Hidekazu Hiroaki - Academia.edu (original) (raw)
Papers by Hidekazu Hiroaki
International journal of molecular sciences, 2015
Intrinsically disordered proteins (IDPs) that lack stable conformations and are highly flexible h... more Intrinsically disordered proteins (IDPs) that lack stable conformations and are highly flexible have attracted the attention of biologists. Therefore, the development of a systematic method to identify polypeptide regions that are unstructured in solution is important. We have designed an "indirect/reflected" detection system for evaluating the physicochemical properties of IDPs using nuclear magnetic resonance (NMR). This approach employs a "chimeric membrane protein"-based method using the thermostable membrane protein PH0471. This protein contains two domains, a transmembrane helical region and a C-terminal OB (oligonucleotide/oligosaccharide binding)-fold domain (named NfeDC domain), connected by a flexible linker. NMR signals of the OB-fold domain of detergent-solubilized PH0471 are observed because of the flexibility of the linker region. In this study, the linker region was substituted with target IDPs. Fifty-three candidates were selected using the predic...
Proceedings of the National Academy of Sciences, 2015
Neurodegeneration correlates with Alzheimer's disease (AD) symptoms, but the molecular 2 identiti... more Neurodegeneration correlates with Alzheimer's disease (AD) symptoms, but the molecular 2 identities of pathogenic amyloid β-protein (Aβ) oligomers and their targets leading to 3 neurodegeneration remain unclear. Amylospheroids (ASPD) are AD patient-derived 10-to 15-nm 4 spherical Aβ oligomers that cause selective degeneration of mature neurons. Here, we show that 5 the ASPD target is neuron-specific Na + /K + -ATPase α3 subunit (NAKα3). ASPD-binding to 6 NAKα3 impaired NAKα3-specific activity, activated N-type voltage-gated calcium channels, 7 and caused mitochondrial calcium dyshomeostasis, tau abnormalities, and neurodegeneration. 8 NMR and molecular modeling studies suggested that spherical ASPD contain 9 N-terminal-Aβ-derived "thorns" responsible for target binding, which are distinct from 10 low-molecular-weight oligomers and dodecamers. The fourth extracellular loop (Ex4) region of 11 NAKα3 encompassing Asn 879 and Trp 880 is essential for ASPD-NAKα3 interaction, because 12 tetrapeptides mimicking this Ex4 region bound to the ASPD surface and blocked ASPD 13 neurotoxicity. Our findings open up new possibilities for knowledge-based design of 14 peptidomimetics that inhibit neurodegeneration in AD by blocking aberrant ASPD-NAKα3 15 interaction.
The Journal of biological chemistry, 2014
Hef is an archaeal protein that probably functions mainly in stalled replication fork repair. The... more Hef is an archaeal protein that probably functions mainly in stalled replication fork repair. The presence of an unstructured region was predicted between the two distinct domains of the Hef protein. We analyzed the interdomain region of Thermococcus kodakarensis Hef and demonstrated its disordered structure by CD, NMR, and high speed atomic force microscopy (AFM). To investigate the functions of this intrinsically disordered region (IDR), we screened for proteins interacting with the IDR of Hef by a yeast two-hybrid method, and 10 candidate proteins were obtained. We found that PCNA1 and a RecJ-like protein specifically bind to the IDR in vitro. These results suggested that the Hef protein interacts with several different proteins that work together in the pathways downstream from stalled replication fork repair by converting the IDR structure depending on the partner protein.
Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 2004
Myeloid differentiating factor 88 (MyD88) is one of a critical adaptor molecule in the Toll-like ... more Myeloid differentiating factor 88 (MyD88) is one of a critical adaptor molecule in the Toll-like receptor (TLR) signaling pathway. The TIR domain of MyD88 serves
Nuclear magnetic resonance (NMR) applications in drug discovery are classified into two categorie... more Nuclear magnetic resonance (NMR) applications in drug discovery are classified into two categories: ligand-based methods and protein-based methods. The latter is based on the observation of the (1)H-(15)N HSQC spectra of a protein with and without lead compounds. However, in order to take this strategy, isotopic labeling is an absolute necessity. Given that each (1)H-(15)N HSQC signal corresponds to a residue of the target protein, signal changes provide specific information on whether a compound will fit into a pocket. Thus, this protein-based method is particularly suitable for fragment-based approaches, such as "SAR-by-NMR" and "fragment-growing." Alternatively, the information from a protein interface may be used to develop inhibitors for protein-protein interactions. This review discusses at the experimental procedures for preparing isotopically labeled protein and introduces selected topics on atom-specific and residue-selective isotope labeling, which may facilitate the development of PPI/PA inhibitors. Furthermore, the author reviews the recent applications of "in-cell" NMR spectroscopy, which is now considered as an important tool in drug delivery research. Many recent advances in labeling methods have succeeded in expanding NMR's potential for drug discovery. In addition to those methods, another new technique called "in-cell NMR" allows the observation of protein-ligand interactions inside living cells. In other words, "in-cell NMR" may become a pharmaceutical NMR technique for drug delivery.
