YscU recognizes translocators as export substrates of the Yersinia injectisome

Abstract

YscU is an essential component of the export apparatus of the Yersinia injectisome. It consists of an N-terminal transmembrane domain and a long cytoplasmic C-terminal domain, which undergoes auto-cleavage at a NPTH site. Substitutions N263A and P264A prevented cleavage of YscU and abolished export of LcrV, YopB and YopD but not of Yop effectors. As a consequence, yscU(N263A) mutant bacteria made needles without the LcrV tip complex and they could not form translocation pores. The graft of the export signal of the effector YopE, at the N-terminus of LcrV, restored LcrV export and assembly of the tip complex. Thus, YscU cleavage is required to acquire the conformation allowing recognition of translocators, which represent an individual category of substrates in the hierarchy of export. In addition, yscU(N263A) mutant bacteria exported reduced amounts of the YscP ruler and made longer needles. Increasing YscP export resulted in needles with normal size, depending on the length of the ruler. Hence, the effect of the yscU(N263A) mutation on needle length was the consequence of a reduced YscP export.

Atomic resolution structure of the cytoplasmic domain of Yersinia pestis YscU, a regulatory switch involved in type III secretion

“…9 The YscU N263A/P264A and YscU N263A mutants yielded tetragonal crystals that diffracted X-rays to 1.30 and 1.53 Å, respectively. Hexagonal crystals of YscU cleaved (residues 220-342) were obtained that diffracted X-rays up to a resolution of 1.13 Å using synchrotron radiation.…”

“…7 The cytoplasmic domain of YscU undergoes auto-cleavage of the N263-P264 peptide bond at the conserved NPTH site, resulting in an N-terminal fragment, YscU CN (residues 211-263), and a C-terminal fragment, YscU CC (residues 264-354), that remain tightly intertwined and copurify together. 8,9 Site-directed mutagenesis of N263 or P264 to alanine abolishes auto-cleavage of YscU, which blocks the export of translocators (LcrV, YopB, and YopD) but not effector proteins via the T3SS. [8][9][10] Therefore, it has been proposed that autocleavage of the YscU cytoplasmic domain results in a conformational change that triggers the recognition and export of translocators at the proper time during the assembly of the type III secretion apparatus.…”

“…8,9 Site-directed mutagenesis of N263 or P264 to alanine abolishes auto-cleavage of YscU, which blocks the export of translocators (LcrV, YopB, and YopD) but not effector proteins via the T3SS. [8][9][10] Therefore, it has been proposed that autocleavage of the YscU cytoplasmic domain results in a conformational change that triggers the recognition and export of translocators at the proper time during the assembly of the type III secretion apparatus. 9,[11][12][13] We report here the crystal structures of two noncleaved forms of YscU, YscU N263A and YscU N263A/ P264A , and an atomic resolution structure of the wildtype cleaved form, YscU cleaved , to elucidate structural changes that occur upon cleavage.…”

“…[8][9][10] Therefore, it has been proposed that autocleavage of the YscU cytoplasmic domain results in a conformational change that triggers the recognition and export of translocators at the proper time during the assembly of the type III secretion apparatus. 9,[11][12][13] We report here the crystal structures of two noncleaved forms of YscU, YscU N263A and YscU N263A/ P264A , and an atomic resolution structure of the wildtype cleaved form, YscU cleaved , to elucidate structural changes that occur upon cleavage. These crystallographic studies reveal the conformational changes induced upon auto-cleavage of YscU and provide structural insights into the cleavage mechanism, at very high resolution, that confirm and extend the information gleaned from structures of YscU homologs from Shigella flexneri (Spa40), enteropathogenic Escherichia coli (EscU) and Salmonella typhimurium (SpaS).…”

The type III secretion injectisome, a complex nanomachine for intracellular ‘toxin’ delivery

“…The switch activity is exerted by residues 405-500 (Agrain et al, 2005a), a domain called T3S4 for type 3 secretion substrate specificity switch. This domain is thought to interact with YscU (FlhB in the flagellum), a component of the basal body that is also involved in setting the hierarchy of export (Hirano et al, 1994;Sorg et al, 2007). The central domain of YscP is predicted to be helical and there is a linear correlation between the number of residues in the protein and the needle length, suggesting that YscP acts as a molecular ruler or a molecular timer.…”