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

Abstract

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 (N TAIL) 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 N TAIL variants, we showed that the 490–500 region of N TAIL 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 N TAIL 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 N TAIL-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 N TAIL, in particular the unfolding of the α-helical region when the temperature was increased from 281 K to 310 K.