Biomolecular NMR assignments, 2011
Zonula occludens-1 (ZO-1) is a scaffolding molecule critical to the formation of intercellular ad... more Zonula occludens-1 (ZO-1) is a scaffolding molecule critical to the formation of intercellular adhesion structures, such as tight junctions (TJs) and adherens junctions (AJs). ZO-1 contains three PDZ domains followed by a GUK domain and a ZU5 domain. The first PDZ of ZO-1 (ZO-1(PDZ1)) serves as a protein-protein interaction module and interacts with the C-termini of almost all claudins to initiate the formation of a belt-like structure on the lateral membranes, thereby promoting TJ formation. It has been recently reported that approximately 15% of all PDZ domains bind phosphoinositides, and ZO-1(PDZ1) is the one of these. Here we report the (15)N, (13)C, and (1)H chemical shift assignments of the first PDZ domain of mouse ZO-1. The resonance assignments obtained in this work may contribute in clarifying the interplay between the two binary interactions, ZO-1(PDZ1)-claudins and ZO-1(PDZ1)-phospholipids, and suggesting a novel regulation mechanism underlying the formation and maintena...
Biomolecular NMR assignments, 2012
Stomatin, a 288-residue protein, is a component of the membrane skeleton of red blood cells (RBCs... more Stomatin, a 288-residue protein, is a component of the membrane skeleton of red blood cells (RBCs), which helps to physically support the membrane and maintains its function. In RBCs, stomatin binds to the glucose transporter GLUT-1 and may regulate its function. Stomatin has a stomatin/prohibitin/flotillin/HflK (SPFH) domain at the center of its polypeptide chain. There are 12 SPFH domain-containing proteins, most of which are localized at the cellular or subcellular membranes. Although the molecular function of the SPFH domain has not yet been established, the domain may be involved in protein oligomerization. The SPFH domain of the archaeal stomatin homolog has been shown to form unique oligomers. Here we report the (15)N, (13)C, and (1)H chemical shift assignments of the SPFH domain of human stomatin [hSTOM(SPFH)]. These may help in determining the structure of hSTOM(SPFH) in solution as well as in clarifying its involvement in protein oligomerization.
The Journal of Cell Biology, 2011
FEBS Journal, 2012
Katanin p60 (p60-katanin) is a microtubule (MT)-severing enzyme and its activity is regulated by ... more Katanin p60 (p60-katanin) is a microtubule (MT)-severing enzyme and its activity is regulated by the p80 subunit (adaptor-p80). p60-katanin consists of an N-terminal domain, followed by a single ATPase associated with various cellular activities (AAA) domain. We have previously shown that the N-terminal domain serves as the binding site for MT, the substrate of p60-katanin. In this study, we show that the same domain shares another interface with the C-terminal domain of adaptor-p80. We further show that Ca 2+ ions inhibit the MT-severing activity of p60-katanin, whereas the MT-binding activity is preserved in the presence of Ca 2+ . In detail, the basal ATPase activity of p60-katanin is stimulated twofold by both MTs and the C-terminal domain of adaptor-p80, whereas Ca 2+ reduces elevated ATPase activity to the basal level. We identify the Ca 2+ -binding site at the end of helix 2 of the N-terminal domain, which is different from the MT-binding interface. On the basis of these observations, we propose a speculative model in which spatial rearrangement of the N-terminal domain relative to the C-terminal AAA domain may be important for productive ATP hydrolysis towards MT-severing. Our model can explain how Ca 2+ regulates both severing and ATP hydrolysis activity, because the Ca 2+ -binding site on the N-terminal domain moves close to the AAA domain during MT severing.
Structure, 2005
The ubiquitin-associated (UBA) domain is one of the most frequently occurring motifs that recogni... more The ubiquitin-associated (UBA) domain is one of the most frequently occurring motifs that recognize ubiquitin tags. Dsk2p, a UBA-containing protein from Saccharomyces cerevisiae, is involved in the ubiquitin-proteasome proteolytic pathway and has been implicated in spindle pole duplication. Here we present the solution structure of the UBA domain of Dsk2p (Dsk2(UBA)) in complex with ubiquitin. The structure reveals that the UBA domain uses a mode of ubiquitin recognition that is similar to that of the CUE domain, another ubiquitin binding motif that shares low sequence homology but high structural similarity with UBA domains. These two domains, as well as the structurally unrelated ubiquitin binding motif UIM, provide a common, crucial recognition site for ubiquitin, comprising a hydrogen-bonding acceptor for the amide group of Gly-47, and a methyl group that packs against the hydrophobic pocket of ubiquitin formed by Leu-8, Ile-44, His-68, and Val-70.
