The 20‐kDa TOM (translocase of outer mitochondrial membrane) subunit, Tom20, is the first receptor of the protein import pathway into mitochondria. Tom20 recognizes the mitochondrial targeting signal embedded in the presequences attached... more
The 20‐kDa TOM (translocase of outer mitochondrial membrane) subunit, Tom20, is the first receptor of the protein import pathway into mitochondria. Tom20 recognizes the mitochondrial targeting signal embedded in the presequences attached to mature mitochondrial proteins, as an N‐terminal extension. Consequently, ~1,000 different mitochondrial proteins are sorted into the mitochondrial matrix, and distinguished from non‐mitochondrial proteins. We previously reported the MPRIDE (multiple partial recognitions in dynamic equilibrium) mechanism to explain the structural basis of the promiscuous recognition of presequences by Tom20. A subset of the targeting signal features is recognized in each pose of the presequence in the binding state, and all of the features are collectively recognized in the dynamic equilibrium between the poses. Here, we changed the volumes of the hydrophobic side chains in the targeting signal, while maintaining the binding affinity. We tethered the mutated presequences to the binding site of Tom20 and placed them in the crystal contact‐free space (CCFS) created in the crystal lattice. The spatial distributions of the mutated presequences were visualized as smeared electron densities in the low‐pass filtered difference maps obtained by X‐ray crystallography. The mutated presequence ensembles shifted their positions in the binding state to accommodate the larger side chains, thus providing positive evidence supporting the use of the MPRIDE mechanism in the promiscuous recognition by Tom20.
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The structure determination of the PX (phox homology) domain of the Saccharomyces cerevisiae Vps17p protein presented a challenging case for molecular replacement because it has noncrystallographic symmetry close to a crystallographic... more
The structure determination of the PX (phox homology) domain of the Saccharomyces cerevisiae Vps17p protein presented a challenging case for molecular replacement because it has noncrystallographic symmetry close to a crystallographic axis. The combination of diffraction-quality crystals grown under microgravity on the International Space Station and a highly accurate template structure predicted by AlphaFold2 provided the key to successful crystal structure determination. Although the structure of the Vps17p PX domain is seen in many PX domains, no basic residues are found around the canonical phosphatidylinositol phosphate (PtdIns-P) binding site, suggesting an inability to bind PtdIns-P molecules.
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Oligosaccharyltransferase (OST) is responsible for the first step in the N-linked glycosylation, transferring an oligosaccharide chain onto asparagine residues to create glycoproteins. In the absence of an acceptor asparagine, OST... more
Oligosaccharyltransferase (OST) is responsible for the first step in the N-linked glycosylation, transferring an oligosaccharide chain onto asparagine residues to create glycoproteins. In the absence of an acceptor asparagine, OST hydrolyzes the oligosaccharide donor, releasing free N-glycans (FNGs) into the lumen of the endoplasmic reticulum (ER). Here, we established a purification method for mutated OSTs using a high-affinity epitope tag attached to the catalytic subunit Stt3, from yeast cells co-expressing the wild-type OST to support growth. The purified OST protein with mutations is useful for wide-ranging biochemical experiments. We assessed the effects of mutations in the Stt3 subunit on the two enzymatic activities in vitro, as well as their effects on the N-glycan attachment and FNG content levels in yeast cells. We found that mutations in the first DXD motif increased the FNG generation activity relative to the oligosaccharyl transfer activity, both in vitro and in vivo, while mutations in the DK motif had the opposite effect; the decoupling of the two activities may facilitate future deconvolution of the reaction mechanism. The isolation of the mutated OSTs also enabled us to identify different enzymatic properties in OST complexes containing either the Ost3 or Ost6 subunit and to find a 15-residue peptide as a better quality substrate than shorter peptides. This toolbox of mutants, substrates, and methods will be useful for investigations of the molecular basis and physiological roles of the OST enzymes in yeast and other organisms.
