functional mutation of multiple solvent-exposed loops in the ecballium elaterium trypsin inhibitor-ii cystine knot miniprotein功能性突变的多重solvent-exposed回路ecballium西洋苦瓜汁胰蛋白酶inhibitor-ii miniprotein胱氨酸结.pdfVIP

Functional Mutation of Multiple Solvent-Exposed Loops in the Ecballium elaterium Trypsin Inhibitor-II Cystine Knot Miniprotein 1 2 1 1 1 2 Richard H. Kimura , Douglas S. Jones , Lei Jiang , Zheng Miao , Zhen Cheng *, Jennifer R. Cochran * 1 Department of Radiology, Molecular Imaging Program at Stanford (MIPS), Cancer Center, Bio-X Program, Stanford University, Stanford, California, United States of America, 2 Department of Bioengineering, Cancer Center, Bio-X Program, Stanford University, Stanford, California, United States of America Abstract Background: The Ecballium elaterium trypsin inhibitor (EETI-II), a 28-amino acid member of the knottin family of peptides, contains three interwoven disulfide bonds that form multiple solvent-exposed loops. Previously, the trypsin binding loop of EETI-II has been engineered to confer binding to several alternative molecular targets. Here, EETI-II was further explored as a molecular scaffold for polypeptide engineering by evaluating the ability to mutate two of its structurally adjacent loops. Methodology/Principal Findings: Yeast surface display was used to engineer an EETI-II mutant containing two separate integrin binding epitopes. The resulting knottin peptide was comprised of 38 amino acids, and contained 11- and 10- residue loops compared to wild-type EETI-II, which naturally contains 6- and 5-residue loops, respectively. This knottin peptide bound to a b and a b integrins with affinities in the low nanomolar range, but bound weakly to the related v 3 v 5 integrins a b and a b . In addition, the engineered knottin peptide inhibited tumor cell adhesion to vitronec