Dephospho-coenzyme A kinase catalyzes the final step in CoA biosynthesis, the phosphorylation of the T-hydroxyl group of ribose using ATP as a phosphate donor. The protein from Haemophilus influenzae was cloned and expressed, and its crystal structure was determined at 2.0-Angstrom resolution in complex with ATP. The protein molecule consists of three domains: the canonical nucleotide-binding domain with a five-stranded parallel beta-sheet, the substrate-binding a-helical domain, and the lid domain formed by a pair of alpha-helices. The overall topology of the protein resembles the structures of nucleotide kinases. ATP binds in the beta-loop in a manner observed in other kinases. The CoA-binding site is located at the interface of all three domains. The double-pocket structure of the substrate-binding site is unusual for nucleotide kinases. Amino acid residues implicated in substrate binding and catalysis have been identified. The structure analysis suggests large domain movements during the catalytic cycle.