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Natsuko Goda

Publications and source records attributed to Natsuko Goda.

10 recordsLinked to original sources
PubMed2006

Fine-tuning of protein domain boundary by minimizing potential coiled coil regions.

Structural determination of individual protein domains isolated from multidomain proteins is a common approach in the post-genomic era. Novel and thus uncharacterized domains liberated from intact proteins often self-associate due to incorrectly defined domain boundaries. Self-association results in missing signals, poor signal dispersion and a low signal-to-noise ratio in (1)H-(15)N HSQC spectra. We have found that a putative, non-canonical coiled coil region close to a domain boundary can cause transient hydrophobic self-association and monomer-dimer equilibrium in solution. Here we propose a rational method to predict putative coiled coil regions adjacent to the globular core domain using the program COILS. Except for the amino acid sequence, no preexisting knowledge concerning the domain is required. A small number of mutant proteins with a minimized coiled coil region have been rationally designed and tested. The engineered domains exhibit decreased self-association as assessed by (1)H-(15)N HSQC spectra with improved peak dispersion and sharper cross peaks. Two successful examples of isolating novel N-terminal domains from AAA-ATPases are demonstrated. Our method is useful for the experimental determination of domain boundaries suited for structural genomics studies.

Adenosine Triphosphatases↗
PubMed2006

LBT/PTD dual tagged vector for purification, cellular protein delivery and visualization in living cells.

Cellular protein delivery is an emerging technique, by which exogenous recombinant proteins are delivered into mammalian cells across the membrane. We have developed an E. coli expression vector suited for protein cellular delivery experiments. The plasmid is designed to generate a C-terminal fusion with the 12 amino acid HIV-Tat peptide as a protein transduction domain (PTD), whereas the protein N-terminus is fused to an 17-residue peptide lanthanide-binding tag (LBT). LBT is used for both purification by affinity chromatography and fluorescent detection with Tb(3+) as a coordinating metal. We have employed the TA-cloning site between the two tags, LBT and PTD, according to the PRESAT-vector methodology [N. Goda, T. Tenno, H. Takasu, H. Hiroaki, M. Shirakawa, The PRESAT-vector: asymmetric T-vector for high-throughput screening of soluble protein domains for structural proteomics, Protein Sci. 13 (2004) 652-658], which facilitates unidirectional cloning of any PCR-amplified DNA fragments corresponding to the protein of interest. A simple three-step protocol consisting of affinity purification of LBT/PTD dual-tagged proteins has also been developed, in which the proteins are purified by heparin-, then immobilized Ni(2+)-, and then heparin-affinity chromatography, in this order. The purified protein is ready for protein delivery experiment, and the delivered protein is visible by fluorescent microscopy. Our LBT/PTD dual-tagged PRESAT-vector provides a powerful research tool for exploring cellular functions of proteins in the post-genomic era.

Amino Acid Sequence↗
PubMed2006

The common phospholipid-binding activity of the N-terminal domains of PEX1 and VCP/p97.

PEX1 is a type II AAA-ATPase that is indispensable for biogenesis and maintenance of the peroxisome, an organelle responsible for the primary metabolism of lipids, such as beta-oxidation and lipid biosynthesis. Recently, we demonstrated a striking structural similarity between its N-terminal domain and those of other membrane-related AAA-ATPases, such as valosine-containing protein (p97). The N-terminal domain of valosine-containing protein serves as an interface to its adaptor proteins p47 and Ufd1, whereas the physiologic interaction partner of the N-terminal domain of PEX1 remains unknown. Here we found that N-terminal domains isolated from valosine-containing protein, as well as from PEX1, bind phosphoinositides. The N-terminal domain of PEX1 appears to preferentially bind phosphatidylinositol 3-monophosphate and phosphatidylinositol 4-monophosphate, whereas the N-terminal domain of valosine-containing protein displays broad and nonspecific lipid binding. Although N-ethylmaleimide-sensitive fusion protein, CDC48 and Ufd1 have structures similar to that of valosine-containing protein, they displayed lipid specificity similar to that of the N-terminal domain of PEX1 in the assays. By mutational analysis, we demonstrate that a conserved arginine surrounded by hydrophobic residues is essential for lipid binding, despite very low sequence similarity between PEX1 and valosine-containing protein.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2005

Structural characterization of the MIT domain from human Vps4b.