Protein Science, 2000
We previously reported the de novo design of an amphiphilic peptide @YGG~IEKKIEA! 4 # that forms ... more We previously reported the de novo design of an amphiphilic peptide @YGG~IEKKIEA! 4 # that forms a native-like, parallel triple-stranded coiled coil. Starting from this peptide, we sought to regulate the assembly of the peptide by a metal ion. The replacement of the Ile18 and Ile22 residues with Ala and Cys residues, respectively, in the hydrophobic positions disrupted of the triple-stranded a-helix structure. The addition of Cd~II!, however, resulted in the reconstitution of the triple-stranded a-helix bundle, as revealed by circular dichroism~CD! spectroscopy and sedimentation equilibrium analysis. By titration with metal ions and monitoring the change in the intensity of the CD spectra at 222 nm, the dissociation constant K d was determined to be 1.5 6 0.8 mM for Cd~II!. The triple-stranded complex formed by the 113 Cd~II! ion showed a single 113 Cd NMR resonance at 572 ppm whose chemical shift was not affected by the presence of Cl Ϫ ions. The 113 Cd NMR resonance was connected with the bH protons of the cysteine residue by 1 H-113 Cd heteronuclear multiple quantum correlation spectroscopy. These NMR results indicate that the three cysteine residues are coordinated to the cadmium ion in a trigonal-planar complex. Hg~II! also induced the assembly of the peptide into a triple-stranded a-helical bundle below the Hg~II!0peptide ratio of 103. With excess Hg~II!, however, the a-helicity of the peptide was decreased, with the change of the Hg~II! coordination state from three to two. Combining this construct with other functional domains should facilitate the production of artificial proteins with functions controlled by metal ions.
Protein Science, 2008
Nodulation formation efficiency D (NfeD) is a member of a class of membrane-anchored ClpP-class p... more Nodulation formation efficiency D (NfeD) is a member of a class of membrane-anchored ClpP-class proteases. There is a second class of NfeD homologs that lack the ClpP domain. The genes of both NfeD classes usually are part of an operon that also contains a gene for a prokaryotic homolog of stomatin. (Stomatin is a major integral-membrane protein of mammalian erythrocytes.) Such NfeD/stomatin homolog gene pairs are present in more than 290 bacterial and archaeal genomes, and their protein products may be part of the machinery used for quality control of membrane proteins. Herein, we report the structure of the isolated C-terminal domain of PH0471, a Pyrococcus horikoshii NfeD homolog, which lacks the ClpP domain. This C-terminal domain (termed NfeDC) contains a five-strand b-barrel, which is structurally very similar to the OB-fold (oligosaccharide/oligonucleotide-binding fold) domain. However, there is little sequence similarity between it and previously characterized OB-fold domains. The NfeDC domain lacks the conserved surface residues that are necessary for the binding of an OB-fold domain to DNA/RNA, an ion. Instead, its surface is composed of residues that are uniquely conserved in NfeD homologs and that form the structurally conserved surface turns and b-bulges. There is also a conserved tryptophan present on the surface. We propose that, in general, NfeDC domains may interact with other spatially proximal membrane proteins and thereby regulate their activities. .
Protein Science, 2004
A rapid unidirectional method for cloning PCR-amplified cDNA fragments into virtually any fusion ... more A rapid unidirectional method for cloning PCR-amplified cDNA fragments into virtually any fusion protein expression vector is described. The method, termed PRESAT-vector cloning, is based on a T-vector technique that does not require restriction endonuclease digestion of the PCR product. Subsequently, we applied a novel ORF selection method of the ligated plasmid products. This second step involves restriction endonuclease treatment that eliminates the plasmids containing an ORF in the wrong orientation prior to transformation into the bacterial host for further protein expression studies. To achieve this selection, we customized the 5Ј-sequence of the "rear" PCR primer corresponding to the C terminus of the protein to be expressed. The colonies harbored only the ligated products of the desired orientation at >90% efficiency. This method is applied to a GST fusion expression system, and an HTS system for soluble proteins from an expression library was tested.
Protein Engineering Design and Selection, 2003
Drosophila GCM (glial cell missing) is a novel DNA-binding protein that determines the fate of gl... more Drosophila GCM (glial cell missing) is a novel DNA-binding protein that determines the fate of glial precursors from the neural default to glia. The GCM protein contains the functional domain that is essential for recognition of the upstream sequence of the repo gene. In the DNA-binding region of this GCM protein, there is a cysteine-rich region with which divalent metal ions such as Zn(2+) must bind and other proteins belonging to the GCM family have a corresponding region. To obtain a more detailed insight into the structural and functional features of this DNA-binding region, we have determined the minimal DNA-binding domain and obtained inductively coupled plasma atomic emission spectra and (1)H-(15)N, (1)H-(15)N-(13)C and (113)Cd(2+) NMR spectra, with or without its specific DNA molecule. Considering the results, it was concluded that the minimal DNA-binding domain includes two Zn(2+)-binding sites, one of which is adjacent to the interface for DNA binding. Systematic mutational analyses of the conserved cysteine residues in the minimal DNA-binding domain revealed that one Zn(2+)-binding site is indispensable for stabilization of the higher order structure of this DNA-binding domain, but that the other is not.
