A Combined NMR and Computational Approach to Determine the RGDechi-hCit-alpha(v)beta(3) Integrin Recognition Mode in Isolated Cell Membranes

The critical role of integrins in tumor progression and metastasis has stimulated intense efforts to identify pharmacological agents that can modulate integrin function. In recent years, alpha(v)beta(3) and alpha(v)beta(5) integrin antagonists were demonstrated to be effective in blocking tumor progression. RGDechi-hCit, a chimeric peptide containing a cyclic RGD motif linked to an echistatin C-terminal fragment, is able to recognize selectively alpha(v)beta(3) integrin both in vitro and in vivo. High-resolution molecular details of the selective alpha(v)beta(3) recognition of the peptide are certainly required, nonetheless RGDechi-hCit internalization limited the use of classical in cell NMR experiments. To overcome such limitations, we used WM266 isolated cellular membranes to accomplish a detailed NMR interaction study that, combined with a computational analysis, provides significant structural insights into alpha(v)beta(3) molecular recognition by RGDechi-hCit. Remarkably, on the basis of the identified molecular determinants, we design a RGDechi-hCit mutant that is selective for alpha(v)beta(5) integrin.