spectroscopy, 13C NMR paramagnetic shifts induced by freely diffusing 4-hydroxy-2, 2, 6, 6-tetramethyl-piperidine-1-oxyl (TEMPOL) are promising, because TEMPOL affects distinct 13C NMR chemical shifts of the solvent accessible nuclei belonging to proteins of interest, while 13C nuclei within the interior of the proteins may be distinguished by a lack of such shifts. Method: We measured the 13C NMR paramagnetic shifts induced by TEMPOL by recording 13C-13C TOCSY spectra for ubiquitin in the free state and the complex state with yeast ubiquitin hydrolase1 (YUH1). Results: Upon complexation of ubiquitin with YUH1, 13C NMR paramagnetic shifts associated with the protein binding interface were reduced by 0.05 ppm or more. The identified interfacial atoms agreed with the prior X-ray crystallographic data. Conclusions: The TEMPOL-induced 13C chemical shift perturbation is useful to determine precise protein-protein interfaces. General significance: The present method is a useful method to determine protein-protein interface by NMR, because it has advantages in easy sample preparations, simple data analyses, and wide applicabilities. © 2009 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbagen.2009.06.001

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Lipoproteins that play critical roles in various cellular functions of Gram-negative bacteria are localized in the cells inner and outer membranes. Lol proteins (LolA, LolB, LolC, LolD, and LolE) are involved in the transportation of outer membrane-directed lipoproteins from the inner to the outer membrane. LolA is a periplasmic chaperone that transports lipoproteins, and LolB is an outer membrane receptor that accepts lipoproteins. To clarify the structural basis for the lipoprotein transfer from LolA to LolB, we examined the interaction between LolA and mLolB, a soluble mutant of LolB, using solution NMR spectroscopy. We determined the interaction mode between LolA and mLolB with conformational changes of LolA. Based upon the observations, we propose that the LolA center dot LolB complex forms a tunnel-like structure, where the hydrophobic insides of LolA and LolB are connected, which enables lipoproteins to transfer from LolA to LolB.

DOI： 10.1074/jbc.M109.001149

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD  

DOI： 10.1016/j.pnmrs.2008.07.001

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Biochemistry & Molecular Biology (ASBMB)  

Macrophage galactose-type C-type lectins 1 and 2 (MGL1/2) are expressed on the surfaces of macrophages and immature dendritic cells. Despite the high similarity between the primary sequences of MGL1 and MGL2, they display different ligand specificities. MGL1 shows high affinity for the Lewis X trisaccharide, whereas MGL2 shows affinity for N- acetylgalactosamine. To elucidate the structural basis for the ligand specificities of the MGLs, we performed NMR analyses of the MGL1-Lewis X complex. To identify the LewisX binding site on MGL1, a saturation transfer experiment for the MGL1-LewisX complex where sugar-CH/CH2-selective saturation was applied was carried out. To obtain sugar moiety-specific information on the interface between MGL1 and the LewisX trisaccharide, saturation transfer experiments where each of galactose-H5-, fucose-CH3-, and N-acetylglucosamine-CH 3-selective saturations was applied to the MGL1-LewisX complex were performed. Based on these results, we present a LewisX binding mode on MGL1 where the galactose moiety is bound to the primary sugar binding site, including Asp-94, Trp-96, and Asp-118, and the fucose moiety interacts with the secondary sugar binding site, including Ala-89 and Thr-111. Ala-89 and Thr-111 in MGL1 are replaced with arginine and serine in MGL2, respectively. The hydrophobic environment formed by a small side chain of Ala-89 and a methyl group of Thr-111 is a requisite for the accommodation of the fucose moiety of the LewisX trisaccharide within the sugar binding site of MGL1. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI： 10.1074/jbc.M804067200

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Binding specificities of mouse macrophage galactose-type C-type lectin 1 (MGL1/CD301a) and 2 (MGL2/CD301b) toward various oligosaccharides were compared by frontal affinity chromatography. MGL1 preferentially bound oligosaccharides containing LewisX (LeX) trisaccharides among 111 oligosaccharides tested, whereas MGL2 preferentially bound globoside Gb4. The important amino acids for the preferential bindings were investigated by pair-wise site-directed mutagenesis at positions 61, 89, 97, 100, 110-113, 115, 124, and 125 in the soluble recombinant carbohydrate recognition domains (CRD) prepared in Escherichia coli and purified with galactose-Sepharose. Mutations of Val, Ala, Thr, and Phe at positions 61, 89, 111 and 125 on MGL1 CRD caused reductions in LeX binding. Mutations of MGL2 CRD at Leu, Arg, Arg, and Tyr at positions 61, 89, 115 and 125 were implicated in the preference for β-GalNAc. LeX binding was observed with MGL2 mutants of Arg89Ala and Arg89Ala/Ser111Thr. MGL1 mutants of Ala89Arg and Ala89Arg/Pro115Arg showed β-GalNAc bindings. Molecular modeling illustrated potential direct molecular interactions of Leu61, Arg89, and His109 in MGL2 CRD with GalNAc. © 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbagen.2007.10.017

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

LolB, which is anchored to the outer membrane of Gram-negative bacteria, receives outer-membrane-directed lipoproteins from LolA, and incorporates them into the outer membrane. Weestablished backbone resonance assignments of H-2/C-13/N-15 labeled LolB from Escherichia coli.

