Conformational analysis by CD and NMR spectroscopy of a peptide encompassing the amphipathic domain of YopD from Yersinia

Abstract

To establish an infection, Yersinia pseudotuberculosis utilizes a plasmid-encoded type III secretion machine that permits the translocation of several anti-host factors into the cytosol of target eukaryotic cells. Secreted YopD is essential for this process. Pre-secretory stabilization of YopD is mediated by an interaction with its cognate chaperone, LcrH. YopD possesses LcrH binding domains located in the N-terminus and in a predicted amphipathic domain located near the C-terminus. This latter domain is also critical for Yersinia virulence. In this study, we designed synthetic peptides encompassing the C-terminal amphipathic domain of YopD. A solution structure of YopD 278)300 , a peptide that strongly interacted with LcrH, was obtained by NMR methods. The structure is composed of a well-defined amphipathic a helix ranging from Phe280 to Tyr291, followed by a type I b turn between residues Val292 and His295. The C-terminal truncated peptides, YopD 278)292 and YopD 271)292 , lacked helical structure, implicating the b turn in helix stability. An interaction between YopD 278)300 and its cognate chaperone, LcrH, was observed by NMR through line-broadening effects and chemical shift differences between the free peptide and the peptide-LcrH complex. These effects were not observed for the unstructured peptide, YopD 278)292 , which confirms that the a helical structure of the YopD amphipathic domain is a critical binding region of LcrH.Keywords: YopD; amphipathic helix; LcrH; NMR solution structure; 2,2,2-trifluoroethanol.Injection of anti-host factors into eukaryotic cells by numerous economically important animal-and plant-interacting Gram-negative bacteria is achieved by functionally homologous Ôtype III secretion systemsÕ (TTSS) [1,2]. This TTSS-dependent process is essential to establish bacterial infections. The enteropathogen Yersinia pseudotuberculosis is a model system used to study the basic molecular mechanisms of type III secretion. All pathogenic Yersinia spp. harbor a 70-kb virulence plasmid that encodes numerous Yop (Yersinia outer protein) and Lcr (low calcium response) virulence determinants that are secreted by the Ysc (Yersinia secretion) type III apparatus [3,4]. Two protein classes are secreted by the Ysc apparatus; antihost Yop-effector proteins and those required for their efficient injection into target cells. Collectively, these determinants co-operate to allow Yersinia to resist uptake by both professional and nonprofessional cells [5][6][7] and subvert host cell signalling that would normally lead to effective bacterial clearance [8].YopD is a crucial TTSS component during a Yersinia infection being essential for the injection of antihost Yop-effectors into target cells, possibly through stabilization of a YopB-LcrV pore complex in the plasma membrane through which Yop-effectors are injected into host cells [4,9]. However, involvement of YopD in pore formation is only transitory, because a portion of YopD is also localized to the host cell cytosol [10]. In addition, we and others observed t...

Yersinia pestis YopD 150–287 fragment is partially unfolded in the native state

“…The full-length and Nterminal binding regions of YopD are insoluble and in vitro biophysical characterization has not been achieved. To the contrary, the C-terminal YopD 278-292 peptide is readily soluble and forms an amphipathic a-helix in solution [18]. The binding site of YopD was postulated to localize to a large surface on the model of SycD, the opposite side of the YopB interactions [19], in agreement with data for a common chaperone of YopD and YopB [16].…”

“…Overall, the behavior is consistent with a partially unfolded peptide. The YopD 150-287 peptide contains the first 10 residues of the region proposed to bind the SycD chaperone [18]. It was of interest to investigate if the truncated region was sufficient to bind the SycD chaperone in a stable complex.…”

YopD Self-assembly and Binding to LcrV Facilitate Type III Secretion Activity by Yersinia pseudotuberculosis

“…Site-directed Mutagenesis of the YopD C Terminus-Phenotypic analysis of YopD variants lacking a putative coiled-coil domain between residues 248 and 277 (36) and the amphipathic domain encompassing residues 278-292 (35,49) suggested that both features were central to YopD-mediated effector delivery into eukaryotic cells (9,11). Furthermore, the hydrophobic residues on one side of the amphipathic ␣-helix primarily contributed to an interaction with the cognate chaperone LcrH (37) and subsequent yop regulatory control (28,29).…”

Dissection of homologous translocon operons reveals a distinct role for YopD in type III secretion by Yersinia pseudotuberculosis

“…Such a role is known for the C-terminal amphipathic domain (Tengel et al, 2002), which is essential for Yop effector translocation (Francis & Wolf-Watz, 1998). Interestingly, a helical wheel projection (ANTHEPROT version 3.2; G. Deleage, France) over the equivalent region in PopD only predicted an a-helix with marginal amphipathic properties (data not shown).…”