title: Structure and functional characterization of the periplasmic N-terminal polypeptide domain of the sugar-specific ion channel protein (ScrY porin).
abstract: The structure of the sucrose-specific porin (ScrY) from Salmonella typhimurium has been elucidated by X-ray crystallography to consist of 18 antiparallel beta-strands, associated as a trimer complex similar to ion-transport channels. However, the 71-amino-acid-residue N-terminal periplasmic domain was not determined from the crystal structure due to the absence of sufficient electron density. The N-terminal polypeptide contains a coiled-coil structural motif and has been assumed to play a role in the sugar binding of ScrY porin. In this study the proteolytic stability and a specific proteolytic truncation site at the N-terminal domain were identified by the complete primary structure characterization of ScrY porin, using MALDI mass spectrometry and post-source-decay fragmentation. The secondary structure and supramolecular association of the coiled-coil N-terminal domain were determined by chemical synthesis of the complete N-terminal polypeptide and several partial sequences and their spectroscopic, biophysical, and mass spectrometric characterization. Circular dichroism spectra revealed predominant alpha-helical conformation for the putative coiled-coil domain comprising residues 4-46. Specific association to both dimer and trimer complexes was identified by electrospray ionization mass spectra and was ascertained by dynamic light scattering and electrophoresis data. The role of the N-terminal domain in sugar binding was examined by comparative TR-NOE-NMR spectroscopy of the complete ScrY porin and a recombinant mutant, ScrY(delta1-62), lacking the N-terminal polypeptide. The TR-NOE-NMR data showed a strong influence of ScrY porin on the sugar-binding affinity and suggested a possible function of the periplasmic N terminus for supramolecular stabilization and low-affinity sugar binding.
authors.affiliation: Department of Chemistry, University of Konstanz, 78457 Konstanz, Germany.
authors.forename: Jenny
authors.initials: J
authors.lastname: Michels
authors.forename: Armin
authors.initials: A
authors.lastname: Geyer
authors.forename: Viorel
authors.initials: V
authors.lastname: Mocanu
authors.forename: Wolfram
authors.initials: W
authors.lastname: Welte
authors.forename: Alma L
authors.initials: AL
authors.lastname: Burlingame
authors.forename: Michael
authors.initials: M
authors.lastname: Przybylski
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D018360:Crystallography, X-Ray; D008320:Maltose / Q000031:analogs & derivatives / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009844:Oligosaccharides / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D018272:Porins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012486:Salmonella typhimurium / Q000737:chemistry; D017384:Sequence Deletion; D013057:Spectrum Analysis
chemical_list: D001426:Bacterial Proteins; D009844:Oligosaccharides; D010446:Peptide Fragments; D018272:Porins; C079242:ScrY protein, bacteria; D008320:Maltose; C009819:maltotetraose