Biophysical studies with AICD-47 reveal unique binding behavior characteristic of an unfolded domain Samir Das a , Saptaparni Ghosh b , Dipak Dasgupta b , Udayaditya Sen c,⇑ , Debashis Mukhopadhyay a,⇑ a Structural Genomics Division, Saha Institute of Nuclear Physics, 1/AF Bidhan Nagar, Kolkata 700 064, WB, India b Biophysics Division, Saha Institute of Nuclear Physics, 1/AF Bidhan Nagar, Kolkata 700 064, WB, India c Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF Bidhan Nagar, Kolkata 700 064, WB, India article info Article history: Received 4 July 2012 Available online 22 July 2012 Keywords: Alzheimer’s disease Intrinsically unstructured protein Conformational switch AICD-47 Fluorinated alcohol abstract APP intracellular C-terminal domain (AICD-47), generated upon c-secretase cleavage of Amyloid precur- sor’s protein (APP), bears the signature of a classical intrinsically unstructured domain (IUD). Comparing the recent crystal structures of AICD-47 peptides bound to its different adaptors such as protein-tyrosine- binding domain-2 (PTB2) of Fe65 and Src homology 2 (SH2) domain of growth factor receptor binding protein 2 (Grb2), the ‘‘conformational switching’’ of AICD-47 becomes evident. In order to understand dif- ferent binding processes undertaken by this flexible molecule, upon recognizing different interfaces resulting in different 3D conformations, spectroscopic and calorimetric studies have been done. CD spec- troscopy has revealed an overall random coil like structure in different pHs while TFE (2 0 -2 0 -2 0 -trifluoro ethanol) and HFIP (Hexa fluoro isopropanol) induced a-helicity to a certain extent. Binding of Tyr phos- phorylated AICD-47 ( P AICD-47) to Grb2-SH2 domain was carried out by a favorable enthalpic change (DH= 197.5 ± 6.2 kcal mole 1 at 25 °C) and an unfavorable entropic contribution (DS= 631 cal mol 1 deg 1 at 25 °C). Alternative conformation of AICD-47 in different biological contexts is another remarkable feature of IUDs which presumably has definitive roles in regulating Alzheimer’s disease phenotype. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction Though the classical paradigm of ‘‘sequence–structure–func- tion’’ is valid for most of the proteins, ‘‘intrinsically unstructured domains’’ (IUDs) are exceptions, being devoid of any compact globular folds and being capable of innumerable functional possi- bilities [1]. IUDs possess a large net charge at neutral pH with low abundance of hydrophobic amino acids [2]. The structural ran- domness or ‘entropy’ decreases dramatically upon binding to spe- cific partners where coupled folding and binding allows burial of an exposed surface area even with small interacting domains and generate complexes with high specificity and relatively low affinity, a feature of these proteins [3–5]. Amyloid precursor’s protein (APP), a type-I transmembrane protein, with a large extracellular amino-terminal domain and a shorter carboxy-terminal cytosolic tail, is involved in Alzheimer’s disease (AD) pathogenesis. Comparing the crystal structures of the Carboxy terminal tail (AICD-47 or APP 649–695, as of APP- 695 isoform numbering) peptides bound to its different adaptors like Fe65-PTB2 and Grb2-SH2 domains, a ‘‘conformational switch- ing’’ of AICD-47 is reckoned [6,7]. Both in acidic and alkaline pH, AICD-47 does not possess any tertiary contacts, typical of an IUD, and shows somewhat folded stable conformation with only hydro- phobic side chain clusters. The domain is significantly enriched in Glu, Lys, His and other charged amino acids and depleted of hydro- phobic or order promoting residues like Trp and Cys. Moreover, the AICD-47 fragment possesses several short eukaryotic linear motifs (ELM), responsible for associating with different binding partners. These transitory conformers are stabilized and rearranged upon binding [8]. The available NMR conformations do point out the inherent flexibility of the molecule, although prove to be inade- quate in terms of explaining the molten-globule formation and the underlying thermodynamics [8]. The crystal structures of Grb2-SH2 domain bound peptides have revealed the consensus recognition motif as ‘pY-X-N-X’ (where ‘X’ is any amino acid) where the protein–peptide interactions are stabilized by a network of hydrogen bonds mediated by conserved Arg, Ser, Thr with PTR and backbone atoms of peptide. With the exception of AICD-47, where Pro is present at pY + 3 position, all other peptides bound to Grb2-SH2 domain possess Val at the equivalent position [6]. 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbrc.2012.07.067 Abbreviations: APP, amyloid precursor’s protein; AICD, APP-intracellular C- terminal domain; AD, Alzheimer’s disease; SH2, Src homology 2; PTR and pY, phosphorylated tyrosine; IUD, intrinsically unstructured domain; Grb2, growth factor receptor binding protein 2, TFE, 2 0 -2 0 -2 0 -trifluoro ethanol; HFIP, hexa fluoro isopropanol. ⇑ Corresponding authors. Fax: +91 33 2337 4637. E-mail addresses: udayaditya.sen@saha.ac.in (U. Sen), debashis.mukhopad hyay@saha.ac.in (D. Mukhopadhyay). Biochemical and Biophysical Research Communications 425 (2012) 201–206 Contents lists available at SciVerse ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc