Interaction between the C‐terminal domains of measles virus nucleoprotein and phosphoprotein: A tight complex implying one binding site

The intrinsically disordered C‐terminal domain (N TAIL ) of the measles virus (MeV) nucleoprotein undergoes α‐helical folding upon binding to the C‐terminal X domain (XD) of the phosphoprotein. The N TAIL region involved in binding coupled to folding has been mapped to a conserved region (Box2) encompassing residues 489–506. In the previous studies published in this journal, we obtained experimental evidence supporting a K D for the N TAIL –XD binding reaction in the n M range and also showed that an additional N TAIL region (Box3, aa 517–525) plays a role in binding to XD. In striking contrast with these data, studies published in this journal by Kingston and coworkers pointed out a much less stable complex ( K D in the μ M range) and supported lack of involvement of Box3 in complex formation. The objective of this study was to critically re‐evaluate the role of Box3 in N TAIL –XD binding. Since our previous studies relied on N TAIL ‐truncated forms possessing an irrelevant Flag sequence appended at their C‐terminus, we, herein, generated an N TAIL devoid of Box3 and any additional C‐terminal residues, as well as a form encompassing only residues 482–525. We then used isothermal titration calorimetry to characterize the binding reactions between XD and these N TAIL forms. Results effectively argue for the presence of a single XD‐binding site located within Box2, in agreement with the results by Kingston et al. , while providing clear experimental support for a high‐affinity complex. Altogether, the present data provide mechanistic insights into the replicative machinery of MeV and clarify a hitherto highly debated point.