8 research outputs found
Purification, crystallization and preliminary X-ray diffraction studies of the soluble domain of the oligosaccharyltransferase STT3 subunit from the thermophilic archaeon Pyrococcus furiosus
The C-terminal soluble domain of the catalytic subunit (STT3) of the oligosaccharyltransferase from P. furiosus was purified and crystallized. A native crystal and a SeMet derivative have been analyzed using X-ray diffraction
Crystallization and preliminary X-ray analysis of mitochondrial presequence receptor Tom20 in complexes with a presequence from aldehyde dehydrogenase
A presequence peptide derived from rat aldehyde dehydrogenase was tethered to the cytosolic domain of rat Tom20 protein via an intermolecular disulfide bond. Two crystal forms were obtained with different linker designs and diffracted to 2.1 and 1.9 Å
Tethering an N‑Glycosylation Sequon-Containing Peptide Creates a Catalytically Competent Oligosaccharyltransferase Complex
Oligosaccharyltransferase
(OST) transfers an oligosaccharide chain
to the Asn residue in the Asn-X-Ser/Thr sequon in proteins, where
X is not proline. A sequon was tethered to an archaeal OST enzyme
via a disulfide bond. The positions of the cysteine residues in the
OST protein and the sequon-containing acceptor peptide were selected
by reference to the eubacterial OST structure in a noncovalent complex
with an acceptor peptide. We determined the crystal structure of the
cross-linked OST–sequon complex. The Ser/Thr-binding pocket
recognizes the Thr residue in the sequon, and the catalytic structure
termed the “carboxylate dyad” interacted with the Asn
residue. Thus, the recognition and the catalytic mechanism of the
sequon are conserved between the archaeal and eubacterial OSTs. We
found that the tethered peptides in the complex were efficiently glycosylated
in the presence of the oligosaccharide donor. The stringent requirements
are greatly relaxed in the cross-linked state. The two conserved acidic
residues in the catalytic structure were each dispensable, although
the double mutation abolished the activity. A Gln residue at the Asn
position in the sequon functioned as an acceptor, and the hydroxy
group at position +2 was not required. In the standard assay using
short free peptides, strong amino acid preferences were observed at
the X position, but the preferences, except for Pro, completely disappeared
in the cross-linked state. By skipping the initial binding process
and stabilizing the complex state, the catalytically competent cross-linked
complex offers a unique system for studying the oligosaccharyl transfer
reaction
Crystal Structure of the C-Terminal Globular Domain of Oligosaccharyltransferase from <i>Archaeoglobus fulgidus</i> at 1.75 Ă… Resolution
Protein N-glycosylation occurs in the three domains of
life. Oligosaccharyltransferase
(OST) transfers glycan to asparagine in the N-glycosylation sequon.
The catalytic subunit of OST is called STT3 in eukaryotes, AglB in
archaea, and PglB in eubacteria. The genome of a hyperthermophilic
archaeon, <i>Archaeoglobus fulgidus</i>, encodes three AglB
paralogs. Two of them are the shortest AglBs across all domains of
life. We determined the crystal structure of the C-terminal globular
domain of the smallest AglB to identify the minimal structural unit.
The <i>Archaeoglobus</i> AglB lacked a β-barrel-like
structure, which had been found in other AglB and PglB structures.
In agreement, the deletion in a larger <i>Pyrococcus</i> AglB confirmed its dispensability for the activity. By contrast,
the <i>Archaeoglobus</i> AglB contains a kinked helix bearing
a conserved motif, called DK/MI motif. The lysine and isoleucine residues
in the motif participate in the Ser/Thr recognition in the sequon.
The <i>Archaeoglobus</i> AglB structure revealed that the
kinked helix contained an unexpected insertion. A revised sequence
alignment based on this finding identified a variant type of the DK
motif with the insertion. A mutagenesis study of the <i>Archaeoglobus</i> AglB confirmed the contribution of this particular type of the DK
motif to the activity. When taken together with our previous results,
this study defined the classification of OST: one group consisting
of eukaryotes and most archaea possesses the DK-type Ser/Thr pocket,
and the other group consisting of eubacteria and the remaining archaea
possesses the MI-type Ser/Thr pocket. This classification provides
a useful framework for OST studies
Crystallographic Snapshots of Tom20–Mitochondrial Presequence Interactions with Disulfide-Stabilized Peptides
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
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