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

Abstract

The intrinsically disordered C-terminal domain (NTAIL) of the measles virus (MeV) nucleoprotein undergoes α-helical folding upon binding to the C-terminal X domain (XD) of the phosphoprotein. The NTAIL 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 KD for the NTAIL-XD binding reaction in the nM range and also showed that an additional NTAIL 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 μM 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 NTAIL-XD binding. Since our previous studies relied on NTAIL-truncated forms possessing an irrelevant Flag sequence appended at their C-terminus, we, herein, generated an NTAIL 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 NTAIL 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. © 2012 The Protein Society.