Protein Engineering Design and Selection, 1998
Recent studies in the field of de novo protein design have focused on the construction of native-... more Recent studies in the field of de novo protein design have focused on the construction of native-like structures. Here we describe the design and characterization of an isoleucine zipper peptide intended to form a parallel triple-stranded coiled coil. To obtain the native-like structural uniqueness, the hydrophobic interface of the peptide consists of β-branched Ile residues for complementary side chain packing. The peptide forms a stable triple-stranded coiled coil, as determined by circular dichroism and sedimentation equilibrium analyses. A fluorescence quenching assay after the incorporation of acridine revealed a parallel orientation of the peptides. The structural uniqueness of the coiled coil was confirmed by proton-deuterium amide hydrogen exchange and hydrophobic dye binding. The peptide contains amide protons with hydrogen exchange rates that are approximately an order of magnitude slower than those expected if the exchange occurred via global unfolding. A hydrophobic dye does not bind to the peptide. These results strongly suggest that the peptide folds into a well-packed structure that is very similar to the native state of a natural protein. Thus, Ile residues in the hydrophobic interface can improve the side chain packing, which can impart native-like structural uniqueness to the designed coiled coil. Keywords: coiled coil/de novo design/hydrogen exchange/ native-like/side chain packing
Protein Engineering Design and Selection, 2004
Fusion protein constructs for labeled peptides were generated with the 114 amino acid thioredoxin... more Fusion protein constructs for labeled peptides were generated with the 114 amino acid thioredoxin (TRX), coupled with the incorporation of a histidine tag for affinity purification. Two tandem AhdI sites were designed in the multiple cloning site of the fusion vector according to our novel unidirectional TA cloning methodology named PRESAT-vector, allowing one-step background-free cloning of DNA fragments. Constructs were designed to incorporate the four residue sequence Ile-Asp-Gly-Arg to generate pure peptides following Factor Xa cleavage of the fusion protein. The system is efficient and cost-effective for isotopic labeling of peptides for heteronuclear NMR studies. Seven peptides of varying length, including pituitary adenylate cyclase activating polypeptide (PACAP), vasoactive intestinal peptide (VIP) and ubiquitin interacting motif (UIM), were expressed using this TRX fusion system to give soluble fusion protein constructs in all cases. Three alternative methods for the preparation of DNA fragments were applied depending on the length of the peptides, such as polymerase chain reaction, chemical synthesis or a 'semi-synthetic method', which is a combination of chemical synthesis and enzymatic extension. The ability easily to construct, express and purify recombinant peptides in a high-throughput manner will be of enormous benefit in areas of biomedical research and drug discovery.
Proceedings of the National Academy of Sciences, 2003
Protein-phosphoinositide interaction participates in targeting proteins to membranes where they f... more Protein-phosphoinositide interaction participates in targeting proteins to membranes where they function correctly and is often modulated by phosphorylation of lipids. Here we show that protein phosphorylation of p47 phox , a cytoplasmic activator of the microbicidal phagocyte oxidase (phox), elicits interaction of p47 phox with phosphoinositides. Although the isolated phox homology (PX) domain of p47 phox can interact directly with phosphoinositides, the lipid-binding activity of this protein is normally suppressed by intramolecular interaction of the PX domain with the C-terminal Src homology 3 (SH3) domain, and hence the wild-type full-length p47 phox is incapable of binding to the lipids. The W263R substitution in this SH3 domain, abrogating the interaction with the PX domain, leads to a binding of p47 phox to phosphoinositides. The findings indicate that disruption of the intramolecular interaction renders the PX domain accessible to the lipids. This conformational change is likely induced by phosphorylation of p47 phox , because protein kinase C treatment of the wild-type p47 phox but not of a mutant protein with the S303͞ 304͞328A substitution culminates in an interaction with phosphoinositides. Furthermore, although the wild-type p47 phox translocates upon cell stimulation to membranes to activate the oxidase, neither the kinase-insensitive p47 phox nor lipid-bindingdefective proteins, one lacking the PX domain and the other carrying the R90K substitution in this domain, migrates. Thus the protein phosphorylation-driven conformational change of p47 phox enables its PX domain to bind to phosphoinositides, the interaction of which plays a crucial role in recruitment of p47 phox from the cytoplasm to membranes and subsequent activation of the phagocyte oxidase.
