Phosphopantetheine adenylyltransferase (PPAT) catalyses the penultimate step in coenzyme A biosynthesis in bacteria and is therefore a candidate target for antibacterial drug development. We randomly mutated the residues in the Helicobacter pylori PPAT sequence to identify those that govern protein folding and ligand binding, and we describe the crystal structure of one of these mutants (I4V/N76Y) that contains the mutations I4 → V and N76 → Y. Unlike other PPATs, which are homohexamers, I4V/N76Y is a domain-swapped homotetramer. The protomer structure of this mutant is an open conformation in which the 65 C-terminal residues are intertwined with those of a neighbouring protomer. Despite structural differences between wild-type PPAT and IV4/N76Y, they had similar ligand-binding properties. ATP binding to these two proteins was enthalpically driven, whereas that for Escherichia coli PPAT is entropically driven. The structural packing of the subunits may affect the thermal denaturation of wild-type PPAT and I4V/N76Y. Mutations in hinge regions often induce domain swapping, i.e. the spatial exchange of portions of adjacent protomers, but residues 4 and 76 of H. pylori PPAT are not located in or near to the hinge region. However, one or both of these residues is responsible for the large conformational change in the C-terminal region of each protomer. To identify the residue(s) responsible, we constructed the single-site mutant, N76Y, and found a large displacement of α-helix 4, which indicated that its flexibility allowed the domain swap to occur.