Interaction between the C‐terminal domains of measles virus nucleoprotein and phosphoprotein: A tight complex implying one binding site |
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Authors: | Stéphanie Costanzo Anthony Doizy Michael Oglesbee Sonia Longhi |
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Institution: | 1. CNRS, Aix‐Marseille Université, Architecture et Fonction des Macromolécules Biologiques (AFMB) UMR 7257, 13288 Marseille, France;2. Department of Veterinary Biosciences, Ohio State University, Columbus, Ohio 43210 |
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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 (KD 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. |
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Keywords: | isothermal titration calorimetry intrinsically disordered proteins folding coupled to binding entropic penalty entropic stabilization paramyxoviruses nucleoprotein phosphoprotein |
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