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 nM 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 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 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.
CNRS, Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques (AFMB) UMR 7257, 13288 Marseille, France.
David
D
Blocquel
Johnny
J
Habchi
Stéphanie
S
Costanzo
Anthony
A
Doizy
Michael
M
Oglesbee
Sonia
S
Longhi
D002151:Calorimetry; D003461:Crystallography; D008958:Models, Molecular; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins / Q000737:chemistry* / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism*
D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; C055859:P protein, Sendai virus; D010750:Phosphoproteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C058314:nucleoprotein, Measles virus