42 43 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 0 / 2 1 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 0 / 2 1 Biological Cryo-Imaging Group eBIC Visualising a bacterial multi-substrate recruitmentmachine in action Related publication: Meir A., Macé K., Lukoyanova N., Chetrit D., Hospenthal M. K., Redzej A., Roy C. &Waksman G. Mechanismof effector capture and delivery by the type IV secretion system fromLegionella pneumophila. Nat. Commun. 11 , 2864 (2020). DOI: 10.1038/s41467-020-16681-z Publication keywords: Type IV secretion system;T4SS; Host-pathogen interaction; Legionella pneumophila B acteria use a wide range of trans-membrane machineries, called secretion systems, to transfer proteins into target cells. These 'bacterial effector proteins' interfere with host cell functions. Gaining a better understanding of how secretion systems work will provide a basis for novel design strategies of antibiotics to combat bacterial infectious diseases. The pathogen that causes Legionnaires’ disease, Legionella pneumophila ( L. pneumophila ) , uses a Type IV Secretion System (T4SS) to infect human cells. To understand itsmechanism, an international teamof researchers purified the part of the T4SS responsible for the recruitment and delivery of effector proteins from the Legionella membrane, the 'Type IV Coupling Complex (T4CC)'. They were then able to determine the structure of T4CC using single-particle cryo-electron microscopy (cryo-EM) with data collected at the Electron Bio-Imaging Centre (eBIC) at Diamond Light Source and the ISMB cryo-EM facility at Birkbeck, University of London. The atomic resolution structure revealed a five- protein core complex fromwhich the T4CC recruitment module interacts with via a flexible linker. Six of these protein complexes combine to form a large nanomachine, containing a channel in the membrane, that effector molecules can pass through. Analysis of multiple cryo-EM maps, further modelling and mutagenesis provided working hypotheses for the mechanism of binding and delivery of two of the classes of Legionella effectors. L. pneumophila bacteria secrete over 300 effectors during infection and blocking their recruitment is known to stop the disease. The high- resolution structureof the Legionella T4CCprovides essential information for designingT4CC-blockingantibiotics.Mechanistic understanding of the T4CCs gained in this study could also provide the basis for engineering T4SSs capable of injecting therapeutic drugs into human cells. The gram-negative bacterium L. pneumophila, the causative agent of Legionnaires’disease, infects human alveolar macrophages, as well as diverse protozoan hosts 1 . L. pneumophila injects over 300 effector proteins (most pathogenic bacteria settle for less than a dozen) into host cells by utilising the Dot/Icm T4SS. The large number of effector proteins that L. pneumophila is required to secrete in order to invade host organisms makes its T4SS a particularly interesting model system to study effector recruitment and translocation in pathogenic bacteria. L. pneumophila employs a protein complex embedded in the inner membrane called T4CC, that acts as a recruitment platform for effectors and translocates them through the secretion channel 2 . This complex was thought to be formed by three proteins, the AAA+ ATPase DotL, DotM and DotN. How this T4CC orchestrates the recruitment and transfer of so many different effectors was unclear prior to the work described in Meir et al . (2020), the subject of this review. Meir et al . (2020) were fascinated by this functionally essential complex, and in order to elucidate its mechanism of action, they sought to solve its cryo-EM structure. Their initial approach, to purify the complex recombinantly in Escherichia coli , failed despite extensive attempts. A creative solution to overcome this obstaclewas the introduction of an affinity tag at the C-terminus of DotL directly in the L. pneumophila genome. This enabled the purification of a stable complex from L. pneumophila cells. The purified apparatus revealed surprising findings. First, as mentioned above, a complex of three proteins was expected. However, the purified complex consisted of eight components: the expected DotL, DotM and DotN proteins, as well as the chaperone module IcmS, IcmW (IcmSW), LvgA and two previously uncharacterised proteins, DotY and DotZ.The second surprising outcome was that the T4CC, although it contains a AAA+ ATPase that would usually form hexamers, purified as a stable complex of monomeric nature where one copy only of each protein was present. The initial EM data (Fig. 1a) suggested a dynamic structure with a stable core and a more flexible U-shape density with various orientations relative to the core. Thanks to multiple data collections (at eBIC and at the ISMB EM facility at Birkbeck), a 3D density map for the core was obtained at a nominal resolution of 3.7 Å. It revealed that the core is composed of five proteins: DotL, DotM, DotN and the two newly discovered proteins, DotY and DotZ (Fig. 1b). A specific analysis of the EM data focusing on the flexible U-shape density (at mid-range resolution of 9.7 Å) revealed that it consists of IcmSW bound to a part