Architecture of the type IV coupling protein complex of Legionella pneumophila

Abstract

Many bacteria, including Legionella pneumophila, rely on the type IV secretion system to translocate a repertoire of effector proteins into the hosts for their survival and growth. Type IV coupling protein (T4CP) is a hexameric ATPase that links translocating substrates to the transenvelope secretion conduit. Yet, how a large number of effector proteins are selectively recruited and processed by T4CPs remains enigmatic. DotL, the T4CP of L. pneumophila, contains an ATPase domain and a C-terminal extension whose function is unknown. Unlike T4CPs involved in plasmid DNA translocation, DotL appeared to function by forming a multiprotein complex with four other proteins. Here, we show that the C-terminal extension of DotL interacts with DotN, IcmS, IcmW and an additionally identified subunit LvgA, and that this pentameric assembly binds Legionella effector proteins. We determined the crystal structure of this assembly and built an architecture of the T4CP holocomplex by combining a homology model of the ATPase domain of DotL. The holocomplex is a hexamer of a bipartite structure composed of a membrane-proximal ATPase domain and a membrane-distal substrate-recognition assembly. The presented information demonstrates the architecture and functional dissection of the multiprotein T4CP complexes and provides important insights into their substrate recruitment and processing.

Biological Diversity and Evolution of Type IV Secretion Systems

“…Many VirD4 subunits also possess sequence-variable C-terminal domains (CTDs) that are typically enriched in acidic residues (Alvarez-Martinez and Christie 2009; Kwak et al 2017). When present, these CTDs also play important roles in substrate recruitment, as shown by studies of the F and pKM101 conjugation systems (see Figs.…”

“…2.2). Recently, an X-ray structure showed that DotL’s C-terminal domain (CTD) interacts with the stabilizing subunit DotN and three adaptors IcmS, IcmW, and LvgA sequentially along its length (Kwak et al 2017; Xu et al 2017). The findings underscore the importance of the CTDs of T4CPs for effector diversification and spatiotemporal control of effector presentation to the T4SS channel.…”

“…For both systems, substrates are delivered through an OMCC channel to the cell surface. For the Dot/Icm system, the DotL—adaptor complex involved in substrate recruitment is shown (Kwak et al 2017)…”

DecipheringLegionellaeffector delivery by Icm/Dot secretion system reveals a new role for c-diGMP signaling

“…Crucial for hijacking host-cell vesicles trafficking necessary for LCV biogenesis, and subsequently for intracellular multiplication of L. pneumophila, is the Type 4 Secretion System (T4SS) Icm/Dot (for Intracellular multiplication and Defect in organelle trafficking) (2, 3). The Icm/Dot system is a complex machinery located at the bacterial pole (4-6) and composed of 27 proteins involved in (i) a multiprotein apparatus for secretion (7), (ii) a coupling protein complex (DotL/IcmO; DotM/IcmP; DotN/IcmJ) (8,9) and (iii) chaperone proteins that associate with the coupling protein complex and are involved in some specific substrate recognition for presentation to the translocon (IcmS; IcmW; LvgA) (10-12). This machinery translocates an exceptionally large repertoire of effectors, over 300 proteins, into the host-cell cytosol (13).…”

“…Within amoeba and human macrophages, L. pneumophila evades endocytic degradation and triggers the biogenesis of a Legionella-containing vacuole (LCV), a rough endoplasmic reticulum-like compartment permissive for its intracellular multiplication (1).Crucial for hijacking host-cell vesicles trafficking necessary for LCV biogenesis, and subsequently for intracellular multiplication of L. pneumophila, is the Type 4 Secretion System (T4SS) Icm/Dot (for Intracellular multiplication and Defect in organelle trafficking) (2, 3). The Icm/Dot system is a complex machinery located at the bacterial pole (4-6) and composed of 27 proteins involved in (i) a multiprotein apparatus for secretion (7), (ii) a coupling protein complex (DotL/IcmO; DotM/IcmP; DotN/IcmJ) (8,9) and (iii) chaperone proteins that associate with the coupling protein complex and are involved in some specific substrate recognition for presentation to the translocon (IcmS; IcmW; LvgA) (10-12). This machinery translocates an exceptionally large repertoire of effectors, over 300 proteins, into the host-cell cytosol (13).Despite major progress on the Icm/Dot structure thanks to electron cryotomography technology (5,14,15), much still needs to understand the functioning of the machinery, in particular regarding the secretion control of so many effectors.…”