Engineered staphylococcal protein A’s IgG-binding domain with cathepsin L inhibitory activity Tomazˇ Bratkovicˇ a,b, * , Alesˇ Berlec c , Tatjana Popovicˇ c , Mojca Lunder a , Samo Kreft a , Urosˇ Urleb b , Borut S ˇ trukelj a,c a Faculty of Pharmacy, Department of Pharmaceutical Biology, University of Ljubljana, Asˇkercˇeva 7, SI-1000 Ljubljana, Slovenia b Lek Pharmaceuticals, Drug Discovery, Verovsˇkova 57, SI-1526 Ljubljana, Slovenia c Jozˇef Stefan Institute, Department of Biochemistry and Molecular Biology, Jamova 39, SI-1000 Ljubljana, Slovenia Received 7 August 2006 Available online 22 August 2006 Abstract Inhibitory peptide of papain-like cysteine proteases, affinity selected from a random disulfide constrained phage-displayed peptide library, was grafted to staphylococcal protein A’s B domain. Scaffold protein was additionally modified in order to allow solvent exposed display of peptide loop. Correct folding of fusion proteins was confirmed by CD-spectroscopy and by the ability to bind the Fc-region of rabbit IgG, a characteristic of parent domain. The recombinant constructs inhibited cathepsin L with inhibitory constants in the low- micromolar range. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Cysteine proteases; Inhibitory peptide; Constrained loop; Staphylococcal protein A; Protein scaffold; Dual functionality Peptides are often looked upon as prototype modulators of biological function, since they are found in all living beings performing functions of hormones, antigens, bio- chemical inhibitors, growth factors, transmembrane carri- ers, etc. [1]. They have also attracted attention of medicinal chemists ever since it became evident they gener- ally form highly specific interactions with (protein) targets. Linear peptides, however, are usually hampered by rela- tively low affinity for receptor proteins due to their pro- nounced flexibility and—as a direct consequence of that—considerable entropy change on receptor binding. Cyclization of linear peptides is thus frequently used to provide conformationally restricted analogues [1]. We have recently reported on small peptide inhibitors of cysteine cathepsins’ affinity selected through phage display [2]. Conformational freedom of these peptides was restrict- ed by an intramolecular disulfide bond, resulting in looped peptides possessing higher inhibitory activity compared to opened linear peptides. Unfortunately, cysteine proteases require reductive media for optimal enzymatic activity, pre- venting oxidation of the active site thiol group. The incom- patibility of our peptides’ structural features (unstable cyclic form) was overcome by preparing head-to-tail cyc- lized peptide GNWTLGGYKGG, an analogue of the rela- tively broadly acting inhibitor CNWTLGGYKC. The former peptide turned out to have increased affinity for cathepsins L and K (26- and 2.7-fold, respectively), but was also more selective (it no longer inhibited cathepsin H and had diminished activity towards papain). In this paper we describe the grafting of inhibitory peptide sequence NWTLGGYK to a small three helical protein, domain B from staphylococcal protein A (SpA). B domain was chosen as a scaffold for numerous reasons: it is com- posed of only 58 AA and is cysteine free (thus resistant to reductants) but nevertheless possesses a stable fold, it is high- ly soluble, easily expressed in Escherichia coli, fairly resistant to proteases, its 3D structure is well-known, and, last but not least, its IgG-Fc-binding property is an attractive feature for isolation purposes or use as Fc-affinity marker. 0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.08.078 * Corresponding author. Fax: +386 1 42 58 031. E-mail address: tomaz.bratkovic@ffa.uni-lj.si (T. Bratkovicˇ). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 349 (2006) 441–445 BBRC