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The specific inhibition of serine proteinases, which are crucial switches in many important physiological processes, is of great value both for basic research and for therapeutic applications. In this study, we report the molecular cloning of the sso0767 gene from Sulfolobus solfataricus, and the functional characterization of its product, SsCEI, which represents the first archaeal phosphatidylethanolamine-binding protein (PEBP)-serine proteinase inhibitor, reported to date. SsCEI is a monomer protein with a molecular mass of 19.0 kDa and a pI of 6.7, which is able to inhibit the serine proteases alpha-chymotrypsin and elastase with K(i) values of 0.08 and 0.1 microM, respectively. Moreover SsCEI is extremely resistant to both thermal inactivation and proteolytic attack suggesting compact folding of the protein. Within the I51 family, the archaeal inhibitor shows strong similarity to the human and murine members. The three-dimensional model of SsCEI revealed a general beta-fold and the presence of an anion-binding pocket, the hallmark of the PEBP family. Moreover SsCEI binds the cognate proteases according to a common, substrate-like standard mechanism. Point mutation experiments supported the prediction of the protease-binding site located on the surface at the C- terminal region of the protein. Interestingly, searches based on preidentified structural reactive loop motifs revealed the occurrence of a sequence (T123-N130) that is not represented in all serine-protease inhibitor families. This unique motif may provide new insights into both the inhibitor/protease binding mode and the specific biological functions of SsCEI within the PEBP family.