Phage display selection of hairpin loop soyacystatin variants that mediatehigh affinity inhibition of a cysteine proteinase

Two hairpin-loop domains in cystatin family proteinase inhibitors form an i nterface surface region that slots into the active site cleft of papain-lik e cysteine proteinases, and determine binding affinity. The slot region sur face architecture of the soybean cysteine proteinase inhibitor (soyacystati n N, scN) was engineered using techniques of in vitro molecular evolution t o define residues that facilitate interaction with the proteinase cleft and modulate inhibitor affinity and function. Combinatorial phage display libr aries of scN variants that contain mutations in the essential motifs of the first (QVVAG) and second (EW) hairpin-loop regions were constructed. Appro ximately 10(10)-10(11) phages expressing recombinant scN proteins were subj ected to biopanning selection based on binding affinity to immobilized papa in. The QVVAG motif in the first hairpin loop was invariant in all function al scN proteins. All selected variants (30) had W79 in the second hairpin-l oop motif, but there was diversity for hydrophobic and basic amino acids in residue 78. Kinetic analysis of isolated scN variants identified a novel s cN isoform scN(LW) with higher papain affinity than the wild-type molecule. The variant contained an E78L substitution and had a twofold lower K-i (2. 1 pM) than parental scN, due to its increased association rate constant (2. 6 +/- 0.09 X 10(7) M(-1)sec(-1)). These results define residues in the firs t and second hairpin-loop regions which are essential for optimal interacti on between phytocystatins and papain, a prototypical cysteine proteinase. F urthermore, the isolated variants are a biochemical platform for further in tegration of mutations to optimize cystatin affinity for specific biologica l targets.