The peptide transporter from a cold-adapted bacterium has never been reported. In the present study, the dpp operon from the psychrophilic bacterium Pseudoalteromonas sp. strain SM9913 was cloned and analyzed. The dipeptide binding protein DppA of SM9913 was overexpressed in Escherichia coli, and its cold adaptation characteristics were studied. The recombinant DppA of SM9913 (PsDppA) displayed the highest ligand-binding affinity at 15 degrees C, whereas the recombinant DppA of E. coli (EcDppA) displayed the highest ligand-binding affinity at 35 degrees C. Thermal and guanidium hydrochloride unfolding analyses indicated that PsDppA has more structural instability than EcDppA. Six domain-exchanged mutants of PsDppA were expressed and purified. Analyses of these mutants indicated that domains III, I-2, and I-3 of PsDppA were less stable than those from EcDppA and that domains III and I-2 made a significant contribution to the high binding affinity of PsDppA at low temperatures. Structural and sequence analyses suggested that the state transition-involved regions in domain III and the alpha part of domain I-2 are the hot spots of optimization during cold adaptation and that decreasing the side-chain size in these regions is an important strategy for the cold adaptation of PsDppA.