International journal of molecular sciences, 2015
Intrinsically disordered proteins (IDPs) that lack stable conformations and are highly flexible h... more Intrinsically disordered proteins (IDPs) that lack stable conformations and are highly flexible have attracted the attention of biologists. Therefore, the development of a systematic method to identify polypeptide regions that are unstructured in solution is important. We have designed an "indirect/reflected" detection system for evaluating the physicochemical properties of IDPs using nuclear magnetic resonance (NMR). This approach employs a "chimeric membrane protein"-based method using the thermostable membrane protein PH0471. This protein contains two domains, a transmembrane helical region and a C-terminal OB (oligonucleotide/oligosaccharide binding)-fold domain (named NfeDC domain), connected by a flexible linker. NMR signals of the OB-fold domain of detergent-solubilized PH0471 are observed because of the flexibility of the linker region. In this study, the linker region was substituted with target IDPs. Fifty-three candidates were selected using the predic...
Proceedings of the National Academy of Sciences, 2015
Neurodegeneration correlates with Alzheimer's disease (AD) symptoms, but the molecular 2 identiti... more Neurodegeneration correlates with Alzheimer's disease (AD) symptoms, but the molecular 2 identities of pathogenic amyloid β-protein (Aβ) oligomers and their targets leading to 3 neurodegeneration remain unclear. Amylospheroids (ASPD) are AD patient-derived 10-to 15-nm 4 spherical Aβ oligomers that cause selective degeneration of mature neurons. Here, we show that 5 the ASPD target is neuron-specific Na + /K + -ATPase α3 subunit (NAKα3). ASPD-binding to 6 NAKα3 impaired NAKα3-specific activity, activated N-type voltage-gated calcium channels, 7 and caused mitochondrial calcium dyshomeostasis, tau abnormalities, and neurodegeneration. 8 NMR and molecular modeling studies suggested that spherical ASPD contain 9 N-terminal-Aβ-derived "thorns" responsible for target binding, which are distinct from 10 low-molecular-weight oligomers and dodecamers. The fourth extracellular loop (Ex4) region of 11 NAKα3 encompassing Asn 879 and Trp 880 is essential for ASPD-NAKα3 interaction, because 12 tetrapeptides mimicking this Ex4 region bound to the ASPD surface and blocked ASPD 13 neurotoxicity. Our findings open up new possibilities for knowledge-based design of 14 peptidomimetics that inhibit neurodegeneration in AD by blocking aberrant ASPD-NAKα3 15 interaction.
The Journal of biological chemistry, 2014
Hef is an archaeal protein that probably functions mainly in stalled replication fork repair. The... more Hef is an archaeal protein that probably functions mainly in stalled replication fork repair. The presence of an unstructured region was predicted between the two distinct domains of the Hef protein. We analyzed the interdomain region of Thermococcus kodakarensis Hef and demonstrated its disordered structure by CD, NMR, and high speed atomic force microscopy (AFM). To investigate the functions of this intrinsically disordered region (IDR), we screened for proteins interacting with the IDR of Hef by a yeast two-hybrid method, and 10 candidate proteins were obtained. We found that PCNA1 and a RecJ-like protein specifically bind to the IDR in vitro. These results suggested that the Hef protein interacts with several different proteins that work together in the pathways downstream from stalled replication fork repair by converting the IDR structure depending on the partner protein.
Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 2004
Myeloid differentiating factor 88 (MyD88) is one of a critical adaptor molecule in the Toll-like ... more Myeloid differentiating factor 88 (MyD88) is one of a critical adaptor molecule in the Toll-like receptor (TLR) signaling pathway. The TIR domain of MyD88 serves
Nuclear magnetic resonance (NMR) applications in drug discovery are classified into two categorie... more Nuclear magnetic resonance (NMR) applications in drug discovery are classified into two categories: ligand-based methods and protein-based methods. The latter is based on the observation of the (1)H-(15)N HSQC spectra of a protein with and without lead compounds. However, in order to take this strategy, isotopic labeling is an absolute necessity. Given that each (1)H-(15)N HSQC signal corresponds to a residue of the target protein, signal changes provide specific information on whether a compound will fit into a pocket. Thus, this protein-based method is particularly suitable for fragment-based approaches, such as "SAR-by-NMR" and "fragment-growing." Alternatively, the information from a protein interface may be used to develop inhibitors for protein-protein interactions. This review discusses at the experimental procedures for preparing isotopically labeled protein and introduces selected topics on atom-specific and residue-selective isotope labeling, which may facilitate the development of PPI/PA inhibitors. Furthermore, the author reviews the recent applications of "in-cell" NMR spectroscopy, which is now considered as an important tool in drug delivery research. Many recent advances in labeling methods have succeeded in expanding NMR's potential for drug discovery. In addition to those methods, another new technique called "in-cell NMR" allows the observation of protein-ligand interactions inside living cells. In other words, "in-cell NMR" may become a pharmaceutical NMR technique for drug delivery.
