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Daisuke Kohda

Publications and source records attributed to Daisuke Kohda.

26 records · Page 2Linked to original sources
PubMed2003

Peptide library approach with a disulfide tether to refine the Tom20 recognition motif in mitochondrial presequences.

Many mitochondrial matrix and inner-membrane proteins are synthesized in the cytosol as precursor proteins with an N-terminal presequence, and are imported into the mitochondria. Although no distinct sequence homology has been found among mitochondrial presequences, Tom20, a general import receptor in the outer mitohcondrial membrane, binds to presequences, and distinguishes mitochondrial proteins from non-mitochonrial proteins. The recently determined structure of the cytosolic domain of Tom20 (DeltaTom20) in a complex with the presequence of rat aldehyde dehydrogenase (ALDH) showed that a short stretch of the presequence forms an amphiphilic helix, and its hydrophobic surface interacts with the hydrophobic-binding groove of Tom20. The following NMR analyses revealed a common five-residue pattern for Tom20 binding in five different presequences. To refine the common amino acid motif for the recognition by Tom20, we introduced a new peptide library approach in this study: we prepared a mixture of ALDH presequence variants, tethered these peptides to DeltaTom20 in a competitive manner by an intermolecular disulfide bond, and determined the relative affinities by MALDI-TOF mass spectrometry. We successfully deduced a refined, common motif for the recognition by Tom20, and found that the segment consisting of residues 14-20 of the ALDH presequence was locally optimized in the sequence space, with respect to Tom20 binding.

Aldehyde Dehydrogenase↗
PubMed2003

Phosphorylation of p47phox directs phox homology domain from SH3 domain toward phosphoinositides, leading to phagocyte NADPH oxidase activation.

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 S303304328A 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-binding-defective 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.

Amino Acid Substitution↗
PubMed2003

NMR and ICP spectroscopic analysis of the DNA-binding domain of the Drosophila GCM protein reveals a novel Zn2+ -binding motif.

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.

Amino Acid Sequence↗
PubMed2002

Functions of outer membrane receptors in mitochondrial protein import.

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins and are imported into mitochondria. The targeting signals for mitochondria are encoded in the presequences or in the mature parts of the precursor proteins, and are decoded by the receptor sites in the translocator complex in the mitochondrial outer membrane. The recently determined NMR structure of the general import receptor Tom20 in a complex with a presequence peptide reveals that, although the amphiphilicity and positive charges of the presequence is essential for the import ability of the presequence, Tom20 recognizes only the amphiphilicity, but not the positive charges. This leads to a new model that different features associated with the mitochondrial targeting sequence of the precursor protein can be recognized by the mitochondrial protein import system in different steps during the import.

Amino Acid Sequence↗
PubMed2002

Diverse recognition of non-PxxP peptide ligands by the SH3 domains from p67(phox), Grb2 and Pex13p.

The basic function of the Src homology 3 (SH3) domain is considered to be binding to proline-rich sequences containing a PxxP motif. Recently, many SH3 domains, including those from Grb2 and Pex13p, were reported to bind sequences lacking a PxxP motif. We report here that the 22 residue peptide lacking a PxxP motif, derived from p47(phox), binds to the C-terminal SH3 domain from p67(phox). We applied the NMR cross-saturation method to locate the interaction sites for the non-PxxP peptides on their cognate SH3 domains from p67(phox), Grb2 and Pex13p. The binding site of the Grb2 SH3 partially overlapped the conventional PxxP-binding site, whereas those of p67(phox) and Pex13p SH3s are located in different surface regions. The non-PxxP peptide from p47(phox) binds to the p67(phox) SH3 more tightly when it extends to the N-terminus to include a typical PxxP motif, which enabled the structure determination of the complex, to reveal that the non-PxxP peptide segment interacted with the p67(phox) SH3 in a compact helix-turn-helix structure (PDB entry 1K4U).

Adaptor Proteins, Signal Transducing↗
PubMed2002

DNA binding of PriA protein requires cooperation of the N-terminal D-loop/arrested-fork binding and C-terminal helicase domains.

PriA protein is essential for RecA-dependent DNA replication induced by stalled replication forks in Escherichia coli. PriA is a DEXH-type DNA helicase, ATPase activity of which depends on its binding to structured DNA including a D-loop-like structure. Here, we show that the N-terminal 181-amino acid polypeptide can form a complex with D-loop in gel shift assays and have identified a unique motif present in the N-terminal segment of PriA that plays a role in its DNA binding. We have also identified residues in the C terminus proximal helicase domain essential for D-loop binding. PriA proteins mutated in this domain do not bind to D-loop, despite the presence of the N-terminal DNA-binding motif. Those mutants that cannot bind to D-loop in vitro do not support a recombination-dependent mode of DNA replication in vivo, indicating that binding to a D-loop-like structure is essential for the ability of PriA to initiate DNA replication and repair from stalled replication forks. We propose that binding of the PriA protein to stalled replication forks requires proper configuration of the N-terminal fork-recognition and C-terminal helicase domains and that the latter may stabilize binding and increase binding specificity.

Adenosine Triphosphatases↗