In mammals, numerous precursors of antibacterial peptides with unrelated sequences share a similar prosequence of 94-114 residues, termed the cathelin-like domain. The cathelin-like domain of protegrin-3 (ProS) was overexpressed in Escherichia coli and uniformly labeled with (15)N or (15)N and (13)C, and its three-dimensional structure was determined by heteronuclear NMR at pH 6.2. Under these conditions and due to the cis-trans isomerization of the R(87)-P(88) and D(118)-P(119) amide bonds, the ProS structure was found to adopt four almost equally populated conformations in slow exchange on the NMR chemical shift time scale. The ProS structure consists of an N-terminal alpha-helix (Y(34)-N(48)) cradled by a four-stranded antiparallel beta-sheet (beta1, N(53)-L(60); beta2, K(74)-P(86); beta3, V(104)-V(111); and beta4, I(122)-C(124)). The solution structure of ProS, which is monomeric, allowed us to determine the structure of the L1 and L2 loops, which are too mobile in the crystal structure. The regions common to the solution and X-ray structures were found to be very similar. Finally, since the overall fold of ProS is very similar to that of cystatins despite a low degree of sequence identity, the ProS solution structure was compared to the solution and X-ray structures of the chicken cystatin. This comparison revealed that the structures of the L1 and L2 loops as well as that of the appending domain are quite different in the two proteins. These differences are mainly due to the high proline residue content (10%) which disorganizes the hydrogen bond network of a part of the ProS beta-sheet in contrast to that of the chicken cystatin structure.