Structure of the Dimerization Domain of the Rabies Virus Phosphoprotein

Abstract

The crystal structure of the dimerization domain of rabies virus phosphoprotein was determined. The monomer consists of two ␣-helices that make a helical hairpin held together mainly by hydrophobic interactions. The monomer has a hydrophilic and a hydrophobic face, and in the dimer two monomers pack together through their hydrophobic surfaces. This structure is very different from the dimerization domain of the vesicular stomatitis virus phosphoprotein and also from the tetramerization domain of the Sendai virus phosphoprotein, suggesting that oligomerization is conserved but not structure.Rabies virus is a negative-strand RNA virus of the Rhabdovirus family. Its genomic RNA is encapsidated by numerous copies of the viral nucleoprotein (N) that binds to the sugar phosphate backbone of the RNA with a stoichiometry of nine nucleotides per N protomer (1). RNA replication and transcription take place on this N-RNA template (2) and are catalyzed by the viral RNA-dependent RNA polymerase (L). L binds to the N-RNA template with the help of the polymerase cofactor, the phosphoprotein (P) (7). The phosphoproteins of the Rhabdoviridae and of the Paramyxoviridae are oligomers (5, 8); P of the Rhabdoviridae (rabies virus and vesicular stomatitis virus [VSV]) form dimers (6, 9), whereas P of the Paramyxoviridae (Sendai virus) form tetramers (21). These phosphoproteins are modular proteins that have an N-terminal domain that keeps newly produced N (called N 0 ) in a soluble, RNAfree form, a central oligomerization domain and a C-terminal domain that binds to N-RNA. The three domains are connected by two intrinsically disordered regions (10, 13). The atomic structures of the oligomerization domains of the phosphoproteins of VSV and Sendai virus have been determined, and it was found that these structures were quite different (6, 21). Because the secondary structure prediction of the oligomerization domain of rabies virus P was different again from that of Sendai virus and VSV (10), we decided to determine its structure.The sequence analysis of rabies virus P suggests that the oligomerization domain stretches from amino acid residues 92 to 131 (10). The DNA corresponding to residues 91 to 133 was cloned into a pET22b vector and expressed in the Escherichia coli BL21 (DE3-RIL) strain as a His tag fusion protein. The protein was purified by nickel resin and size-exclusion chromatography (S75). In solution the protein behaves as a dimer with a molecular mass of 13.9 kDa (monomer, 6,501 Da) (10). Monomers or higher-order oligomers have never been observed.The protein was crystallized at 20°C in 50 mM sodium cacodylate (pH 6.5)-10 mM MgSO 4 plus 2 M (NH 4 ) 2 SO 4 . Diffraction data on frozen crystals were obtained on beam lines ID14-4 and BM14 (ESRF, Grenoble, France). The crystals diffracted to a 1.5-Å resolution and belong to space group I4 1 22 with one dimer in the asymmetric unit. The phases were solved by the Sulfur-MAD method (Table 1). Integration and scaling of the data were performed with XDS (12). Three methionine sites ...

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

“…This has already been well illustrated in the case of the P proteins from measles (44) and Sendai viruses (83, 99 -102) as well as for the phosphoproteins from Rhabdoviridae members (103)(104)(105)(106)(107), and, more generally, for viral proteins targeted by large scale structural genomics projects (for examples see Refs. 108 -116).…”

Plasticity in Structural and Functional Interactions between the Phosphoprotein and Nucleoprotein of Measles Virus

“…The exact oligomeric state of MeV P is not known. By analogy with the closely related Sendai virus (13,14), it is thought to be tetrameric, contrary to the dimeric P proteins of rabies and vesicular stomatitis viruses (15,16). The P oligomers simultaneously bind to L (via the P multimerization domain) and to the exposed C-terminal domain of N (N TAIL ; amino acids 400 -525) via the C-terminal X domain of P (XD; amino acids 459 -507) (17)(18)(19)(20)(21)(22)(23)(24)(25)(26).…”

Structure of the C-Terminal Domain of Lettuce Necrotic Yellows Virus Phosphoprotein

“…A central domain is responsible for the dimerization of the protein, whereas a C-terminal domain binds to the N-RNA template. The structures of both domains of VSV and RAV P, as well as the C-terminal domain of Mokola virus (MOKV), were solved recently (20)(21)(22)(23)(24). Despite a lack of sequence conservation, the structures of the three C-terminal domains are similar (24,25), suggesting that they are homologous.…”

“…Despite a lack of sequence conservation, the structures of the three C-terminal domains are similar (24,25), suggesting that they are homologous. Conversely, the structures of the central domains of VSV and RAV are so different that it is impossible to conclude that they have a common ancestry (22). The N-terminal region of the protein is globally disordered, while a flexible linker connects the central and C-terminal domains, making the protein highly flexible and structurally heterogeneous.…”