Binding of flexible and constrained ligands to the Grb2 SH2 domain: structural effects of ligand preorganization

Abstract

Structures of the Grb2 SH2 domain complexed with a series of pseudopeptides containing flexible (benzyl succinate) and constrained (aryl cyclopropanedicarboxylate) replacements of the phosphotyrosine (pY) residue in tripeptides derived from Ac-pYXN-NH(2) (where X = V, I, E and Q) were elucidated by X-ray crystallography. Complexes of flexible/constrained pairs having the same pY + 1 amino acid were analyzed in order to ascertain what structural differences might be attributed to constraining the phosphotyrosine replacement. In this context, a given structural dissimilarity between complexes was considered to be significant if it was greater than the corresponding difference in complexes coexisting within the same asymmetric unit. The backbone atoms of the domain generally adopt a similar conformation and orientation relative to the ligands in the complexes of each flexible/constrained pair, although there are some significant differences in the relative orientations of several loop regions, most notably in the BC loop that forms part of the binding pocket for the phosphate group in the tyrosine replacements. These variations are greater in the set of complexes of constrained ligands than in the set of complexes of flexible ligands. The constrained ligands make more direct polar contacts to the domain than their flexible counterparts, whereas the more flexible ligand of each pair makes more single-water-mediated contacts to the domain; there was no correlation between the total number of protein-ligand contacts and whether the phosphotyrosine replacement of the ligand was preorganized. The observed differences in hydrophobic interactions between the complexes of each flexible/constrained ligand pair were generally similar to those observed upon comparing such contacts in coexisting complexes. The average adjusted B factors of the backbone atoms of the domain and loop regions are significantly greater in the complexes of constrained ligands than in the complexes of the corresponding flexible ligands, suggesting greater thermal motion in the crystalline state in the former complexes. There was no apparent correlation between variations in crystal packing and observed structural differences or similarities in the complexes of flexible and constrained ligands, but the possibility that crystal packing might result in structural variations cannot be rigorously excluded. Overall, it appears that there are more variations in the three-dimensional structure of the protein and the ligand in complexes of the constrained ligands than in those of their more flexible counterparts.

Probing the Effect of Conformational Constraint on Phosphorylated Ligand Binding to an SH2 Domain Using Polarizable Force Field Simulations

“…Structural variations in the BC loop (GluBC1–GluBC4) were observed upon comparing the crystal structures of complexes of the Grb2 SH2 domain with constrained and unconstrained ligands, although comparable variations in the BC loops were also found in coexisting complexes in the asymmetric unit. 11,14,102 In our B-factor calculations, the fluctuations (1.5 to 2.0 Å) in the BC loops of the four complexes are the highest among all other loops. This observation suggests that the variations of the BC loop are more likely to result from the intrinsic flexibility of the loop rather than from differences in the binding modes of the constrained and unconstrained ligands.…”

Thermodynamics of binding by calmodulin correlates with target peptide α‐helical propensity

“…Thus, ligands that are predominantly in the bound conformation prior to complex formation would have a higher binding affinity than those that are not. In studies of ligands binding to the SH2 domain from Grb2, Martin and coworkers26, 27 have found that this common assumption does not always hold. Furthermore, Bachmann et al 28.…”

Design and Optimisation of Bioactive Cyclic Peptides: Generation of a Down-Regulator of TNF Secretion

“…In such cases, introduction of conformation rigidity will be advantageous, and this can often, also, increase the strength of the desired binding interaction. In a series of studies conducted in the labs of Martin and coworkers [6,7], it is shown that in general a reduction in conformational flexibility is associated with an increase in Gibbs free energy of binding to ligands, although, surprisingly, it is often the change in enthalpy which contributes most to this effect, rather than entropy, as was originally assumed. A standard approach directed towards strengthening binding interactions in this way is to employ peptides in a cyclic form, since the conformational flexibility of the peptide backbone is reduced.…”