Biomolecular NMR assignments, 2011
Zonula occludens-1 (ZO-1) is a scaffolding molecule critical to the formation of intercellular ad... more Zonula occludens-1 (ZO-1) is a scaffolding molecule critical to the formation of intercellular adhesion structures, such as tight junctions (TJs) and adherens junctions (AJs). ZO-1 contains three PDZ domains followed by a GUK domain and a ZU5 domain. The first PDZ of ZO-1 (ZO-1(PDZ1)) serves as a protein-protein interaction module and interacts with the C-termini of almost all claudins to initiate the formation of a belt-like structure on the lateral membranes, thereby promoting TJ formation. It has been recently reported that approximately 15% of all PDZ domains bind phosphoinositides, and ZO-1(PDZ1) is the one of these. Here we report the (15)N, (13)C, and (1)H chemical shift assignments of the first PDZ domain of mouse ZO-1. The resonance assignments obtained in this work may contribute in clarifying the interplay between the two binary interactions, ZO-1(PDZ1)-claudins and ZO-1(PDZ1)-phospholipids, and suggesting a novel regulation mechanism underlying the formation and maintena...
Biomolecular NMR assignments, 2012
Stomatin, a 288-residue protein, is a component of the membrane skeleton of red blood cells (RBCs... more Stomatin, a 288-residue protein, is a component of the membrane skeleton of red blood cells (RBCs), which helps to physically support the membrane and maintains its function. In RBCs, stomatin binds to the glucose transporter GLUT-1 and may regulate its function. Stomatin has a stomatin/prohibitin/flotillin/HflK (SPFH) domain at the center of its polypeptide chain. There are 12 SPFH domain-containing proteins, most of which are localized at the cellular or subcellular membranes. Although the molecular function of the SPFH domain has not yet been established, the domain may be involved in protein oligomerization. The SPFH domain of the archaeal stomatin homolog has been shown to form unique oligomers. Here we report the (15)N, (13)C, and (1)H chemical shift assignments of the SPFH domain of human stomatin [hSTOM(SPFH)]. These may help in determining the structure of hSTOM(SPFH) in solution as well as in clarifying its involvement in protein oligomerization.
The Journal of Cell Biology, 2011
FEBS Journal, 2012
Katanin p60 (p60-katanin) is a microtubule (MT)-severing enzyme and its activity is regulated by ... more Katanin p60 (p60-katanin) is a microtubule (MT)-severing enzyme and its activity is regulated by the p80 subunit (adaptor-p80). p60-katanin consists of an N-terminal domain, followed by a single ATPase associated with various cellular activities (AAA) domain. We have previously shown that the N-terminal domain serves as the binding site for MT, the substrate of p60-katanin. In this study, we show that the same domain shares another interface with the C-terminal domain of adaptor-p80. We further show that Ca 2+ ions inhibit the MT-severing activity of p60-katanin, whereas the MT-binding activity is preserved in the presence of Ca 2+ . In detail, the basal ATPase activity of p60-katanin is stimulated twofold by both MTs and the C-terminal domain of adaptor-p80, whereas Ca 2+ reduces elevated ATPase activity to the basal level. We identify the Ca 2+ -binding site at the end of helix 2 of the N-terminal domain, which is different from the MT-binding interface. On the basis of these observations, we propose a speculative model in which spatial rearrangement of the N-terminal domain relative to the C-terminal AAA domain may be important for productive ATP hydrolysis towards MT-severing. Our model can explain how Ca 2+ regulates both severing and ATP hydrolysis activity, because the Ca 2+ -binding site on the N-terminal domain moves close to the AAA domain during MT severing.
Structure, 2005
The ubiquitin-associated (UBA) domain is one of the most frequently occurring motifs that recogni... more The ubiquitin-associated (UBA) domain is one of the most frequently occurring motifs that recognize ubiquitin tags. Dsk2p, a UBA-containing protein from Saccharomyces cerevisiae, is involved in the ubiquitin-proteasome proteolytic pathway and has been implicated in spindle pole duplication. Here we present the solution structure of the UBA domain of Dsk2p (Dsk2(UBA)) in complex with ubiquitin. The structure reveals that the UBA domain uses a mode of ubiquitin recognition that is similar to that of the CUE domain, another ubiquitin binding motif that shares low sequence homology but high structural similarity with UBA domains. These two domains, as well as the structurally unrelated ubiquitin binding motif UIM, provide a common, crucial recognition site for ubiquitin, comprising a hydrogen-bonding acceptor for the amide group of Gly-47, and a methyl group that packs against the hydrophobic pocket of ubiquitin formed by Leu-8, Ile-44, His-68, and Val-70.
