First Archaeal PEPB-Serine Protease Inhibitor from Sulfolobus solfataricus with Noncanonical Amino Acid Sequence in the Reactive-Site Loop Gianna Palmieri, †,§ Giuliana Catara, †,§ Michele Saviano,* ,‡ Emma Langella, ‡ Marta Gogliettino, † and Mose ` Rossi † Istituto di Biochimica delle Proteine, Via P. Castellino 111, 80131 Napoli, Italy, and Istituto di Biostrutture e Bioimmagini, Consiglio Nazionale delle Ricerche, Via Mezzocannone 16, 80134 Napoli, Italy Received August 1, 2008 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 R-chymotrypsin and elastase with K i values of 0.08 and 0.1 µM, 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 -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. Keywords: Serine protease inhibitor • PEBP • Sulfolobus solfataricus • protease inhibitor family • protease binding site • homology modeling Introduction Organized into a number of distinct families, the protease inhibitors are ubiquitous proteins which have been identified in a wide variety of organisms. Considerable attention to date has been devoted to the eukaryotic serine protease inhibitors, which are assembled in gene families including from 10 up to 500 members. 1 Comparative genome analyses have revealed that only three families of protease inhibitors are shared by prokaryotes and eukaryotes as well as the Archaea: these are the serpins (I4), chagasins (I42) and phosphatidylethanolamine- binding proteins (PEBP) (I51). The I51 family constitutes a distinct cluster characterized by the presence of a PEBP binding domain, 2 and show no sequence similarity to other known proteinase inhibitors. The PEBP family contains over 500 members, only a small number of which have been individually characterized in terms of their specific biological function. They are multifunctional proteins, shown to be involved in signaling mechanisms during cell growth and differentiation via the modulation of catalytic activity of kinases or serine proteases, and by the recognition of lipids or nucleotides. 3-7 Among the PEBP class, the TFS1 inhibitor from Saccharomyces cerevisiae 8 and the mouse PEBP, 2,9 the most studied members of the I51 family, are considered archetypal serine proteinase inhibitors. Recently, the human PEBP has been included as new member of this family as an inhibitor of proteasome chymotrypsin-like activity. 10 The current study has been undertaken with the aim to isolate novel thermophilic serine-proteinase inhibitors from Archaea, in particular from the hyperthermophile Sulfolobus solfataricus, both for evolutionary studies and potential bio- technological applications. A computational genome analysis of S. solfataricus P2 revealed the occurrence of a unique and putative PEBP-protease inhibitor-encoding gene, sso0767. Through extensive S. solfataricus proteome investigation ob- tained via combined separation approaches, 11 the Sso0767- product was shown to be expressed in S. solfataricus cells in midexponential growth phase, representing one of the 1323 * To whom correspondence should be addressed. Michele Saviano, Phone: (39) 0812536648, Fax: (39) 0812534560, E-mail: msaviano@unina.it † Istituto di Biochimica delle Proteine. ‡ Istituto di Biostrutture e Bioimmagini. § These authors equally contributed to this paper. 10.1021/pr800587t CCC: $40.75  2009 American Chemical Society Journal of Proteome Research 2009, 8, 327–334 327 Published on Web 12/04/2008