Structural Disorder within Henipavirus Nucleoprotein and Phosphoprotein: From Predictions to Experimental Assessment

Abstract

Henipaviruses are newly emerged viruses within the Paramyxoviridae family. Their negative-strand RNA genome is packaged by the nucleoprotein (N) within α-helical nucleocapsid that recruits the polymerase complex made of the L protein and the phosphoprotein (P). To date structural data on Henipaviruses are scarce, and their N and P proteins have never been characterized so far. Using both computational and experimental approaches we herein show that Henipaviruses N and P proteins possess large intrinsically disordered regions. By combining several disorder prediction methods, we show that the N-terminal domain of P (PNT) and the C-terminal domain of N (NTAIL) are both mostly disordered, although they contain short order-prone segments. We then report the cloning, the bacterial expression, purification and characterization of Henipavirus PNT and NTAIL domains. By combining gel filtration, dynamic light scattering, circular dichroism and nuclear magnetic resonance, we show that both NTAIL and PNT belong to the premolten globule sub-family within the class of intrinsically disordered proteins. This study is the first reported experimental characterization of Henipavirus P and N proteins. The evidence that their respective N-terminal and C-terminal domains are highly disordered under native conditions is expected to be invaluable for future structural studies by helping to delineate N and P protein domains amenable to crystallization. In addition, following previous hints establishing a relationship between structural disorder and protein interactivity, the present results suggest that Henipavirus PNT and NTAIL domains could be involved in manifold protein-protein interactions.

Characterization of the Interactions between the Nucleoprotein and the Phosphoprotein of Henipavirus

“…The Henipavirus pDest14/N TAILHN constructs encoding residues 400 -532 of N with a hexahistidine tag fused to their N termini have been described (14). The NiV and HeV N TAIL F527W constructs, encoding N-terminally hexahistidine tagged N TAIL bearing a F527W substitution, were obtained by PCR using pDest14/N TAILHN (encoding for either NiV or HeV N TAIL, respectively) as template, Turbo-Pfu polymerase (Stratagene), and a pair of complementary mutagenic primers of 39 nucleotides in length (Operon).…”

“…Overall, the proteins of henipaviruses share the same features as cognate proteins in the Paramyxovirinae subfamily. The Henipavirus P protein however is significantly larger, with a larger P N-terminal domain accounting for the extra length (14).…”

“…Indeed high-resolution structural data are limited to their surface proteins, where crystallographic studies led to the determination of the three-dimensional structure of Henipavirus fusion (F) and attachment (G) proteins (15)(16)(17)(18). As for the N and P proteins, the only available data come from studies carried out by Chan et al (12) and from our recently published studies (14). Although Chan et al (12) reported the bacterial expression of Henipavirus N and P proteins and performed interaction studies by protein-blotting protein-overlay assays that led to the mapping of the reciprocal N-P binding sites, those studies did not embrace any biochemical characterization of the proteins.…”

“…Although Chan et al (12) reported the bacterial expression of Henipavirus N and P proteins and performed interaction studies by protein-blotting protein-overlay assays that led to the mapping of the reciprocal N-P binding sites, those studies did not embrace any biochemical characterization of the proteins. On the other hand, in our previous studies, by combining computational and experimental approaches, we deciphered the modular organization of Henipavirus N and P proteins and showed that the C-terminal region of N (N TAIL ; amino acids 400 -532) and the N-terminal region of P (amino acids 1-404/406) belong to the family of intrinsically disordered proteins (IDPs), although they both contain short, orderprone segments (14). The occurrence of some residual transient secondary and/or tertiary structure within the N TAIL and the N-terminal disordered domain of P led to their classification within the premolten globules (PMG) subfamily within the family of IDPs (19 -24).…”

“…IDPs recognize their partner(s) through molecular recognition elements (MoREs); these are short, order-prone regions within intrinsically disordered regions with a propensity to undergo induced folding upon binding to partners (40 -43). Using bioinformatics approaches, we have previously identified, within both the HeV and NiV N TAIL domains, four putative MoREs, with at least two of them (amino acids 408 -422 and 473-493) exhibiting a clear ␣-helical nature (14). By analogy with the closely related measles virus (MeV) (44 -55), we have previously speculated that the C-terminal X domain of the Henipavirus P protein (P XD ) may constitute a possible partner of Henipavirus N TAIL , with the MoREs of this latter being involved in partner recognition (14).…”