Takayuki Obita

Structure‐guided identification of a new catalytic motif of oligosaccharyltransferase

Asn‐glycosylation is widespread not only in eukaryotes but also in archaea and some eubacteria. Oligosaccharyltransferase (OST) catalyzes the co‐translational transfer of an oligosaccharide from a lipid donor to an asparagine residue in nascent polypeptide chains. Here, we report that a thermophilic archaeon, Pyrococcus furiosus OST is composed of the STT3 protein alone, and catalyzes the transfer of a heptasaccharide, containing one hexouronate and two pentose residues, onto peptides in an Asn‐X‐Thr/Ser‐motif‐dependent manner. We also determined the 2.7‐Å resolution crystal structure of the C‐terminal soluble domain of Pyrococcus STT3. The structure‐based multiple sequence alignment revealed a new motif, DxxK, which is adjacent to the well‐conserved WWDYG motif in the tertiary structure. The mutagenesis of the DK motif residues in yeast STT3 revealed the essential role of the motif in the catalytic activity. The function of this motif may be related to the binding of the pyrophosphate group of lipid‐linked oligosaccharide donors through a transiently bound cation. Our structure provides the first structural insights into the formation of the oligosaccharide–asparagine bond.

Tom20 recognizes mitochondrial presequences through dynamic equilibrium among multiple bound states.

Most mitochondrial proteins are synthesized in the cytosol and imported into mitochondria. The N‐terminal presequences of mitochondrial‐precursor proteins contain a diverse consensus motif (ϕχχϕϕ, ϕ is hydrophobic and χ is any amino acid), which is recognized by the Tom20 protein on the mitochondrial surface. To reveal the structural basis of the broad selectivity of Tom20, the Tom20–presequence complex was crystallized. Tethering a presequence peptide to Tom20 through a disulfide bond was essential for crystallization. Unexpectedly, the two crystals with different linker designs provided unique relative orientations of the presequence with respect to Tom20, and neither configuration could fully account for the hydrophobic preference at the three hydrophobic positions of the consensus motif. We propose the existence of a dynamic equilibrium in solution among multiple states including the two bound states. In accordance, NMR 15N relaxation analyses suggested motion on a sub‐millisecond timescale at the Tom20–presequence interface. We suggest that the dynamic, multiple‐mode interaction is the molecular mechanism facilitating the broadly selective specificity of the Tom20 receptor toward diverse mitochondrial presequences.

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.

Purification, crystallization and preliminary X-ray diffraction studies of the soluble domain of the oligosaccharyltransferase STT3 subunit from the thermophilic archaeon Pyrococcus furiosus.

Oligosaccharyltransferase catalyzes the transfer of preassembled oligosaccharides onto asparagine residues in nascent polypeptide chains. The STT3 subunit is thought to bear the catalytic site. The C-terminal domain of the STT3 protein of Pyrococcus furiosus was expressed in Escherichia coli cells. STT3 protein prepared from two different sources, the soluble fraction and the inclusion bodies, produced crystals that diffracted to 2.7 A. During crystallization screening, cocrystals of P. furiosus STT3 with an E. coli 50S ribosomal protein, L7/L12, were accidentally obtained. This cross-species interaction is not biologically relevant, but may be used to design a built-in polypeptide substrate for the STT3 crystals.

Determination of the secondary structure in solution of the Escherichia coli DnaA DNA-binding domain

DnaA protein binds specifically to a group of binding sites collectively called as DnaA boxes within the bacterial replication origin to induce local unwinding of duplex DNA. The DNA-binding domain of DnaA, domain IV, comprises the C-terminal 94 amino acid residues of the protein. We overproduced and purified a protein containing only this domain plus a methionine residue. This protein was stable as a monomer and maintained DnaA box-specific binding activity. We then analyzed its solution structure by CD spectrum and heteronuclear multi-dimensional NMR experiments. We established extensive assignments of the 1H, 13C, and 15N nuclei, and revealed by obtaining combined analyses of chemical shift index and NOE connectivities that DnaA domain IV contains six alpha-helices and no beta-sheets, consistent with results of CD analysis. Mutations known to reduce DnaA box-binding activity were specifically located in or near two of the alpha-helices. These findings indicate that the DNA-binding fold of DnaA domain IV is unique among origin-binding proteins.