of the DotL C-terminal tail (this partial structure had been previously solved by Kwak et al 3 ) (Fig.1b). IcmSW is known to bind a particular class of effectors called the IcmSW-dependent effectors, so-called because of their dependence on IcmSW for recruitment. Density for the LvgA protein was not found. The main protein of the complex is the ATPase DotL, which has a very long C-terminal tail onto which all other components assemble like pearls on a string, including IcmSW which is found bound to the C-terminus of the DotL tail. Because AAA+ ATPases function as channel-forming hexamers, Meir et al . (2020) proposed a hexameric model of the entire T4CC based on a close homologue of DotL (Fig. 2a). Hexamerisation produces a large complex resembling a starfish 26 nm in diameter, with a central channel large enough for effectors to go through. A previous mutational analysis provided experimental data that showed that the hexameric interface is biologically relevant, likely suggesting that the T4CC functions as a hexamer of DotLMNZY- IcmSW heptameric units. As mentioned previously, the U-shape IcmSW is positioned flexibly relative to the core. Meir et al . (2020) were able to show that the IcmSWmodule bound to the DotL tail moves along a defined trajectory (Fig. 2b) which maximises delivery of IcmSW-bound effectors to the central T4CC hexameric channel formed by DotL (Fig. 2c). DotM is also known to form a platform for recruitment of a second class of effectors, the DotM-dependent effectors 4 . The T4CC structure revealed a binding region on DotM where these effectors bind, thereby providing a mechanism for their uptake by the T4CC channel. In conclusion, the results elucidated the overall architecture of the Legionella T4CC recruitment platform, revealed the binding regions for two different classes of effectors, provided information regarding the dynamics of the system, and delineated a potential mechanism for the sequential and selective recruitment of effectors of various size and function. More generally, this study greatly enhanced our scientific understanding of effector recruitment platforms in bacterial secretion systems. References: 1. Mondino S. et al. Legionnaires’Disease: State of the Art Knowledge of Pathogenesis Mechanisms of Legionella. Annu. Rev. Pathol. Mech. Dis. 15 , 439–466 (2020). DOI: 10.1146/annurev-pathmechdis-012419-032742 2. Vincent C. D. et al. Identification of the DotL coupling protein subcomplex of the Legionella Dot/Icm type IV secretion system. Mol. Microbiol. 85 , 378–391 (2012). DOI: 10.1111/j.1365-2958.2012.08118.x 3. Kwak M. J. et al. Architecture of the type IV coupling protein complex of Legionella pneumophila. Nat. Microbiol. 2 , 17114 (2017). DOI: 10.1038/ nmicrobiol.2017.114 4. Meir A. et al. Legionella DotM structure reveals a role in effector recruiting to the Type 4B secretion system. Nat. Commun. 9 , 507 (2018). DOI: 10.1038/s41467-017-02578-x Funding acknowledgement: This work was funded by ERC grant 321630 andWellcome grant 098302 to G.W. and NIAID grants R21AI130671 and R37AI041699 to CR. Most of the cryo-EM data for this investigation were collected at the ISMB EM facility at Birkbeck College, University of London with financial support fromWellcome (202679/Z/16/Z and 206166/Z/17/Z). We would also like to thank Diamond Light Source for access to the cryo-EM facilities at the UK National electron bio-imaging centre (eBIC, proposal EM14704) funded by theWellcome Trust, the Medical Research Council UK and the Biotechnology and Biological Sciences Research Council. Corresponding authors: Dr Amit Meir, Yale University,
[email protected]; Dr Kevin Macé, Birkbeck, University of London,
[email protected] ; Prof. Gabriel Waksman, Birkbeck, University of London,
[email protected] a 6 nm a DotL DotM DotN DotZ DotY IcmS IcmW b core Flexible IcmSW module Figure 1: (a) Representative 2D class averages of the T4CC showing IcmSW in various orientations. The IcmSWmodule U-shaped density is indicated in white arrows; (b) Ribbon diagram of the T4CC core and IcmSW flexible module. All proteins are indicated in a different colour and labelled. The DotLMNZY core as well as the flexible IcmSWmodule are indicated. As shown in panel (a), the IcmSW module is flexible relative to the core and therefore panel (b) here reports on only one configuration of this module. Panel (a) is reproduced fromMeir et al. (2020). b DotL channel a c 1 2 3 4 5 6 7 Figure 2: (a) Ribbon diagram of the hexameric form of the T4CC including the IcmSWmodules in the relative orientation shown in Fig. 1b; (b) Positional flexibility and trajectory of the IcmSWmodule, as indicated by the superposition of 7 low resolution EMmaps of the DotLMNZY-IcmSW complex; (c) Two diametrically opposite DotLMNZY-IcmSW complexes of the T4CC hexamer are shown, one of them includes the 7 flexible conformations of the IcmSWmodules as shown in (b). The T4CC channel is indicated as 'DotL channel'. In (b) and (c), the double arrow indicates the flexible orientation of IcmSW relative to the core in the context of the monomeric (b) or hexameric (c) assembly. Colour-coding is as in Fig. 1. Panels (b) and (c) are similar, but not identical to those published in Meir et al. (2020).