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.
Department of Physical Chemistry and Institute of Biotechnology, Faculty of Sciences, University of Granada, 18071, Spain.
Eva S
ES
Cobos
M Teresa
MT
Pisabarro
M Cristina
MC
Vega
Emmanuel
E
Lacroix
Luis
L
Serrano
Javier
J
Ruiz-Sanz
Jose C
JC
Martinez
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