Structural Biological Studies On The Regulation Of C-di-GMP Metabolism By Legionella Pneumophila Protein Lpg1168

Legionella pneumophila is a gram-negative bacterium commonly found in aquatic environments that can infect host alveolar macrophages and cause severe atypical pneumonia.In recent years,the reported cases of Legionella pneumophila infection have gradually increased,and many Legionella pneumophila strains have been found to be resistant to antibiotics,we thus have committed to the research on Legionella pneumophila,hoping to provide more information for the development of related therapeutic drugs through further understanding of Legionella pneumophila.c-di-GMP is a second messenger widely present in bacteria,which is involved in various physiological functions such as regulation of cell cycle and differentiation,cell motility,biofilm formation,production of pathogenic factors and quorum sensing.Therefore,the c-di-GMP-related signaling pathway plays an important role in bacterial life.Lpg1168 is a complex protein of conserved GGDEF and EAL domain encoded in Legionella pneumophila.The GGDEF domain is known to be a conserved domain located in guanylate cyclases involved in c-di-GMP synthesis,the EAL domain is a conserved domain located in phosphodiesterases involved in c-di-GMP degradation.This study intends to take Lpg1168 as the main research object,combining biochemical and structural biology methods to explore the molecular mechanism of Lpg1168 involved in c-di-GMP metabolism in Legionella pneumophila.In this study,the Lpg1168_{（470-755）}truncated protein containing the EAL domain has a strong affinity for c-di-GMP by ITC experiments and fluorescence thermal drift experiments.The structure of Lpg1168_{（470-755）}/c-di-GMP complex was successfully resolved by X-ray crystallography.The structural analysis found that:（1）Similar to the classical phosphodiesterases with EAL domains,Lpg1168_{（470-755）}has a barrel-like structure and has a c-di-GMP binding pocket at its C-terminus.Further analysis identified the key residues of Lpg1168_{（470-755）}that involved in the interaction with c-di-GMP and metal ions mainly occurred on theβ2 sheet inside the barrel structure.（2）In addition,unlike the classical EAL conserved domain,the conserved motif in Lpg1168_{（470-755）}protein consists of E49-I50-L51 residues located on theβ2 sheet,and the E49 residue in the EIL motif plays a key role in binding of c-di-GMP.（3）Finally,the site-directed mutagenesis of the interaction site with c-di-GMP in Lpg1168_{（470-755）}confirmed that E49 residue,N108 residue,E140 residue mutation can affect the interaction of Lpg1168_{（470-755）}with c-di-GMP.In conclusion,we found that Lpg1168 with phosphodiesterase activity in Legionella pneumophila,and elucidated the molecular mechanism of its phosphodiesterase activity specifically binding to c-di-GMP.This study provides an important basis for us to reveal that Lpg1168 exerts phosphodiesterase activity and participates in c-di-GMP related signaling pathway in Legionella pneumophila.It also lays a foundation for further exploring that Lpg1168 inhibits biofilm formation involved in the regulatory network of c-di-GMP in Legionella pneumophila,which provides insights for the understanding to the Legionella pneumophila infection.