Calorimetric and Structural Studies of 1,2,3-Trisubstituted Cyclopropanes as Conformationally Constrained Peptide Inhibitors of Src SH2 Domain Binding

Abstract

Isothermal titration calorimetry and X-ray crystallography have been used to determine the structural and thermodynamic consequences associated with constraining the pTyr residue of the pYEEI ligand for the Src Homology 2 domain of the Src kinase (Src SH2 domain). The conformationally constrained peptide mimics that were used are cyclopropane-derived isosteres whereby a cyclopropane ring substitutes to the N-Calpha-Cbeta atoms of the phosphotyrosine. Comparison of the thermodynamic data for the binding of the conformationally constrained peptide mimics relative to their equivalent flexible analogues as well as a native tetrapeptide revealed an entropic advantage of 5-9 cal mol(-1) K(-1) for the binding of the conformationally constrained ligands. However, an unexpected drop in enthalpy for the binding of the conformationally constrained ligands relative to their flexible analogues was also observed. To evaluate whether these differences reflected conformational variations in peptide binding modes, we have determined the crystal structure of a complex of the Src SH2 domain bound to one of the conformationally constrained peptide mimics. Comparison of this new structure with that of the Src SH2 domain bound to a natural 11-mer peptide (Waksman et al. Cell 1993, 72, 779-790) revealed only very small differences. Hence, cyclopropane-derived peptides are excellent mimics of the bound state of their flexible analogues. However, a rigorous analysis of the structures and of the surface areas at the binding interface, and subsequent computational derivation of the energetic binding parameters, failed to predict the observed differences between the binding thermodynamics of the rigidified and flexible ligands, suggesting that the drop in enthalpy observed with the conformationally constrained peptide mimic arises from sources other than changes in buried surface areas, though the exact origin of the differences remains unclear.

Preorganization in biological systems: Are conformational constraints worth the energy?

“…The thermodynamic parameters (∆G, ∆H, ∆S) for forming complexes of 12 and 13 with the Src SH2 domain were determined by isothermal titration microcalorimetry (ITC) [11]. Consistent with our expectations, an entropic advantage of ~9 eu/mol arose from preorganizing 13.…”

“…In a preliminary attempt to elucidate the origin of the observed enthalpic penalty in this series of test ligands, the structure of the complex of 12 with the Src SH2 domain was determined [11], and this structure was compared with that of an 11-mer peptide derivative bound to the Src SH2 domain [13]. The pYEEI segment of both ligands bound in similar extended conformations, and the interatomic distances between the domain and the ligands in each of the two structures were in close agreement.…”

Phosphate Recognition in Structural Biology

“…Figure 12 shows the X-ray crystal structure of the complex of the Src homology domain of Src kinase and an isostere of an O-phosphorylated YEEI tetrapeptide (PDB code: 1IS0). [67] To probe the topography of the binding pocket, a cyclopropane residue has been introduced to stiffen the backbone of the peptide chain. The ligand binds on the surface of the protein and only the O-phosphotyrosine fragment can reach into a shallow cavity formed by the N terminus of an a helix and a loop connecting two b strands.…”

Ligand Preorganization May Be Accompanied by Entropic Penalties in Protein–Ligand Interactions

“…[17] In one study, constrained and flexible derivatives of the tetrapeptide Ac-pTyr-Glu-Glu-Ile-OH, the consensus sequence in Src SH2 domain binding ligands, [18,19] were prepared that contained substituted cyclopropane and succinyl replacements for the phosphotyrosine residue. [20,21] The thermodynamic parameters for complex formation of these ligands with the Src SH2 domain were determined by isothermal titration calorimetry (ITC). Although the constrained ligands bound with more favorable entropies of binding than their flexible counterparts, this expected entropic advantage was always offset by a corresponding enthalpic penalty that resulted in comparable binding affinities of the various ligand pairs.…”