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A miniprotein scaffold used to assemble the polyproline II binding epitope recognized by SH3 domains.

Abstract：

: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.

journal_name

J Mol Biol

journal_title

Journal of molecular biology

authors

Cobos ES,Pisabarro MT,Vega MC,Lacroix E,Serrano L,Ruiz-Sanz J,Martinez JC

doi

10.1016/j.jmb.2004.06.078

keywords:

subject

Has Abstract

pub_date

2004-09-03 00:00:00

pages

355-65

issue

eissn

0022-2836

issn

1089-8638

pii

S0022-2836(04)00677-1

journal_volume

342

pub_type

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A miniprotein scaffold used to assemble the polyproline II binding epitope recognized by SH3 domains.