Structural And Enzymatic Characterization Of Choline Kinase LicA And Phosphocholine Transferase LicD2from Streptococcus Pneumoniae

The major human pathogen Streptococcus pneumoniae is responsible for many infectious diseases, such as lobar pneumoniae, meningitis and bronchitis. It leads to approximately1.6million deaths annually in the world. The major virulence factors include pneumococcal teichoic acid, capsular polysaccharide and hemolysin. Choline molecule is an important component of pneumococcal cell wall and capsular. In meantime, choline is required for the pneumococcal growth, normal transformability and cell division. It can also anchor a family of surface proteins (called as choline binding proteins), which play important roles in pneumococcal pathogenesis. The pneumococcal choline catabolism pathway consists of eight proteins encoded by a gene cluster, termed lic. The investigation of their biological functions will help us to better underdtanding the mechanism of pneumococcal choline catabolism and the pathogenesis. Moreover, the work will provide insights into the rational development of novel inhibitors and vaccines.LicA, a putative choline kinase catalyses the choline phosphorylation in the cytoplasm to phosphocholine in S. pneumoniae. Here we determined the crystal structures of LicA apo-form at1.94A and three complex forms LicA-choline, LicA-AMP-MES and LicA-ADP-HEPES at2.01,1.45and1.86A, respectively. The overall structure adopts a canonical protein kinase-like fold with the active site located at the crevice of the N-and C-terminal kinase domains, which undergo significant conformational changes during catalysis. Structural analysis together with site-directed mutagenesis enabled us to assign key residues which are indispensable for catalysis. Furthermore, structure comparison and sequence analysis revealed that the loopα7-α8at the choline-binding pocket might modulate the substrate affinity and catalytic activity. Taken together, our work provides structural insights into the mechanism of pneumococcal choline kinase and might directs the rational design of new anti-pneumococcal drugs.LicD2is a pneumoooccal phosphocholine transferase. It transfers phosphocholine from CDP-choline to the GalNAc moiety of teichoic acid. LicD2is a membrane associated protein. By using fluorescence spectrum, we determined the KD value of LicD2to its substrates. The KD of LicD2towards CDP-choline and GalNAc was about145μM and34μM, respectively. Crystallization screens of apo-LicD2and substrate-complexs give the initial condition for futher crystal improvement.