Cryo-EM Structural studies on the Xanthomonadaceae Type IV secretion systems

Abstract

Bacteria use a variety of secretion systems to translocate macromolecules across the bacterial cell envelope. The type IV secretion system (T4SS) of the phytopathogen Xanthomonas citri is responsible for the secretion of toxins that kill other Gram-negative bacteria. The T4SS are very large molecular assemblies formed by two subcomplexes, the core complex (~ 1 MDa) and the inner membrane complex. Preliminary results from our group in single particle electron microscopy studies on the X. citri core complex show that the 14 repeats of a VirB7-VirB9-VirB10 trimer come together to form a tetradecameric ring. Each one of these subunits can be divided into N-terminal and C-terminal domains (B7NT, B7CT, B9NT, B9CT, B10NT, B10CT). In this structure, the only density available for the VirB10 N-terminal domain (predicted to be intrinsically disordered) corresponds to a helix composed of residues 150-161. The tetradecameric ring can be divided into two ring layers: the upper layer is associated with the bacterial outer membrane and is made up of B7NT, B7CT, B9CT and B10CT and a lower layer made up of B9NT and B10NT. Within each layer the interactions between subunit domains are strong. On the other hand, interactions between layers are seemingly weak and they are connected by flexible linkers between the N- and C-terminal domains of VirB9 and VirB10. Furthermore, the interactions between B7CT with the main body of the upper layer are also tenuous. These observations suggest that the core complex is intrinsically dynamic, and what we are observing is most likely only one of perhaps multiple conformations that are available for the core complex. Intra-subunit, inter-subunit, and inter-layer motions in various time scales could be essential for the function of this system. Therefore, to obtain information on the dynamics of the X. citri core complex we plan to run molecular dynamics (MD) simulations using our structure as starting point. The molecular simulations will be complemented with Cryo-electron microscopy studies. Since Cryo-EM preserves the conformational heterogeneity of the molecules under study (upon rapid freezing in amorphous ice) it can potentially provide structural information on different conformational states available to the protein in solution. These conformational states can be detected by 3D classification of whole particles or by focused 3D classification of specific domains. We plan to use this latter focussed classification technique to detect conformational heterogeneity among chemically identical VirB7-VirB9-VirB10 trimers in the core complex tetradecamer.