The type II secretion system (T2SS) releases large folded exoproteins across the envelope of many Gram-negative pathogens. This secretion process therefore requires specific gating, interacting, and dynamics properties mainly operated by a bipartite outer membrane channel called secretin. We have a good understanding of the structure-function relationship of the pore-forming C-terminal domain of secretins. In contrast, the high flexibility of their periplasmic N-terminal domain has been an obstacle in obtaining the detailed structural information required to uncover its molecular function. In Pseudomonas aeruginosa, the Xcp T2SS plays an important role in bacterial virulence by its capacity to deliver a large panel of toxins and degradative enzymes into the surrounding environment. Here, we revealed that the N-terminal domain of XcpQ secretin spontaneously self-assembled into a hexamer of dimers independently of its C-terminal domain. Furthermore, and by using multidisciplinary approaches, we elucidate the structural organization of the XcpQ N domain and demonstrate that secretin flexibility at interdimer interfaces is mandatory for its function.

In many Gram-negative bacteria, the type 2 secretion system (T2SS) plays an important role in virulence because of its capacity to deliver a large amount of fully folded protein effectors to the extracellular milieu. Despite our knowledge of most T2SS components, the mechanisms underlying effector recruitment and secretion by the T2SS remain enigmatic. Using complementary biophysical and biochemical approaches, we identified here two direct interactions between the secreted effector CbpD and two components, XcpY L and XcpZ M , of the T2SS assembly platform (AP) in the opportunistic pathogen Pseudomonas aeruginosa. Competition experiments indicated that CbpD binding to XcpY L is XcpZ M -dependent, suggesting sequential recruitment of the effector by the periplasmic domains of these AP components. Using a bacterial two-hybrid system, we then tested the influence of the effector on the AP protein-protein interaction network. Our findings revealed that the presence of the effector modifies the AP interactome and, in particular, induces XcpZ M homodimerization and increases the affinity between XcpY L and XcpZ M . The observed direct relationship between effector binding and T2SS dynamics suggests an additional synchronizing step during the type 2 secretion process, where the activation of the AP of the T2SS nanomachine is triggered by effector binding.Type IV filament (Tff) 3 nanomachines are membrane-embedded macromolecular complexes organized around a char-acteristic helical pilus-like structure emerging from an assembly platform at the cytoplasmic membrane (1). Tffs are widespread in prokaryotes where they fulfill diverse cellular functions, in particular the type 2 secretion system (T2SS) that is found in many pathogenic Gram-negative proteobacteria (2). It is dedicated to the secretion of large folded periplasmic exoproteins via a piston-like apparatus called the secreton. Secretons are constituted by 12-15 different components organized into three subcomplexes: the outer-membrane pore belonging to the secretin family, the assembly platform (AP) or motor of the system, and the typical pseudopilus emerging from the AP up to the secretin pore (for the most recent reviews, see Refs. 3 and 4).T2SS secretins are homomultimers assembled into a giant gated ␤-barrel pore in the outer membrane connected to the AP by an N-terminal periplasmic extension (5-7). The Tff-specific structure of the T2SS is called the pseudopilus. It is constituted by the helical assembly of the five pseudopilins of the system. Chronologically, the four minor pseudopilins, GspHIJK, are first assembled by the AP and thus form the head of the structure. This step is followed by the addition, from the bottom of the filament, of the major pseudopilin GspG into a pseudopilus (8 -11). Pseudopilus assembly is energized and promoted by the AP, which is constituted by the oligomeric assembly of four membrane proteins, including the three bitopic proteins GspC, GspL, and GspM and the integral membrane protein GspF. The energy for pseudopilus assembly is p...