Phage display selection of hairpin loop soyacystatin variants that mediate high affinity inhibition of a cysteine proteinase.
Two hairpin-loop domains in cystatin family proteinase inhibitors form an interface surface region that slots into the active site cleft of papain-like cysteine proteinases, and determine binding affinity. The slot region surface architecture of the soybean cysteine proteinase inhibitor (soyacystatin N, scN) was engineered using techniques of in vitro molecular evolution to define residues that facilitate interaction with the proteinase cleft and modulate inhibitor affinity and function. Combinatorial phage display libraries of scN variants that contain mutations in the essential motifs of the first (QVVAG) and second (EW) hairpin-loop regions were constructed. Approximately 1010-1011 phages expressing recombinant scN proteins were subjected to biopanning selection based on binding affinity to immobilized papain. The QVVAG motif in the first hairpin loop was invariant in all functional scN proteins. All selected variants (30) had W79 in the second hairpin-loop motif, but there was diversity for hydrophobic and basic amino acids in residue 78. Kinetic analysis of isolated scN variants identified a novel scN isoform scN(LW) with higher papain affinity than the wild-type molecule. The variant contained an E78L substitution and had a twofold lower Ki (2.1 pM) than parental scN, due to its increased association rate constant (2.6 +/- 0.09 x 107 M-1sec-1). These results define residues in the first and second hairpin-loop regions which are essential for optimal interaction between phytocystatins and papain, a prototypical cysteine proteinase. Furthermore, the isolated variants are a biochemical platform for further integration of mutations to optimize cystatin affinity for specific biological targets.
Koiwa
H
H
Center for Plant Environmental Stress Physiology, Purdue University, West Lafayette, IN 47907-1165, USA.
D'Urzo
M P
MP
Assfalg-Machleidt
I
I
Zhu-Salzman
K
K
Shade
R E
RE
An
H
H
Murdock
L L
LL
Machleidt
W
W
Bressan
R A
RA
Hasegawa
P M
PM
D001483:Base Sequence; D015891:Cystatins / Q000235:genetics / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000235:genetics / Q000378:metabolism*; D019020:Directed Molecular Evolution; D014644:Genetic Variation; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010206:Papain / Q000037:antagonists & inhibitors*; D019151:Peptide Library; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D030262:Soybean Proteins
D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D019151:Peptide Library; D011994:Recombinant Proteins; D030262:Soybean Proteins; C414109:scN protein, soybean; D010206:Papain