A miniprotein scaffold used to assemble the polyproline II binding epitope recognized by SH3 domains.
SH3 domains are molecular-recognition modules that function by interacting with proteins containing sequences in polyproline II (PPII) conformation. The main limitation in designing short-ligand peptides to interact with these domains is the preservation of this helical arrangement, for which a high content of proline is needed. We have overcome this limitation by using a protein scaffold provided by the avian pancreatic polypeptide (APP), a natural hormone of 36 amino acid residues. The APP protein contains a PPII stretch packed against an alpha-helix. We have designed a structure in which some residues of the APP PPII helix are replaced by a sequence motif, named RP1, which interacts with the SH3 domain of the Abelson tyrosine kinase (Abl-SH3). This design, which we call APP-RP1, is folded and, as shown by circular dichroism, has a structural content similar to that of natural APP (APP-WT). The stability of both miniproteins has been compared by unfolding experiments; the designed APP-RP1 is almost 20 deg. C more stable than the wild-type and has a higher Gibbs energy function. This increase in stability has an entropic origin. Isothermal titration calorimetry and fluorescence spectroscopy show that the thermodynamics of the binding of the APP-RP1 molecule to Abl-SH3 is comparable to that of the shorter RP1 peptide. Furthermore, the mutation by Tyr of two proline residues in APP-RP1, which are essential for the binding of some linear peptides to Abl-SH3, demonstrates the effectiveness of the scaffold in enhancing the variability in the design of high-affinity and high-specificity ligands for any SH3 domain. The application of this strategy may help in the design of ligands for other polyproline-recognition domains such as WW, PX or EVH1, and even for the in vivo application of these miniproteins.
Cobos
Eva S
ES
Department of Physical Chemistry and Institute of Biotechnology, Faculty of Sciences, University of Granada, 18071, Spain.
Pisabarro
M Teresa
MT
Vega
M Cristina
MC
Lacroix
Emmanuel
E
Serrano
Luis
L
Ruiz-Sanz
Javier
J
Martinez
Jose C
JC
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D000939:Epitopes; D016313:Genes, abl; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010191:Pancreatic Polypeptide / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011392:Proline / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D013816:Thermodynamics; D018909:src Homology Domains
D000939:Epitopes; C009725:pancreatic polypeptide, avian; D010191:Pancreatic Polypeptide; D011392:Proline