Structural investigations of a GYF domain covalently linked to a proline-rich peptide.

Protein structure determination of low affinity complexes of interacting macromolecules is often hampered by a lack of observable NOEs between the binding partners. Covalent linkage offers a way to shift the equilibrium of the interaction partners to the bound state. Here we show that a single-chain protein containing the GYF domain of CD2BP2 and the target peptide SHRPPPPGHRV from CD2 allows for the intramolecular association of the binding partners. We obtained NOEs between the GYF domain and the peptide that could define the principal orientation of the peptide in the complex. In conjunction with general recognition rules for proline-rich sequence recognition these NOEs allowed the accurate modeling of the protein-peptide complex.