AbstractSso7d from the extreme thermophilic crenarchaeon Sulfolobus solfataricus is a multifunctional protein in in vitro assays, whose in vivo role is still puzzling. Crystals of Sso7d in complex with DNA elucidated the protein surface involved in the binding to the nucleic acid, whereas the locations of the Sso7d regions responsible for a chaperone activity in renaturing protein aggregates (i.e., the protein‐binding surface and the site of ATPase activity) are still unknown. We identified the regions of Sso7d involved in protein‐binding by limited proteolysis experiments associated to advanced mass spectrometric procedures performed on isolated Sso7d and Sso7d in complex with the peptide melittin. By affinity labeling of Sso7d with the ATP analogue 5′‐p‐fluorosulfonylbenzoyl adenosine and characterization of the labeled tryptic peptides by tandem mass spectrometry, we found that Y7 and K39 are residues involved in ATP binding/hydrolysis. Insights into the positions of the ligands melittin and ATP were achieved by a molecular modeling study; the models obtained were in agreement with most experimental data. A comparison among the complexes of Sso7d with DNA, with melittin, and with ATP showed that the DNA‐binding surface and the protein‐binding surface overlap, whereas the ATPase site is mostly independent of the binding sites for the nucleic acid and melittin. Proteins 2007. © 2007 Wiley‐Liss, Inc.