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

Characterization of susceptibility variants of poliovirus grown in the presence of favipiravir

Background: T-705 (favipiravir) is a potent inhibitor of RNA-dependent RNA polymerases of influenza viruses and no favipiravir-resistant virus has been isolated. Poliovirus RNA polymerase has been well characterized and isolation of resistant virus was examined in poliovirus. Methods: Susceptibility variants of poliovirus I (Sabin strain) were isolated during passages in the presence of favipiravir and characterized for their susceptibility and the sequence of RNA polymerase. Results: Five variants with 0.47–1.88 times the 50% inhibitory concentration for plaque formation of the parent poliovirus had amino acid variations in the 3D gene of the RNA polymerase. The distribution of amino acid variations was not related to ribavirin resistance, and two amino acid variation sites were found near the finger domain. Conclusion: Favipiravir as a chain terminator would not be incorporated and replicate to cause lethal mutagenesis as a mutagen like ribavirin, and resistant mutants were not isolated. A high replication level would generate mutations leading to favipiravir resistance as ribavirin resistance was generated, but generated mutations would be lethal to the RNA polymerase function. Keywords: Poliovirus, T-705, Favipiravir, Mutants, Resistance, RNA dependent RNA polymeras

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

ESCRT-III binding protein MITD1 is involved in cytokinesis and has an unanticipated PLD fold that binds membranes

The endosomal sorting complexes required for transport (ESCRT) proteins have a critical function in abscission, the final separation of the daughter cells during cytokinesis. Here, we describe the structure and function of a previously uncharacterized ESCRT-III interacting protein, MIT-domain containing protein 1 (MITD1). Crystal structures of MITD1 reveal a dimer, with a microtubule-interacting and trafficking (MIT) domain at the N terminus and a unique, unanticipated phospholipase D-like (PLD) domain at the C terminus that binds membranes. We show that the MIT domain binds to a subset of ESCRT-III subunits and that this interaction mediates MITD1 recruitment to the midbody during cytokinesis. Depletion of MITD1 causes a distinct cytokinetic phenotype consistent with destabilization of the midbody and abscission failure. These results suggest a model whereby MITD1 coordinates the activity of ESCRT-III during abscission with earlier events in the final stages of cell division

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