CD and NMR conformational studies of a peptide encompassing the Mid Loop interface of Ship2–Sam

Abstract

The lipid phosphatase Ship2 is a protein that intervenes in several diseases such as diabetes, cancer, neurodegeneration, and atherosclerosis. It is made up of a catalytic domain and several protein docking modules such as a C-terminal Sam (Sterile alpha motif) domain. The Sam domain of Ship2 (Ship2-Sam) binds to the Sam domains of the EphA2 receptor (EphA2-Sam) and the PI3K effector protein Arap3 (Arap3-Sam). These heterotypic Sam-Sam interactions occur through formation of dimers presenting the canonical "Mid Loop/End Helix" binding mode. The central region of Ship2-Sam, spanning the C-terminal end of α2, the α3 and α4 helices together with the α2α3 and α3α4 interhelical loops, forms the Mid Loop surface that is needed to bind partners Sam domains. A peptide encompassing most of the Ship2-Sam Mid Loop interface (Shiptide) capable of binding to both EphA2-Sam and Arap3-Sam, was previously identified. Here we investigated the conformational features of this peptide, through solution CD and NMR studies in different conditions. These studies reveal that the peptide is highly flexible in aqueous buffer, while it adopts a helical conformation in presence of 2,2,2-trifluoroethanol. The discovered structural insights and in particular the identification of a helical motif, may lead to the design of more constrained and possibly cell permeable Shiptide analogs that could work as efficient antagonists of Ship2-Sam heterotypic interactions and embrace therapeutic applications.

The Sam-Sam interaction between Ship2 and the EphA2 receptor: design and analysis of peptide inhibitors

“…TFE can reveal the intrinsic tendency of an amino-acid sequence to assume a specific secondary structure that may characterize its conformation when interacting with membranes or different peptides/proteins 17 , 18 . Our recent work on several fragments from different Sam domains (Ship2 19 and the adaptor protein Odin 20 ) further shows, in agreement with earliest studies 21 , that native-like helices are well reproduced in solution containing TFE. However, propagation of helical content to the more disordered protein regions may occur at high TFE concentration 20 .…”

Exploring the Ability of Cyclic Peptides to Target SAM Domains: A Computational and Experimental Study

“…CD : CD spectra were recorded on a Jasco J‐810 spectropolarimeter (JASCO Corp, Milan, Italy), as previously reported –. Spectra were obtained by averaging three scans, subtracting blanks, and converting the signal to mean residue ellipticity [deg cm 2 dmol −1 res −1 ].…”

Hunting for Novel Routes in Anticancer Drug Discovery: Peptides against Sam-Sam Interactions

“…Considering that inhibition of heterotypic Sam-Sam interactions involving EphA2 could be a promising route in cancer therapy, during the last few years, we centered our research activities on the design of peptides able to hamper these interactions. To achieve our goal and obtain such peptides we implemented a variety of approaches: (1) we set-up protein dissection strategies involving all Sam domains under study (i.e., EphA2-Sam, Ship2-Sam and Odin-Sam1) [ 187 , 188 , 189 , 190 ], (2) we designed helical peptides, both linear and stapled, to mimic native-like structural motives in Sam domains [ 191 , 192 ], (3) we implemented structure-based computational approaches to generate virtual peptide libraries to be screened in silico against target Sam domains [ 92 , 193 ]. All designed peptides were evaluated through multidisciplinary studies involving diverse computational and experimental techniques such as NMR and CD (Circular Dichroism) for conformational studies, molecular modelling (docking), NMR, ITC, SPR and MST (Microscale Thermophoresis) for binding analyses, and in vitro cell-based assays for evaluation of biological activities.…”

“…As already mentioned in Section 4.1.1 EphA2-Sam interacts with Ship2-Sam following the ML/EH structural topology in which the ML interface is represented by the central regions of Ship2-Sam ( Figure 6 c–e). The Shiptide is a 22 residue long peptide whose sequence ( Table 2 ) encompasses residues from E43 to L64 of Ship2-Sam (residue numbering according to PDB entry 2K4P [ 68 ]) thus comprising most of its ML site ( Figure 8 a) [ 68 , 69 , 187 ]. First, to gain insights into the Shiptide tendency to adopt a native-like fold, when extracted from the whole 3D protein organization, CD and NMR studies were conducted in 10 mM sodium phosphate buffer at pH 7.2.…”

“…Next, as it is very challenging to collect transfer NOE data and determine a bound peptide conformation while working with small proteins like Sam-domains [ 194 ], the co-solvent 2-2-2-trifluoroethanol (TFE) was employed to investigate the ability of the peptide to assume precise secondary structure elements that could mimic bioactive conformations [ 195 ]. A solution structure of the Shiptide was calculated in a H 2 O/TFE mixture containing 70% TFE because, as indicated by CD studies, that amount of co-solvent should allow the peptide to assume the highest structuration level [ 187 ]. The Shiptide NMR structure in TFE is characterized by an α-helix encompassing most of peptide sequence except for the more disordered stretch at the N-terminus ( Figure 8 a).…”