Structural and biochemical study on proteins involved in D-alanylation of teichoic acids

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Teichoic acid is a unique cell wall component found in Gram-positive bacteria. Similar to phosphoribose backbone in nucleic acids, this anionic biopolymer is made of repeating units of phosphoglycerol or phosphoribitol. A ubiquitous modification of this biopolymer is D-alanine esterification, which requires the functioning of four proteins (DltA, DltB, DltC and DltD) coded by the dlt gene. This esterification process partially neutralizes the anionic polymer, and appears to be essential for biofilm formation, bacterial proliferation and host cell infection. The D-alanyl carrier protein ligase DltA is an enzyme resembling the adenylation domains found in acyl-CoA synthetases and firefly luciferases. DltA catalyzes the ATP-driven adenylation of D-alanine and the transfer of the activated D-alanyl to the thiol group of 4'-phosphopantetheine which is covalently attached to a serine side chain of the D-alanyl carrier protein DltC. The role of the integral membrane protein DltB has not been established. DltD, a membrane-bound protein with an N-terminal putative transmembrane anchor, is a possible candidate for catalyzing the final transfer of D-alanyl form DltC to teichoic acid. We have recently determined the 3-dimensional structures of a DltA protein from a Gram-positive bacterium Bacillus cereus in complex with D-alanine adenylate and with ATP. The complex with ATP revealed a more closed conformation previously unknown to similar adenylation domains, which shed light on the catalytic mechanism of such domains. We proposed to further study the structure-function relationship of DltA, to study the possible interactions between the four Dlt proteins in order to understand how they form a functional system, and to verify whether DltD serves as the D-alanyl transferase which catalyzes the last reaction step of D-alanylation of teichoic acid. In the long run, information gleaned from this study would rationalize how these proteins promote this important D-alanylation pathway of teichoic acids in Gram-positive bacteria and aid the design of inhibitors of biofilms formation.