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Research Interests: Crystallization, Mitochondria, Magnetic Resonance Spectroscopy, Biological Sciences, X Rays, and 12 moreAnimals, Peptides, Membrane transport proteins, CHEMICAL SCIENCES, Rats, Cysteine, Protein Conformation, Amino Acid Sequence, Aldehyde Dehydrogenase, Mitochondrion, Molecular Sequence Data, and Protein subunits
The consistency principle represents a physicochemical condition requisite for ideal protein folding. It assumes that any pair of amino acid residues in partially folded structures has an attractive short-range interactiononly ifthe two... more
The consistency principle represents a physicochemical condition requisite for ideal protein folding. It assumes that any pair of amino acid residues in partially folded structures has an attractive short-range interactiononly ifthe two residues are in contact within the native structure. The residue-specific equilibrium constant,K, and the residue-specific rate constant,k(forward and backward), can be determined by NMR and hydrogen-deuterium exchange studies. Linear free energy relationships (LFER) in the rate-equilibrium free energy relationship (REFER) plots (i.e., logkvs. logK) are widely seen in protein-related phenomena, but our REFER plot differs from them in that the data points are derived from one polypeptide chain under a single condition. Here, we examined the theoretical basis of the residue-based LFER. First, we derived a basic equation, ρij= ½(ϕi+ ϕj), from the consistency principle, where ρijis the slope of the line segment that connects residues i and j in the REFER...
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Research Interests: Chemistry and Elongation
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K. NAGATA1,2 , H. HATANAKA1 , D. KOHDA1 , H. ISHIZAKI3 , K. MOMOMURA4 , K. TAMORI4 , T. KADOWAKI4 , M. TANAKA2 , H. KATAOKA2 , H. NAGASAWA2 , A. ISOGAI2 , A. SUZUKI2 and F. INAGAKI1 1 Department of Molecular Physiology, The Tokyo... more
K. NAGATA1,2 , H. HATANAKA1 , D. KOHDA1 , H. ISHIZAKI3 , K. MOMOMURA4 , K. TAMORI4 , T. KADOWAKI4 , M. TANAKA2 , H. KATAOKA2 , H. NAGASAWA2 , A. ISOGAI2 , A. SUZUKI2 and F. INAGAKI1 1 Department of Molecular Physiology, The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan 2 Department of Agricultural Chemistry, The University of Tokyo, Tokyo, Japan 3 Department of Biology, Nagoya University, Nagoya, Japan 4 The Third Department of Internal Medicine, The University of Tokyo, Tokyo, Japan
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Cryo-electron microscopy, Single particle reconstruction, Drug design, Glycan-protein complex, Infectious diseases
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The tertiary structure of mouse epidermal growth factor (EGF) in solution (28 degrees C, pH 2.0) was studied by two-dimensional NMR spectroscopy. Proton-proton distance constraints derived from NOESY spectra were used to construct a... more
The tertiary structure of mouse epidermal growth factor (EGF) in solution (28 degrees C, pH 2.0) was studied by two-dimensional NMR spectroscopy. Proton-proton distance constraints derived from NOESY spectra were used to construct a mechanical molecular model of mouse EGF, which was subsequently checked by means of a preliminary distance geometry calculation. The chain-folds in the two structural domains of mouse EGF were very similar to those previously reported (Montelione et al. (1987) Proc. Natl. Acad. Sci. U.S. 84, 5226-5230). However, the relative orientations of the two domains were different. Because we could assign much more inter-domain NOEs, the relative orientations of the two domains were well determined in our model. The hollow between the two domains may function as a binding site for the EGF receptor.
