The D-Ala-modification of lipoteichoic acidsof gram positive bacteria, which is carried out by proteins of the dlt-operon, is necessary to adopton changing environmental conditions. DltA catalyse the activation of D-alanine by ATP to D-Ala-adenylate and the subsequent transfer of the D-Ala to the carrier protein DltC. At least three DltA conformations could be identified that were dependent on the associated nucleotide. Two crystal structures of the DltA:DltC complex could be solved. The first structure represents a productive complex. The second one shows an alternative binding site for DltC on the large domain of DltA far away from the active centre, with no apparent electron density for the cofactor.Mapping of the DltC interaction surface monitored by NMR titration provides support for both interaction interfaces in solution, butquantitative protein-protein interaction studies and extensive enzyme kinetic analysis could not reveal a possible function of this alternative binding in vitro.