DOI： 10.1007/s12104-007-9033-0

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

LolA is an essential periplasmic protein in Gram-negative bacteria and plays a role in transporting lipoproteins through periplasmic space from the inner to the outer membrane. We established backbone resonance assignments of H-2/C-13/N-15 labeled LolA from Escherichia coli.

DOI： 10.1007/s12104-007-9034-z

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

Catalytic antibodies 6D9 and 9C10, which were induced by immunization with a haptenic transition-state analog (TSA), catalyze the hydrolysis of a nonbioactive chloramphenicol monoester derivative to generate a bioactive chloramphenicol. These antibodies stabilize the transition state to catalyze the hydrolysis reaction, strictly according to the theoretical relationship: for 6D9, k(cat)/k(uncat) 895 and K(S)/K(TSA)=900, and for 9C10, k(cat)/k(uncat)=56 and K(S)/K(TSA)=60. To elucidate the molecular basis of the antibody-catalyzed reaction, the crystal structure of 6D9 was determined, and the binding thermodynamics of 6D9 and 9C10 with both the substrate and the TSA were analyzed using isothermal titration calorimetry. The crystal structure of the unliganded 6D9 Fab was determined at 2.25 angstrom resolution and compared with that of the TSA-liganded 6D9 Fab reported previously, showing that the TSA is bound into the hydrophobic pocket of the antigen-combining site in an "induced fit" manner, especially at the L1 and H3 CDR loops. Thermodynamic analyses showed that 6D9 binds the substrate of the TSA with a positive Delta S, differing from general thermodynamic characteristics of antigen-antibody interactions. This positive Delta S could be due to the hydrophobic interactions between 6D9 and the substrate or the TSA mediated by Trp H100i. The difference in A G between substrate and TSA-binding to 6D9 was larger than that to 9C10, which is in good correlation with the larger k(cat) value of 6D9. Interestingly, the Delta Delta G was mainly because of the Delta Delta H. The correlation between k(cat) and Delta Delta H is suggestive of "enthalpic strain" leading to destabilization of antibody-substrate complexes. Together with X-ray structural analyses, the thermodynamic analyses suggest that upon binding the substrate, the antibody alters the conformation of the ester moiety in the substrate from the planar Z form to a thermodynamically unstable twisted conformation, followed by conversion into the transition state. Enthalpic strain also contributes to the transition-state stabilization by destabilizing the ground state, and its degree is much larger for the more efficient catalytic antibody, 6D9. (c) 2007 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2007.03.023

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

The catalytic antibody 6D9, which was raised against a transition-state analogue (TSA), catalyzes the hydrolysis of a non-bioactive chloramphenicol monoester to generate chloramphenicol. It has been shown that 6D9 utilizes the binding affinity in the catalysis
the differential affinity of the TSA relative to the substrate is equal to the rate enhancement. To reveal the recognition mechanism of 6D9 for the TSA and the substrate, we performed NMR analysis of the Fv fragment of 6D9 (6D9-Fv), together with site-directed mutagenesis and stopped-flow kinetic analyses. Among six 6D9-Fv mutants, Y58(H)A and W100i(H)A displayed significant reductions in their affinities to the TSA, while their substrate-binding affinities were identical with that of the wild-type 6D9-Fv. The stopped-flow kinetic studies revealed that the TSA binding to 6D9-Fv occurred by an induced-fit mechanism. In contrast, no induced-fit type of TSA-binding mechanism was observed for Y58(H)A and W100i(H)A. From NMR experiments, we identified the residues with chemical shifts that were perturbed by the ligand-binding. The residues affected by the TSA binding were located on the TSA-binding site determined by the X-ray study, and on the regions far from the binding site. On the other hand, the residues affected by the substrate binding were localized on the TSA-binding site. As for W100i(H)A, no residue other than those in the binding site was affected by the ligand binding. On the basis of these results and the crystal structure, we concluded that the TSA binding induced a conformational change involving the formation of aromatic-aromatic interactions and a hydrogen bond. These interactions can account for the differential affinity for the TSA relative to the substrate. W100i(H) probably plays an important role in inducing the conformational changes. The present NMR studies have enabled us to visualize the concept of transition-state stabilization in enzymatic catalysis, in which the transition-state contacts are better than those of the substrate. © 2006 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2006.12.037

