title: Yersinia enterocolitica type III secretion chaperone SycH. Recombinant expression, purification, characterisation, and crystallisation.
abstract: All pathogenic Yersinia species (Y. enterocolitica, Y. pestis, and Y. pseudotuberculosis) share a type three secretion system (TTSS) that allows translocation of effector proteins into host cells. Yersinia enterocolitica SycH is a chaperone assisting the transport of the effector YopH and two regulatory components of the TTSS, YscM1 and YscM2. We have recombinantly expressed SycH in Escherichia coli. Purification of tag-free SycH to near homogeneity was achieved by combining ammonium sulfate precipitation, anion exchange chromatography, and gel filtration. Functionality of purified SycH was proven by demonstrating binding to YopH. SycH crystals were grown that diffracted to 2.94A resolution. Preliminary crystallographic data and biochemical findings suggest that SycH forms homotetramers. SycH may therefore represent a novel class of TTSS chaperones. In addition, we found that YopH was enzymatically active in the presence of SycH. This implies that the function of the secretion chaperone SycH is not to keep YopH in a globally unfolded state prior to secretion.
authors.affiliation: Max von Pettenkofer-Institut, Lehrstuhl für Bakteriologie, Ludwig-Maximilians-Universität München, Pettenkoferstr. 9a, D-80336 Munich, Germany.
authors.forename: Wibke
authors.initials: W
authors.lastname: Neumayer
authors.forename: Michael
authors.initials: M
authors.lastname: Groll
authors.forename: Verena
authors.initials: V
authors.lastname: Lehmann
authors.forename: Uladzimir
authors.initials: U
authors.lastname: Antoneka
authors.forename: Susanne
authors.initials: S
authors.lastname: Kähler
authors.forename: Jürgen
authors.initials: J
authors.lastname: Heesemann
authors.forename: Gottfried
authors.initials: G
authors.lastname: Wilharm
mesh_terms: D001483:Base Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003460:Crystallization; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification*; D015008:Yersinia enterocolitica / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D018832:Molecular Chaperones; C090049:SycH protein, Yersinia