Conformational Analysis of the Partially Disordered Measles Virus NTAIL-XD Complex by SDSL EPR Spectroscopy

To characterize the structure of dynamic protein systems, such as partly disordered protein complexes, we propose a novel approach that relies on a combination of site-directed spin-labeled electron paramagnetic resonance spectroscopy and modeling of local rotation conformational spaces. We applied this approach to the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (NTAIL) both free and in complex with the X domain (XD, aa 459–507) of the viral phosphoprotein. By comparing measured and modeled temperature-dependent restrictions of the side-chain conformational spaces of 12 SL cysteine-substituted NTAIL variants, we showed that the 490–500 region of NTAIL is prestructured in the absence of the partner, and were able to quantitatively estimate, for the first time to our knowledge, the extent of the α-helical sampling of the free form. In addition, we showed that the 505–525 region of NTAIL conserves a significant degree of freedom even in the bound form. The latter two findings provide a mechanistic explanation for the reported rather high affinity of the NTAIL-XD binding reaction. Due to the nanosecond timescale of X-band EPR spectroscopy, we were also able to monitor the disordering in the 488–525 region of NTAIL, in particular the unfolding of the α-helical region when the temperature was increased from 281 K to 310 K.