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

CD44, a major cell surface receptor for hyaluronan (HA), contains a functional domain responsible for HA binding at its N terminus (residues 21-178). Accumulating evidence indicates that proteolytic cleavage of CD44 in its extracellular region (residues 21-268) leads to enhanced tumor cell migration and invasion. Hence, understanding the mechanisms underlying the CD44 proteolytic cleavage is important for understanding the mechanism of CD44-mediated tumor progression. Here we present the NMR structure of the HA-binding domain of CD44 in its HA-bound state. The structure is composed of the Link module (residues 32-124) and an extended lobe (residues 21-31 and 125-152). Interestingly, a comparison of its unbound and HA-bound structures revealed that rearrangement of the beta-strands in the extended lobe (residues 143-148) and disorder of the structure in the following C-terminal region (residues 153-169) occurred upon HA binding, which is consistent with the results of trypsin proteolysis studies of the CD44 HA-binding domain. The order-to-disorder transition of the C-terminal region by HA binding may be involved in the CD44-mediated cell migration.

DOI： 10.1074/jbc.M608425200

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

DOI： 10.1007/s10858-005-3870-x

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その他リンク： http://link.springer.com/article/10.1007/s10858-005-3870-x/fulltext.html

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

Using oxygen as a paramagnetic probe, researchers can routinely study topologies and protein-binding interfaces by NMR. The paramagnetic contribution to the amide 1H spin-lattice relaxation rates (R1 P) have been studied for uniformly 2H,15N- labeled FB protein, a 60-residue three-helix bundle, constituting the B domain of protein A. Through TROSY versions of inversion-recovery experiments, R 1P could be determined. R1P was then measured in the presence of a stoichiometric equivalent of an unlabeled Fc fragment of immunoglobulin (Ig) G, and the ratio of R1P of the FB-Fc complex to that of free FB [i.e., R1P(complex)/ R1P(free)] was determined for each observable residue. Regions of helix I and helix II, which were previously known to interact with Fc, were readily identified as belonging to the binding interface by their characteristically reduced values of R1P(complex)/R 1P(free). The method of comparing oxygen-induced spin-lattice relaxation rates of free protein and protein-protein complexes, to detect binding interfaces, offers greater sensitivity than chemical shift perturbation, while it is not necessary to heavily deuterate the labeled protein, as is the case in cross saturation experiments. © 2005 American Chemical Society.

DOI： 10.1021/ja047825j

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

DOI： 10.1023/B:JNMR.0000019465.12250.e0

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

CD44 is the main cell surface receptor for hyaluronic acid (HA) and contains a functional HA-binding domain (HABD) composed of a Link module with N- and C-terminal extensions. The contact residues of human CD44 HABD for HA have been determined by cross-saturation experiments and mapped on the topology of CD44 HABD, which we elucidated by NMR. The contact residues are distributed in both the consensus fold for the Link module superfamily and the additional structural elements consisting of the flanking regions. Interestingly, the contact residues exhibit small changes in chemical shift upon HA binding. In contrast, the residues with large chemical shift changes are localized in the C-terminal extension and the first alpha-helix and are generally inconsistent with the contact residues. These results suggest that, upon ligand binding, the C-terminal extension and the first alpha-helix undergo significant conformational changes, which may account for the broad ligand specificity of CD44 HABD.

DOI： 10.1074/jbc.M308199200

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

The nature of the supramolecular complex between fibrillar collagen and collagen-binding proteins (CBPs) has hindered detailed X-ray and NMR analyses of the ligand-recognition mechanism at atomic resolution because of the lack of appropriate approaches for studying large heterogeneous supramolecular complexes. Recently, we proposed an NMR method, termed transferred cross-saturation (TCS), that enables the rigorous identification of contact residues in a huge protein complex. Here we used TCS to study the supramolecular complex between the A3 domain of von Willebrand factor and fibrillar collagen, which allowed the successful determination of the ligand-binding site of the A3 domain. The binding site of the A3 domain was located at its hydrophobic 'front' surface and was completely different from that of the I domain from the alpha2 subunit of integrin (alpha2-I domain), which was reported to be the hydrophilic 'top' surface of alpha2-I, although the A3 domain and the alpha2-I domain share a similar fold and possess the identical function of collagen binding.

DOI： 10.1038/nsb876

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

In an earlier paper, it was shown that the cross-saturation method enables us to identify the contact residues of large protein complexes in a more rigorous manner than is possible using chemical shift perturbation and hydrogen-deuterium exchange experiments. However, there are limitations within the determination of the contact residues by the cross-saturation method, in that the method is difficult to apply to protein complexes with a molecular mass over 150 kDa and/or with weak binding, since the resonances originating from the complexes should be observed directly in the method. In the present work, to overcome these limitations, we carried out the cross-saturation measurements under conditions of a fast exchange between free and bound states on the NMR time-scale, and determined the contact residues of the complex of the B domain of protein A and intact IgG, which has a molecular mass of 164 kDa and shows weak binding. (C) 2002 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/S0022-2836(02)00018-9

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

DOI： 10.1021/ja982153g

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記述言語：日本語   出版者・発行元：一般社団法人日本生物物理学会  

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