Compaction and binding properties of the intrinsically disordered C-terminal domain of Henipavirus nucleoprotein as unveiled by deletion studies

Henipaviruses are recently emerged severe human pathogens within the Paramyxoviridae family. Their genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that recruits the polymerase complex via the phosphoprotein (P). We have previously shown that in Henipaviruses the N protein possesses an intrinsically disordered C-terminal domain, N-TAIL, which undergoes alpha-helical induced folding in the presence of the C-terminal domain (P-XD) of the P protein. Using computational approaches, we previously identified within N-TAIL four putative molecular recognition elements (MoREs) with different structural propensities, and proposed a structural model for the N-TAIL-P-XD complex where the MoRE encompassing residues 473-493 adopt an alpha-helical conformation at the P-XD surface. In this work, for each N-TAIL protein, we designed four deletion constructs bearing different combinations of the predicted MoREs. Following purification of the N-TAIL truncated proteins from the soluble fraction of E. coli, we characterized them in terms of their conformational, spectroscopic and binding properties. These studies provided direct experimental evidence for the structural state of the four predicted MoREs, and showed that two of them have clear alpha-helical propensities, with the one spanning residues 473-493 being strictly required for binding to P-XD. We also showed that Henipavirus N-TAIL and P-XD form heterologous complexes, indicating that the P-XD binding regions are functionally interchangeable between the two viruses. By combining spectroscopic and conformational analyses, we showed that the content in regular secondary structure is not a major determinant of protein compaction.'