The microtubule interacting and trafficking (MIT) domain is a small protein module of unknown function that is conserved in proteins of diverse function, such as Vps4, sorting nexin 15 (SNX15), and spastin. One non-synonymous single nucleotide polymorphism was reported, which results in a Ile58-to-Met (I58M) substitution in hVps4b. Here, we have determined the solution structure of the MIT domain isolated from the NH(2)-terminus of human Vps4b, an AAA-ATPase involved in multivesicular body formation. The MIT domain adopts an 'up-and-down' three-helix bundle. Comparison with the sequences of other MIT domains clearly shows that the residues involved in inter-helical contacts are well conserved. The Ile58-to-Met substitution resulted a substantial thermal instability. In addition, we found a shallow crevice between helices A and C that may serve as a protein-binding site. We propose that the MIT domain serves as a putative adaptor domain for the ESCRT-III complex involved in endosomal trafficking.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2005

Structure of the UBA domain of Dsk2p in complex with ubiquitin molecular determinants for ubiquitin recognition.

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.

Amino Acid Sequence↗
PubMed2004

Crystallographic characterization of the N-terminal domain of PEX1.

Peroxisomal enzymes are responsible for several primary metabolism pathways, including beta-oxidation and lipid biosynthesis. PEX1 and PEX6 are hexameric AAA-type ATPases and both are necessary for the import of more than 50 peroxisomal resident proteins from the cytosol into peroxisomes. In this study, PEX1 N-terminal domain crystals have been prepared. The crystals belong to space group P3(1) or P3(2), with unit-cell parameters a = b = 63.5 A, c = 33.5 A, and contain one protein molecule per crystallographic asymmetric unit. An intensity data set was collected to a resolution of 2.05 A.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2004

Structure of the N-terminal domain of PEX1 AAA-ATPase. Characterization of a putative adaptor-binding domain.

Peroxisomes are responsible for several pathways in primary metabolism, including beta-oxidation and lipid biosynthesis. PEX1 and PEX6 are hexameric AAA-type ATPases, both of which are indispensable in targeting over 50 peroxisomal resident proteins from the cytosol to the peroxisomes. Although the tandem AAA-ATPase domains in the central region of PEX1 and PEX6 are highly similar, the N-terminal sequences are unique. To better understand the distinct molecular function of these two proteins, we analyzed the unique N-terminal domain (NTD) of PEX1. Extensive computational analysis revealed weak similarity (<10% identity) of PEX1 NTD to the N-terminal domains of other membrane-related type II AAA-ATPases, such as VCP (p97) and NSF. We have determined the crystal structure of mouse PEX1 NTD at 2.05-A resolution, which clearly demonstrated that the domain belongs to the double-psi-barrel fold family found in the other AAA-ATPases. The N-domains of both VCP and NSF are structural neighbors of PEX1 NTD with a 2.7- and 2.1-A root mean square deviation of backbone atoms, respectively. Our findings suggest that the supradomain architecture, which is composed of a single N-terminal domain followed by tandem AAA domains, is a common feature of organellar membrane-associating AAA-ATPases. We propose that PEX1 functions as a protein unfoldase in peroxisomal biogenesis, using its N-terminal putative adaptor-binding domain.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2004

High-throughput construction method for expression vector of peptides for NMR study suited for isotopic labeling.

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.

Amino Acid Sequence↗
PubMed2004

The PRESAT-vector: asymmetric T-vector for high-throughput screening of soluble protein domains for structural proteomics.

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 5'-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.

ATPases Associated with Diverse Cellular Activitie↗