Solution structure and activity of mouse lysozyme M

Abstract. The three-dimensional structure of mouse lysozyme M, glycoside hydrolase, with 130 amino acids has been determined by heteronuclear NMR spectroscopy. We found that mouse lysozyme M had four α-helices, two 310 helices, and a double- and a triple-stranded anti-parallel β-sheet, and its structure was very similar to that of hen lysozyme in solution and in the crystalline state. The pH activity profile of p-nitrophenyl penta N-acetyl-β-D-chitopentaoside hydrolysis by mouse lysozyme M was similar to that of hen lysozyme, but the hydrolytic activity of mouse lysozyme M was lower. From analyses of binding affinities of lysozymes to a substrate analogue and internal motions of lysozymes, we suggest that the lower activity of mouse lysozyme M was due to the larger dissociation constant of its enzyme-substrate complex and the restricted internal backbone motions in the molecule.

Mutations in the PQBP1 gene prevent its interaction with the spliceosomal protein U5–15kD

A loss-of-function of polyglutamine tract-binding protein 1 (PQBP1) induced by frameshift mutations is believed to cause X-linked mental retardation. However, the mechanism by which structural changes in PQBP1 lead to mental retardation is unknown. Here we present the crystal structure of a C-terminal fragment of PQBP1 in complex with the spliceosomal protein U5-15 kD. The U5-15 kD hydrophobic groove recognizes a YxxPxxVL motif in PQBP1, and mutations within this motif cause a loss-of-function phenotype of PQBP1 in vitro. The YxxPxxVL motif is absent in all PQBP1 frameshift mutants seen in cases of mental retardation. These results suggest a mechanism by which the loss of the YxxPxxVL motif could lead to the functional defects seen in this type of mental retardation.

Identification of the region in Escherichia coli DnaA protein required for specific recognition of the DnaA box

DnaA protein binds specifically to a 9-base- pair motif called the DnaA box. Domain IV comprises 94 amino acid residues and is required for DNA binding. Using nuclear magnetic resonance analysis, we investigated the interaction between DnaA domain IV and both a DnaA box and a non-specific oligonucleotide that has a reduced affinity for DnaA. The 1H-15N HSQC spectrum of DnaA domain IV showed prominent chemical shift perturbations on six residues (Arg399, Ala404, Leu422, Asp433, Thr435 and Thr436) in the presence of the DnaA box. Through homology modeling, we located all of these residues on one side surface of the DnaA domain IV molecule. Moreover, we compared the chemical shift perturbation of the 1H-15N HSQC spectrum in the presence of the DnaA box with that in the presence of a non-specific oligonucleotide, and the results suggested that Leu422 imparts specificity in binding with the DnaA box.

Complete Covalent Structure of Nisin Q, New Natural Nisin Variant, Containing Post-Translationally Modified Amino Acids

The third member of the nisin variant, nisin Q, produced by Lactococcus lactis 61-14, is a ribosomally-synthesized antimicrobial peptide, the so-called lantibiotic containing post-translationally modified amino acids such as lanthionine and dehydroalanine. Here, we determined the complete covalent structure of nisin Q, consisting of 34 amino acids, by two-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy. Sequential assignment of nisin Q containing the unusual amino acids was performed by total correlation spectroscopy (TOCSY) and nuclear Overhauser enhancement spectroscopy (NOESY). The observed long range nuclear Overhauser effect (NOE) in nisin Q indicated assignment of all five sets of lanthionines that intramolecularly bridge residues 3–7, 8–11, 13–19, 23–26, and 25–28. Consequently, the covalent structure of nisin Q was determined to hold the same thioether linkage formation as the other two nisins, but to harbor the four amino acid substitutions, in contrast with nisin A.

Crystallization and preliminary X-ray analysis of mitochondrial presequence receptor Tom20 in complexes with a presequence from aldehyde dehydrogenase

Most mitochondrial proteins are synthesized in the cytosol and must be imported into the mitochondria. Many mitochondrial precursor proteins have an extra leader sequence at their N-terminus called a presequence. Presequences are recognized by the Tom20 receptor protein. Based on the previously determined NMR structure of rat Tom20, a fragment corresponding to the core structure was generated. A cysteine residue was added at the C-terminus of the rat aldehyde dehydrogenase presequence to fix the presequence peptide onto the Tom20 fragment via an intermolecular disulfide bond. Two crystal forms of the complex were successfully obtained with different designs of the linker sequence which diffracted to 2.1 and 1.9 A. Crystal dehydration and subsequent annealing was essential to obtain good diffraction data for the 2.1 A crystal form.