期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 102, 期 6, 页码 1889-1894出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0408890102
关键词
de novo protein design; helical bundle; IL-4 receptor; structure-based design
An IL-4 antagonist was designed based on structural and biochemical analysis of unbound IL-4 and IL-4 in complex with its high-affinity receptor (IL-4Ralpha). Our design strategy sought to capture a protein-protein interaction targeting the high affinity that IL-4 has for IL-4Ra. This strategy has impact due to the potential relevance of IL-4Ralpha as a drug target in the treatment of asthma. To mimic the IL-4 binding surface, critical side chains for receptor binding were identified, and these side chains were transplanted onto a previously characterized, de novo-designed four-helix protein called designed helical protein 1 (DHP-1). This first-generation design resolved the ambiguity previously described for the connectivity between helices in DHP-1 and resulted in a protein capable of binding to IL-4Ralpha. The second-generation antagonist was based upon further molecular modeling, and it succeeded in binding IL-4Ralpha better than the first-generation. This protein, termed DHP-14-AB, yielded a protein with a cooperative unfolding transition (DeltaG(u)(0) = 8.1 kcal/mol) and an IC50 of 27 muM when in competition with IL-4 whereas DHP-1 had no affinity for IL-4Ralpha. The crystal structure of DHP-14-AB was determined to 1.9-Angstrom resolution and was compared with IL-4. This comparison revealed how design strategies targeting protein-protein interactions require high-resolution 3D data and the incorporation of orientation-specific information at the level of side-chains and secondary structure element interactions.
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