Targeting metastable coiled-coil domains by computational design.
Approximately 30% of eukaryotic genomes are predicted to encode partially unfolded proteins. Many of these unstructured domains contact multiple partners in short-lived interactions critical for cellular homeostasis. Understanding the functional implications of these transient binding events is a current challenge that could be addressed with designed peptide inhibitors. Most current protein design methodologies, however, target only structurally well-defined, stable structures. To address this limitation, we implemented a computational design strategy that alternates between a fixed backbone sequence search for binding specificity and structural optimization of the designed interfaces. We applied this method to create specific peptide inhibitors of the C-terminal metastable coiled-coil domain of the essential yeast septin Cdc12p. Specific binding of the designed sequences was demonstrated by circular dichroism and equilibrium ultracentrifugation. Our results validate computational methods to design specific peptide ligands to protein domains lacking intrinsic structural stability and set the stage for functional analysis of Cdc12p coiled coil function in vivo.
Barth
Patrick
P
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3220, USA. barthp@u.washington.edu
Schoeffler
Allyn
A
Alber
Tom
T
D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
C099504:CDC12 protein, S cerevisiae; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D029701:Saccharomyces cerevisiae Proteins