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The translocase of the outer membrane (TOM) mediates the membrane permeation of mitochondrial matrix proteins. Tom20 is a subunit of the TOM complex and binds to the N-terminal region (ie, presequence) in mitochondrial matrix precursor... more
The translocase of the outer membrane (TOM) mediates the membrane permeation of mitochondrial matrix proteins. Tom20 is a subunit of the TOM complex and binds to the N-terminal region (ie, presequence) in mitochondrial matrix precursor proteins. Previous experimental studies indicated that the presequence recognition by Tom20 was achieved in a dynamic-equilibrium among multiple bound states of the α-helical presequence. Accordingly, the co-crystallization of Tom20 and a presequence peptide required a disulfide-bond cross-linking. A 3-residue spacer sequence (XAG) was inserted between the presequence and the anchoring Cys residue at the C-terminus to not disturb the movement of the presequence peptide in the binding site of Tom20. Two crystalline forms were obtained according to Ala or Tyr at the X position of the spacer sequence, which may reflect the dynamic-equilibrium of the presequence. Here, we have performed replica-exchange molecular dynamics (REMD) simulations to study the effect of disulfide-bond linker and single amino acid difference in the spacer region of the linker on the conformational dynamics of Tom20-presequence complex. Free energy and network analyses of the REMD simulations were compared against previous simulations of non-tethered system. We concluded that the disulfide-bond tethering did not strongly affect the conformational ensemble of the presequence peptide in the complex. Further investigation showed that the choice of Ala or Tyr at the X position did not affect the most distributions of the conformational ensemble of the presequence. The present study provides a rational basis for the disulfide-bond tethering to study the dynamics of weakly binding complexes.
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Multiprobe measurements, such as NMR and hydrogen exchange study, can provide the equilibrium constant K and kinetic rate constant k of the structural changes of a polypeptide on a per-residue basis. We previously found a linear... more
Multiprobe measurements, such as NMR and hydrogen exchange study, can provide the equilibrium constant K and kinetic rate constant k of the structural changes of a polypeptide on a per-residue basis. We previously found a linear relationship between residue-specific log K values and residuespecific log k values for the two-state topological isomerization of a 27-residue peptide. To test the general applicability of the residue-based linear free energy relationship (rbLEFR), we performed a literature search to collect residue-specific equilibrium and kinetic constants in various exchange processes, including protein folding, coupled folding and binding of intrinsically disordered peptides, and structural fluctuations of folded proteins. The good linearity in a substantial number of log-log plots proved that the rbLFER holds for the structural changes in a wide variety of protein-related phenomena. Protein molecules quickly fold into their native structures and change their conformati...
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Research Interests: Biochemistry, Chemistry, Kinetics, Biology, Biological Chemistry, and 13 moreMagnetic Resonance Spectroscopy, Medicine, Biological Sciences, Escherichia coli, CHEMICAL SCIENCES, Amino Acids, Methionine, Substrate Specificity, Amino Acid Profile, binding sites, Isoleucine, Medical and Health Sciences, and Transfer-RNA
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Research Interests: Chemistry, Cell Biology, Biological Chemistry, Medicine, Biological Sciences, and 15 moreMice, Animals, Male, Biological, CHEMICAL SCIENCES, Epidermal Growth Factor, Epidermal Growth Factor Receptor, Molecular weight, Amino Acid Sequence, Extracellular, Molecular Sequence Data, binding sites, CHO cells, Cricetinae, and Medical and Health Sciences
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SH3 (Src homology 3) domains are found in many signaling proteins and appear to function as binding modules for cytoplasmic target proteins. The solution structure of the SH3 domain of human phospholipase C-gamma (PLC-gamma) was... more
SH3 (Src homology 3) domains are found in many signaling proteins and appear to function as binding modules for cytoplasmic target proteins. The solution structure of the SH3 domain of human phospholipase C-gamma (PLC-gamma) was determined by two-dimensional 1H NMR analysis. This SH3 domain is composed of eight antiparallel beta strands consisting of two successive "Greek key" motifs, which form a barrel-like structure. The conserved aliphatic and aromatic residues form a hydrophobic pocket on the molecular surface, and the conserved carboxylic residues are localized to the periphery. The hydrophobic pocket may serve as a binding site for target proteins. Analysis of the slowly exchanging amide protons by NMR measurements indicates that despite containing a high content of beta structure, the SH3 domain of PLC-gamma is flexible.