Enantiomeric Binding Elements Interacting at the Same Site of an SH3 Protein Receptor

Sensitivity to chirality in protein-ligand interactions is of particular interest in molecular recognition and enzyme catalysis. That enzymes catalyze reactions involving only one enantiomer of a chiral substrate is commonly explained by the inherent chirality of proteins.1-3 However, an apparent lack of stereospecificity has also been observed in biochemical studies of protein-ligand binding.4,5 For example, calmodulin is able to bind two enantiomeric peptides with comparable affinity.5 The structural details of how a protein receptor can recognize ligands having mirror image relationship are scarce. Two ligands relevant to this issue that bind SH3 protein domains were recently discovered using split-pool synthesis and an affinity selection assay.6 Although they contain key SH3binding elements with a mirror image relationship that bind to the same pocket in the protein, these ligands bind the Src SH3 domain with similar affinity. Multidimensional NMR has now been used to uncover the structural basis for this result. A comparison of the structures of two synthetic ligands, NL2 and NL2R (Figure 1), complexed to enantiomerically pure Src SH3 reveals that a subsite on the binding surface maintains key intermolecular contacts with the mirror image elements. The results illustrate a mechanism through which a protein receptor interacts with binding elements having opposite chirality. Similar topographical surfaces of the chiral ligands coupled with a hydrophobic binding site lacking directional hydrogen bonds or charge-charge interactions appear to be important features of a receptor-ligand system having minimal sensitivity to chirality. The NMR structure of the Src SH3-NL2 (Ac-Mn18-Mn1PLPPLP-NH2; P ) Pro, L ) Leu) complex has been reported.7 Since Mn18 is a chiral monomer, its enantiomer Mn20, which was not used in the original library synthesis, was synthesized8,9 in order to investigate the effect of inverting the stereochemistry on binding. The corresponding diastereomeric compound AcMn20-Mn1-PLPPLP-NH2 (NL2R) was found to bind the Src SH3 domain with affinity comparable to that of NL2 (NL2, Kd ) 11 μM; NL2R, Kd ) 5.4 μM; Figure 1).7 Both Mn18 and Mn20 are crucial for high affinity binding to the Src SH3 domain as evidenced by the weak affinities of the two truncation ligands. The ligand Ac-Mn1-PLPPLP-NH2 has a Kd of 220 μM to Src SH3, and the Kd for Ac-PLPPLP-NH2 is larger than 1 mM (the binding was too weak to be measured accurately using fluorescence perturbation6). The structure of the SH3-NL2R complex10 was determined using multidimensional NMR and compared to that of the SH3-NL2 complex. Although the unbound forms of NL2 and NL2R have highly similar 1D NMR spectra (Figure 2A), the two SH3-bound ligands have very different 2D 13C-filtered TOCSY spectra for the respective aromatic protons on Mn18 and Mn20 (the TOCSY spectra were acquired using samples consisting of a 1:1 ratio of the uniformly 13C-labeled SH3 protein and unlabeled ligand) (Figure 2B). The binding site serves as a chiral shift reagent, dispersing otherwise degenerate resonances. The spectra indicate that racemization did not occur during synthesis. The structures reveal how the same receptor binds two mirror image elements. Upon complexation, the common peptidic PLPPLP fragments of the two ligands adopt essentially the same polyproline type II (PPII) helix conformation as expected from studies of other Src SH3 ligands (Figure 3A).7,11-13 Mn1 serves as a bridging element linking the PPII helix to the “monomer” residing in the pocket between the n-Src and RT loops. NL2 and NL2R differ from each other by the chirality of the C10 stereocenters of Mn18 and Mn20 (Figure 1). In the two structures, opposite faces of the tetrahydroisoquinoline group pack against Thr96 and Thr98 in the RT loop as a result of the opposite stereochemistry, but the bound conformations of the two enantiomeric moieties are remarkably similar (Figure 3B). NL2 and NL2R can be interconverted mentally by disconnecting the N11-C10 and C15-C10 bonds in one monomer, flipping the disubstituted phenyl ring by 180°, and rejoining the bonds to form the enantiomeric monomer. Flipping of the tetrahydroisoquinoline rings is accompanied by a 180° rotation of the thiazolyl groups along the C8-C10 bond, thereby preserving the N9-C8-C10-N11 dihedral angle in the two complexes. For