| Type ? secretion system(T6SS)is a versatile multiprotein secretory machine implicated in both interbacterial competition and anti-eukaryotic host activities.It was identified in 2006,as a new secretion system in Pseudomonas aeruginosa and Vibrio cholerae where it was implicated in pathogenicity.The T6SS delivers a broad arsenal of toxins with peptidoglycan,phospholipid or DNA hydrolysis activities into the target cell directly or induces cytoskeleton rearrangements in the cell..The T6SS,which is typically encoded by clusters of contiguous genes,is a complex structure that is composed of 13 conserved core components(TssA-TssM)and a variable complement of accessory elements.The model of T6SS,which exhibits evolutionary and functional similarity to the cell puncturing machinery of contractile bacteriophage tails,has been proposed recently.TssK,as an important core component of the T6SS,plays a critical role in the assembly and secretion of the T6SS machinery.To elucidate the mechanism of TssK in the type ?secretion system,the gene of tssK was amplified and cloned into the expression vector pET24b.The recombinant TssK was successfully expressed in E.coli C43(DE3)and purified by immobilized metal affinity chromatography(IMAC)and gel filtration chromatography.The purified TssK was then used for crystallization screening by vapor diffusion method.The initial condition was further optimized and the best diffracting crystals were obtained with a reservoir solution consisting 0.2 M Li2SO4,0.1 M ADA pH 6.75,12%PEG4000,2%v/v 2-Propanol and 0.2 M Tri-Sodium Citrate at 283K.Finally,a complete 2.5 A diffraction dataset was collected by X-ray.TssK shares very low sequence identity with existing homologous proteins which structures have been resolved,therefore,the selenomethionine-labeled TssK protein was produced to determine the phase,and a 2.97 A dataset was collected for the phase determination.The crystal structure of TssK is finally resolved by Single wavelength anomalous dispersion(SAD)method.Preliminary X-ray diffraction data analysis showed that the crystal of TssK belongs to the space group P6522 with unit-cell parameters a=b=151.8 A,c=315.2 A,?=?=90°,?=120°.The asymmetric unit of the crystal contains three TssK molecules which consists of three distinct domains,the N-terminal domain starts from a 14-amino-acid(residues 2-15)long loop and is formed by three ?-helices and nine ?-hairpins;the central domain is comprised of a series of helical segments,and the long four helixs of them form a central helices bundle;the C-terminal domain is composed of three a-helices(?9-?11)and eight ?-strands including two group antiparallel ?-sheets(?10,12,14-15,17 and ?11,13,16).The N-terminal domain of the trimer cross together forming a triangular pyramid structure and the central domain forming a twelve-helix bundle predominantly contribute to the stability of the trimeric fomation through several intramolecular interactions,including salt bridges as well as hydrogen bonds,whereas the C-terminal domain extends in different directions due to relatively variable C-terminal domain and highly flexible junction.In order to determine the fucntion of TssK of the T6SS in FS14,an in-frame chromosomal deletion mutant FS14? tssK was constructed,and the secretion of Hcp was abolished in FS14?tssK.This secretion defect could be fully complemented by the expression of recommbinant tssK in trans which indicates that TssK makes essential contribution to T6SS function in FS14.However,the secretion could only be partailly complement by the tssKN313(TssK N-terminal 313 residues,including N-terminal domain and central domain),further investigation need to be carried out to elucidate the detailed mechanism of TssK. |
PDF Full Text Request