Protein Science, 2000
We previously reported the de novo design of an amphiphilic peptide @YGG~IEKKIEA! 4 # that forms ... more We previously reported the de novo design of an amphiphilic peptide @YGG~IEKKIEA! 4 # that forms a native-like, parallel triple-stranded coiled coil. Starting from this peptide, we sought to regulate the assembly of the peptide by a metal ion. The replacement of the Ile18 and Ile22 residues with Ala and Cys residues, respectively, in the hydrophobic positions disrupted of the triple-stranded a-helix structure. The addition of Cd~II!, however, resulted in the reconstitution of the triple-stranded a-helix bundle, as revealed by circular dichroism~CD! spectroscopy and sedimentation equilibrium analysis. By titration with metal ions and monitoring the change in the intensity of the CD spectra at 222 nm, the dissociation constant K d was determined to be 1.5 6 0.8 mM for Cd~II!. The triple-stranded complex formed by the 113 Cd~II! ion showed a single 113 Cd NMR resonance at 572 ppm whose chemical shift was not affected by the presence of Cl Ϫ ions. The 113 Cd NMR resonance was connected with the bH protons of the cysteine residue by 1 H-113 Cd heteronuclear multiple quantum correlation spectroscopy. These NMR results indicate that the three cysteine residues are coordinated to the cadmium ion in a trigonal-planar complex. Hg~II! also induced the assembly of the peptide into a triple-stranded a-helical bundle below the Hg~II!0peptide ratio of 103. With excess Hg~II!, however, the a-helicity of the peptide was decreased, with the change of the Hg~II! coordination state from three to two. Combining this construct with other functional domains should facilitate the production of artificial proteins with functions controlled by metal ions.
Protein Science, 2008
Nodulation formation efficiency D (NfeD) is a member of a class of membrane-anchored ClpP-class p... more Nodulation formation efficiency D (NfeD) is a member of a class of membrane-anchored ClpP-class proteases. There is a second class of NfeD homologs that lack the ClpP domain. The genes of both NfeD classes usually are part of an operon that also contains a gene for a prokaryotic homolog of stomatin. (Stomatin is a major integral-membrane protein of mammalian erythrocytes.) Such NfeD/stomatin homolog gene pairs are present in more than 290 bacterial and archaeal genomes, and their protein products may be part of the machinery used for quality control of membrane proteins. Herein, we report the structure of the isolated C-terminal domain of PH0471, a Pyrococcus horikoshii NfeD homolog, which lacks the ClpP domain. This C-terminal domain (termed NfeDC) contains a five-strand b-barrel, which is structurally very similar to the OB-fold (oligosaccharide/oligonucleotide-binding fold) domain. However, there is little sequence similarity between it and previously characterized OB-fold domains. The NfeDC domain lacks the conserved surface residues that are necessary for the binding of an OB-fold domain to DNA/RNA, an ion. Instead, its surface is composed of residues that are uniquely conserved in NfeD homologs and that form the structurally conserved surface turns and b-bulges. There is also a conserved tryptophan present on the surface. We propose that, in general, NfeDC domains may interact with other spatially proximal membrane proteins and thereby regulate their activities. .
Protein Science, 2004
A rapid unidirectional method for cloning PCR-amplified cDNA fragments into virtually any fusion ... more A rapid unidirectional method for cloning PCR-amplified cDNA fragments into virtually any fusion protein expression vector is described. The method, termed PRESAT-vector cloning, is based on a T-vector technique that does not require restriction endonuclease digestion of the PCR product. Subsequently, we applied a novel ORF selection method of the ligated plasmid products. This second step involves restriction endonuclease treatment that eliminates the plasmids containing an ORF in the wrong orientation prior to transformation into the bacterial host for further protein expression studies. To achieve this selection, we customized the 5Ј-sequence of the "rear" PCR primer corresponding to the C terminus of the protein to be expressed. The colonies harbored only the ligated products of the desired orientation at >90% efficiency. This method is applied to a GST fusion expression system, and an HTS system for soluble proteins from an expression library was tested.
Protein Engineering Design and Selection, 2003
Drosophila GCM (glial cell missing) is a novel DNA-binding protein that determines the fate of gl... more Drosophila GCM (glial cell missing) is a novel DNA-binding protein that determines the fate of glial precursors from the neural default to glia. The GCM protein contains the functional domain that is essential for recognition of the upstream sequence of the repo gene. In the DNA-binding region of this GCM protein, there is a cysteine-rich region with which divalent metal ions such as Zn(2+) must bind and other proteins belonging to the GCM family have a corresponding region. To obtain a more detailed insight into the structural and functional features of this DNA-binding region, we have determined the minimal DNA-binding domain and obtained inductively coupled plasma atomic emission spectra and (1)H-(15)N, (1)H-(15)N-(13)C and (113)Cd(2+) NMR spectra, with or without its specific DNA molecule. Considering the results, it was concluded that the minimal DNA-binding domain includes two Zn(2+)-binding sites, one of which is adjacent to the interface for DNA binding. Systematic mutational analyses of the conserved cysteine residues in the minimal DNA-binding domain revealed that one Zn(2+)-binding site is indispensable for stabilization of the higher order structure of this DNA-binding domain, but that the other is not.
