Analysis of Influenza A Virus NS1 Dimer Interfaces in Solution by Pulse EPR Distance Measurements

[Image: see text] Pulsed electron–electron double resonance (PELDOR) is an electron paramagnetic resonance (EPR) spectroscopy technique for nanometer distance measurements between paramagnetic centers such as radicals. PELDOR has been recognized as a valuable tool to approach structural questions in biological systems. In this manuscript, we demonstrate the value of distance measurements for differentiating competing structural models on the dimerization of the effector domain (ED) of the non-structural protein 1 (NS1) of the influenza A virus. Our results show NS1 to be well amenable to nanometer distance measurements by EPR, yielding high quality data. In combination with mutants perturbing protein dimerization and in silico prediction based on crystal structures, we can exclude one of two potential dimerization interfaces. Furthermore, our results lead to a viable hypothesis of a NS1 ED:ED interface which is flexible through rotation around the vector interconnecting the two native cysteines. These results prove the high value of pulse EPR as a complementary method for structural biology.