Conformational stability and differential structural analysis of LcrV, PcrV, BipD, and SipD from type III secretion systems

Abstract

Diverse Gram-negative bacteria use type III secretion systems (T3SS) to translocate effector proteins into the cytoplasm of eukaryotic cells. The type III secretion apparatus (T3SA) consists of a basal body spanning both bacterial membranes and an external needle. A sensor protein lies at the needle tip to detect environmental signals that trigger type III secretion. The Shigella flexneri T3SA needle tip protein, invasion plasmid antigen D (IpaD), possesses two independently folding domains in vitro. In this study, the solution behavior and thermal unfolding properties of IpaD's functional homologs SipD (Salmonella spp.), BipD (Burkholderia pseudomallei), LcrV (Yersinia spp.), and PcrV (Pseudomonas aeruginosa) were examined to identify common features within this protein family. CD and FTIR data indicate that all members within this group are a-helical with properties consistent with an intramolecular coiled-coil. SipD showed the most complex unfolding profile consisting of two thermal transitions, suggesting the presence of two independently folding domains. No evidence of multiple folding domains was seen, however, for BipD, LcrV, or PcrV. Thermal studies, including DSC, revealed significant destabilization of LcrV, PcrV, and BipD after N-terminal deletions. This contrasted with SipD and IpaD, which behaved like two-domain proteins. The results suggest that needle tip proteins share significant core structural similarity and thermal stability that may be the basis for their common function. Moreover, IpaD and SipD possess properties that distinguish them from the other tip proteins.

Deoxycholate Interacts with IpaD of Shigella flexneri in Inducing the Recruitment of IpaB to the Type III Secretion Apparatus Needle Tip

“…Thus, the inability to grasp IpaB probably stems from sequence differences at positions other than the C terminus of the tip proteins. More importantly, however, this suggests that BipD has the information within its structure needed to bind deoxycholate, but that the unusual stability and complexity of its N-terminal domain (10,17) may block deoxycholate binding in vitro, whereas the BipDMxiH needle tip complex is able to recognize the presence of deoxycholate. This once again implicates the central coiled-coil of these tip complex proteins in the recognition of environmental small molecules.…”

“…Protein Expression and Purification-Recombinant IpaD, SipD, and BipD have been prepared by standard methods as previously described (16,17). Briefly, Tuner(DE3) containing D/pET15b, SipD/pET15b, or BipD/pET15b were grown to 0.5 A 600 units, induced to express protein with 1.0 mM isopropyl 1-thio-␤-D-galactopyranoside and incubated a further 3 h. The bacteria were collected by centrifugation, lysed by sonication, and the HisTag protein from the clarified supernatant was purified by standard nickel affinity chromatography.…”

ExoS Controls the Cell Contact-Mediated Switch to Effector Secretion in Pseudomonas aeruginosa

“…This could of course also be due to an inherently weak affinity of PcrV for the translocation pore, requiring either other cofactors or multiple simultaneous interactions as found in the assembled needle tip. The needle tip most likely harbors multiple PcrV molecules (8) that can interact with the translocation pore (purified PcrV, on the other hand, is a monomer [16]). We propose that it is docking of the needle tip to the translocation pore that results in a conformational change in PcrV, which is propagated down the needle and results in the activation of effector secretion.…”

Macrophage Apoptosis Triggered by IpaD from Shigella flexneri

“…4E). These data imply that macrophage killing by T3SA needle tip proteins is limited to the family of tip proteins possessing an N-terminal domain like that of IpaD and SipD (41).…”