Structure of the Paramyxovirus Parainfluenza Virus 5 Nucleoprotein in Complex with an Amino-Terminal Peptide of the Phosphoprotein

Abstract

Parainfluenza virus 5 (PIV5) belongs to the family Paramyxoviridae, which consists of enveloped viruses with a nonsegmented negative-strand RNA genome encapsidated by the nucleoprotein (N). Paramyxovirus replication is regulated by the phosphoprotein (P) through protein-protein interactions with N and the RNA polymerase (L). The chaperone activity of P is essential to maintain the unassembled RNA-free form of N in order to prevent nonspecific RNA binding and premature N oligomerization. Here, we determined the crystal structure of unassembled PIV5 N in complex with a P peptide (N⁰P) derived from the N terminus of P (P50) at 2.65 Å. The PIV5 N⁰P consists of two domains: an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a hinge region. The cleft at the hinge region of RNA-bound PIV5 N was previously shown to be an RNA binding site. The N⁰P structure shows that the P peptide binds to the CTD of N and extends toward the RNA binding site to inhibit N oligomerization and, hence, RNA binding. Binding of P peptide also keeps the PIV5 N in the open form. A molecular dynamics (MD) analysis of both the open and closed forms of N shows the flexibility of the CTD and the preference of the N protein to be in an open conformation. The gradual opening of the hinge region, to release the RNA, was also observed. Together, these results advance our knowledge of the conformational swapping of N required for the highly regulated paramyxovirus replication.IMPORTANCE Paramyxovirus replication is regulated by the interaction of P with N and L proteins. Here, we report the crystal structure of unassembled parainfluenza virus 5 (PIV5) N chaperoned with P peptide. Our results provide a detailed understanding of the binding of P to N. The conformational switching of N between closed and open forms during its initial interaction with P, as well as during RNA release, was analyzed. Our data also show the plasticity of the CTD and the importance of domain movement for conformational switching. The results improve our understanding of the mechanism of interchanging N conformations for RNA replication and release.

Keywords: PIV5; conformational change; crystal structure; nucleoprotein; paramyxovirus; phosphoprotein; replication.

FIG 1

(A) Schematic representation of the different regions of PIV5 N (upper panel) and the different constructs of PIV5 N and P (lower panel) used in this study. (B) Electron microscopy (EM) of various PIV5 N-P proteins. (C) The size exclusion chromatogram of PIV5 N⁰P (N_32–372-P50) is shown. The blue and red curves indicate absorbance at 280 and 260 nm, respectively. The SDS-PAGE profile of PIV5 N⁰P is shown. Lane 1, protein molecular mass marker (kilodaltons); lanes 2 and 3, His-tagged and His-tag-cleaved purified proteins. AU, arbitrary units.

FIG 2

(A) The surface view of the PIV5 N⁰P dimer showing the two points of interaction (white arrows) within the dimer. The right panel shows the cartoon representation of the dimer. Chain A is shown in green (N) and red (P) while chain B is shown in blue (N) and yellow (P). (B) The cartoon view of the monomer shows the presence of the hinge between the NTD (dark green) and CTD (green) with P peptide (red) bound to the CTD. (C) The overlay alignment of one monomer from oligomeric PIV5 N (salmon) and unassembled N⁰P (green and red) is shown. The N-arm (purple), C-arm (marine), and RNA (black) are shown. The conformational change in the CTD is clearly visible in the structures. (D) A zoomed-in view of the dimer interface shows both interacting sites. The interface residues are shown as sticks, and the polar contacts are presented as dashed lines. The zoomed-in view of the two different points of contact is shown, and the residues contributing mostly to the dimer interaction are labeled.

FIG 3

(A) The interaction of the P peptide at the surface of N is shown. The helix in P interacts with helix α11 of N. (B) The 2F_O − F_C electron density map of the P peptide contoured at the 1σ level is shown in gray, with the P peptide residues (red) in sticks and N (green) in cartoon view. (C) The structure superposition of PIV5 N trimer (sand and salmon) and N⁰P (green) is represented in cartoon view. The N-arm (purple) and P peptide (red) are present at the same positions. (D and E) Zoomed-in view of the P binding to N in chains A and B, respectively. The P peptide in chain B completely blocks the RNA binding site, and in chain A, it reaches toward the hinge region. (F) The molecular dynamics analysis of chain A shows the movement of P peptide toward the hinge region. The P peptide in the initial structure is shown in red, and in the final simulated structure it is shown in light brown. (G) The detailed N-P interaction is shown. Red, P peptide; green, N. The interacting residues are presented in sticks. The polar contacts are shown with black dashed lines. The residues involved in either polar or hydrophobic contacts are labeled.

FIG 4

(A) The RNA binding pocket region of PIV5 N⁰P (green) is compared with that of PIV5 N (salmon). RNA is shown in black. The movement of the helix α16 loop region in the CTD of N is shown (black arrow). (B) The RNA binding pocket residues are shown as sticks in both PIV5 N and N⁰P to display the conformational changes. (C) The Q202 interaction with an RNA base is compared between the open and closed forms of PIV5 N. (D) The flexibility of Y260 is demonstrated in both chain A and chain B.

FIG 5

The N⁰P structures from different NNVs (Protein Data Bank accession numbers) are shown in yellow: PIV5 (5WKN), NiV (4CO6), hMPV (5FVD), VSV (3PMK), EBOV (4ZTI), and MeV (5E4V). The P peptide is shown in red, and the C-arm is in marine. The RMSD of PIV5 N⁰P with these different structures is marked. In MeV and EBOV N⁰P structures, the P peptide binds to the other protomer of the dimer.

FIG 6

(A) The molecular dynamics simulation was performed with one monomer of PIV5 N, and the trajectory analysis shows the conformational changes from the closed to open form. The superposition of the initial (yellow) and final (magenta) forms of PIV5 N with PIV5 N⁰P (open form) (green) is displayed. The N-arm and C-arm are highly flexible. (B) Zoomed-in view of the RNA binding site. (C) The MD analysis of the trimer from the PIV5 N ring structure (blue) is shown. The N-arms remain at their initial positions (red circles). (D to F). The zoomed-in view of the hinge region is shown for three protomers of the trimer to show the gradual conformational variations: salmon, N+1; magenta, N; green, and N−1.