Protein Engineering Design and Selection, 1998
Recent studies in the field of de novo protein design have focused on the construction of native-... more Recent studies in the field of de novo protein design have focused on the construction of native-like structures. Here we describe the design and characterization of an isoleucine zipper peptide intended to form a parallel triple-stranded coiled coil. To obtain the native-like structural uniqueness, the hydrophobic interface of the peptide consists of β-branched Ile residues for complementary side chain packing. The peptide forms a stable triple-stranded coiled coil, as determined by circular dichroism and sedimentation equilibrium analyses. A fluorescence quenching assay after the incorporation of acridine revealed a parallel orientation of the peptides. The structural uniqueness of the coiled coil was confirmed by proton-deuterium amide hydrogen exchange and hydrophobic dye binding. The peptide contains amide protons with hydrogen exchange rates that are approximately an order of magnitude slower than those expected if the exchange occurred via global unfolding. A hydrophobic dye does not bind to the peptide. These results strongly suggest that the peptide folds into a well-packed structure that is very similar to the native state of a natural protein. Thus, Ile residues in the hydrophobic interface can improve the side chain packing, which can impart native-like structural uniqueness to the designed coiled coil. Keywords: coiled coil/de novo design/hydrogen exchange/ native-like/side chain packing
Protein Engineering Design and Selection, 2004
Fusion protein constructs for labeled peptides were generated with the 114 amino acid thioredoxin... more Fusion protein constructs for labeled peptides were generated with the 114 amino acid thioredoxin (TRX), coupled with the incorporation of a histidine tag for affinity purification. Two tandem AhdI sites were designed in the multiple cloning site of the fusion vector according to our novel unidirectional TA cloning methodology named PRESAT-vector, allowing one-step background-free cloning of DNA fragments. Constructs were designed to incorporate the four residue sequence Ile-Asp-Gly-Arg to generate pure peptides following Factor Xa cleavage of the fusion protein. The system is efficient and cost-effective for isotopic labeling of peptides for heteronuclear NMR studies. Seven peptides of varying length, including pituitary adenylate cyclase activating polypeptide (PACAP), vasoactive intestinal peptide (VIP) and ubiquitin interacting motif (UIM), were expressed using this TRX fusion system to give soluble fusion protein constructs in all cases. Three alternative methods for the preparation of DNA fragments were applied depending on the length of the peptides, such as polymerase chain reaction, chemical synthesis or a 'semi-synthetic method', which is a combination of chemical synthesis and enzymatic extension. The ability easily to construct, express and purify recombinant peptides in a high-throughput manner will be of enormous benefit in areas of biomedical research and drug discovery.
Proceedings of the National Academy of Sciences, 2003
Protein-phosphoinositide interaction participates in targeting proteins to membranes where they f... more Protein-phosphoinositide interaction participates in targeting proteins to membranes where they function correctly and is often modulated by phosphorylation of lipids. Here we show that protein phosphorylation of p47 phox , a cytoplasmic activator of the microbicidal phagocyte oxidase (phox), elicits interaction of p47 phox with phosphoinositides. Although the isolated phox homology (PX) domain of p47 phox can interact directly with phosphoinositides, the lipid-binding activity of this protein is normally suppressed by intramolecular interaction of the PX domain with the C-terminal Src homology 3 (SH3) domain, and hence the wild-type full-length p47 phox is incapable of binding to the lipids. The W263R substitution in this SH3 domain, abrogating the interaction with the PX domain, leads to a binding of p47 phox to phosphoinositides. The findings indicate that disruption of the intramolecular interaction renders the PX domain accessible to the lipids. This conformational change is likely induced by phosphorylation of p47 phox , because protein kinase C treatment of the wild-type p47 phox but not of a mutant protein with the S303͞ 304͞328A substitution culminates in an interaction with phosphoinositides. Furthermore, although the wild-type p47 phox translocates upon cell stimulation to membranes to activate the oxidase, neither the kinase-insensitive p47 phox nor lipid-bindingdefective proteins, one lacking the PX domain and the other carrying the R90K substitution in this domain, migrates. Thus the protein phosphorylation-driven conformational change of p47 phox enables its PX domain to bind to phosphoinositides, the interaction of which plays a crucial role in recruitment of p47 phox from the cytoplasm to membranes and subsequent activation of the phagocyte oxidase.