Structural Studies Of Human Immune DNA Sensor CGAS And The DotL-Icms-IcmW Coupling Complex Of Legionella Type ⅣB Secretion System

Molecular mechanisms of pathogen-host interactions represent an important field of biological research. In the past two years, I have mainly focused on two important projects. One is to study the crystal structure of human immune DNA sensor cGAS. The other is to crystallize the DotL-IcmS-IcmW coupling protein complex of Legionella type Ⅳ secretion system (T4SS). Cyclic GMP-AMP synthase (cGAS) is a newly identified immune DNA sensor that detects cytosolic double-strand DNAs (dsDNA). Once bound to dsDNA, cGAS synthesizes cyclic GMP-AMP (cGAMP), a new secondary massager found in metazoan, leading to type Ⅰ interferons and inflammatory cytokines expression through activating STING. In my studies, Ⅰ determined the crystal structure of the free-form human cGAS (hcGAS) at 2.6 A resolution. Structural analysis suggested that hcGAS adopts an architecture that is similar to those of other nucleitidyltransferse (NTase) family members and the active center of hcGAS is located in a canonical catalytic pocket. Structural comparison of hcGAS with its homolog OAS1 (double-stranded RNA oligoadenyl-atesynthetase 1) revealed the catalytic similarities and differences between the two nucleitidyltransferses. The crystal structure also revealed the molecular basis for ligand-binding specificity of cGAS to dsDNA, but not RNA or other nucleotides.In my second project, I focus on the Legionella DotL-IcmS-IcmW ternary protein complex. DotL is a component of the type Ⅳ secretion system (T4SS) of Legionella pneumophila, a causative pathogen of human Legionellosis. The Legionella T4SS is assembled by～30 proteins and functions as an injector to deliver more than 200 virulent effector proteins into the host macrophage. The delivered effectors modulate the host vesicular trafficking pathways to build the Legionella-containing vacuoles (LCV) and inhibit the host immune defenses for bacterial survival and proliferation. DotL specifically interacts with the chaperone-like IcmS and IcmW to assemble the coupling protein complex for the Legionella T4SS and mediates the translocation of IcmS-IcmW dependent substrates through its ATPase activity. However, how DotL interacts with IcmS-IcmW and how IcmS-IcmW recognizes the type Ⅳ effectors are unclear. In this study, I purified and crystallized the recombinant DotL-IcmS-IcmW complex. Then we determined the DotL-IcmS-IcmW complex at 2.6 A resolution. From the structure, we found that the DotL-IcmS-IcmW forms letter "n" architecture and the complex is mainly maintained by hydrophobic interactions. Finally, we proposed the competitive and non-competitive models of DotL and substrates binding to IcmS-IcmW to illustrate the role of DotL-IcmS-IcmW in IcmS-IcmW dependent substrates delivering.