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Architecture of the type IV coupling protein complex of Legionella pneumophila

Nature Microbiology volume 2, Article number: 17114 (2017) Cite this article

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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.

Many bacterial species are equipped with a type IV secretion system (T4SS) that delivers DNA–protein conjugates or virulence proteins across the cell envelope and into recipient cells to mediate horizontal gene transfer or to establish a survival niche in hosts1. The T4SS is mainly composed of two components: a transenvelope secretion conduit and a type IV coupling protein (T4CP)2. T4CP is an AAA+ type hexameric transmembrane ATPase that plays a dual role: recruiting substrates and conveying them to the secretion conduit3. A prototypic T4SS is the VirB/VirD4 system, encoded by the Ti plasmid in Agrobacterium tumefaciens4, where VirD4 is the T4CP. It translocates the covalently linked transfer DNA–relaxase complex as well as three effector proteins (VirE2, VirE3 and VirF) into plant cells5,6. After being recruited to VirD4, the substrate has to be processed to pass through the narrow T4SS channel. In addition to VirD4, at least two other ATPases, VirB4 and VirB11, are required for substrate processing, which includes protein unfolding7,8.

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Figure 1: Crystal structures of DotL(656–783)–IcmSW and DotL(590–659)–DotN.
Figure 2: Crystal structure of DotL(656–783)–IcmSW–LvgA.
Figure 3: Structural reconstitution of DotL(590–783)–DotN–IcmSW–LvgA.
Figure 4: Effector protein binding.
Figure 5: Model for the T4CP holocomplex.
Figure 6: C-terminal extension of T4CPs.

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Acknowledgements

This study made use of Beamlines 4C and 5C at Pohang Accelerator Laboratory, Korea, and was supported by the National Research Foundation of Korea (NRF) (grant no. 2008-00576) and KAIST Future Systems Healthcare Project, Ministry of Science, ICT and Future Planning. J.D.K. was supported by a TJ PARK postdoctoral fellowship from POSCO TJ PARK Foundation. The authors thank the Korea Institute for Advanced Study for providing computing resources (KIAS Center for Advanced Computation Linux Cluster).

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  1. Mi-Jeong Kwak and J. Dongun Kim: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biological Sciences, KAIST Institute for the Biocentury, Korea Advanced Institute of Science and Technology, 34141, Daejeon, Korea

    Mi-Jeong Kwak, J. Dongun Kim, Hyunmin Kim, Seonghoon Kim & Byung-Ha Oh

  2. Department of Physics, Pohang University of Science and Technology, Pohang, 37673, Kyungbuk, Korea

    Cheolhee Kim & Nam Ki Lee

  3. Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, 1975 Zonal Avenue, Los Angeles, 90033, California, USA

    James W. Bowman & Jae U. Jung

  4. Center for Advanced Computation, School of Computational Sciences, Korea Institute for Advanced Study, 02455, Seoul, Korea

    Keehyoung Joo & Jooyoung Lee

  5. Pohang Accelerator Laboratory, Pohang University of Science and Technology, Pohang, 37673, Kyungbuk, Korea

    Kyeong Sik Jin & Yeon-Gil Kim

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Contributions

M.-J.K., J.D.K., H.K., S.K. and Y.-G.K. performed X-ray crystallography and biochemical experiments. C.K. conducted the ALEX-FRET experiment, J.W.B. the SiMPull, K.J. and J.L. homology modelling, and K.S.J. the SAXS. B.-H.O., M.-J.K., J.D.K., N.K.L. and J.U.J. conceived the experiments and wrote the manuscript. All authors discussed the results.

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Correspondence to Byung-Ha Oh.

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Kwak, MJ., Kim, J., Kim, H. et al. Architecture of the type IV coupling protein complex of Legionella pneumophila. Nat Microbiol 2, 17114 (2017). https://doi.org/10.1038/nmicrobiol.2017.114

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