Replication of non-segmented negative-strand RNA viruses requires the continuous supply of the nucleoprotein (N) in the form of a complex with the phosphoprotein (P). Here, we present the structural characterization of a soluble, heterodimeric complex between a variant of vesicular stomatitis virus N lacking its 21 N-terminal residues (N_Δ21) and a peptide of 60 amino acids (P₆₀) encompassing the molecular recognition element (MoRE) of P that binds RNA-free N (N⁰). The complex crystallized in a decameric circular form, which was solved at 3.0 Å resolution, reveals how the MoRE folds upon binding to N and competes with RNA binding and N polymerization. Small-angle X-ray scattering experiment and NMR spectroscopy on the soluble complex confirms the binding of the MoRE and indicates that its flanking regions remain flexible in the complex. The structure of this complex also suggests a mechanism for the initiation of viral RNA synthesis. The negative sense RNA genome of the rhabdoviruses is encapsidated by the nucleoprotein, and the replication of the genome requires a continuous supply of RNA-free, monomeric nucleoprotein (N⁰) to encapsidate the newly synthesized (+)RNA intermediate antigenomes and (−)RNA genomes. In this process, the viral phosphoprotein acts as a chaperone, forming a heterodimeric complex, named N⁰-P, which prevents nascent N molecules from self-assembling and from binding to cellular RNAs. We reconstructed the N⁰-P complex of the prototype rhabdovirus, vesicular stomatitis virus, and characterized its structure by crystal X-ray diffraction and solution experiments. Our results show how the N-terminal region of the phosphoprotein folds upon binding to the RNA-free nucleoprotein and how it prevents the non-specific encapsidation of host-cell RNA. This complex is soluble and heterodimeric, but by forcing it to polymerize into a crystal it associated into a circular decamer of heterodimers very similar to the previously crystallized decameric N-RNA ring. On the basis of our results, we propose a model that explains the role of the phosphoprotein in the encapsidation of newly synthesized RNA and in the initiation of RNA synthesis by the viral polymerase.

Interaction of the C-terminal domains of Sendai virus (SeV) P and N proteins is crucial for RNA synthesis by correctly positioning the polymerase complex (L+P) onto the nucleocapsid (N/RNA). To better understand this mechanism within the paramyxovirus family, we have studied the complex formed by the SeV C-terminal domains of P (PX) and N (N _TAIL ) proteins by solution nuclear magnetic resonance spectroscopy. We have characterized SeV N _TAIL , which belongs to the class of intrinsically disordered proteins, and precisely defined the binding regions within this latter domain and within PX. SeV N _TAIL binds with residues 472 to 493, which have a helical propensity (residues 477 to 491) to the surface created by helices α2 and α3 of PX with a 1:1 stoichiometry, as was also found for measles virus (MV). The binding interface is dominated by charged residues, and the dissociation constant was determined to be 57 ± 18 μM under conditions of the experiment (i.e., in 0.5 M NaCl). We have also shown that the extreme C terminus of SeV N _TAIL does not interact with PX, which is in contrast to MV, where a second binding site was identified. In addition, the interaction surfaces of the MV proteins are hydrophobic and a stronger binding constant was found. This gives a good illustration of how selection pressure allowed the C-terminal domains of N and P proteins to evolve concomitantly within this family of viruses in order to lead to protein complexes having the same three-dimensional fold, and thus the same function, but with completely different binding interfaces.

The endophytic Gram-negative bacterium Gluconacetobacter diazotrophicus SRT4 secretes a constitutively expressed levansucrase (LsdA, EC 2.4.1.10), which converts sucrose into fructooligosaccharides and levan. The enzyme is included in GH (glycoside hydrolase) family 68 of the sequence-based classification of glycosidases. The three-dimensional structure of LsdA has been determined by X-ray crystallography at a resolution of 2.5 Å (1 Å=0.1 nm). The structure was solved by molecular replacement using the homologous Bacillus subtilis (Bs) levansucrase (Protein Data Bank accession code 1OYG) as a search model. LsdA displays a five-bladed β-propeller architecture, where the catalytic residues that are responsible for sucrose hydrolysis are perfectly superimposable with the equivalent residues of the Bs homologue. The comparison of both structures, the mutagenesis data and the analysis of GH68 family multiple sequences alignment show a strong conservation of the sucrose hydrolytic machinery among levansucrases and also a structural equivalence of the Bs levansucrase Ca²⁺-binding site to the LsdA Cys³³⁹–Cys³⁹⁵ disulphide bridge, suggesting similar fold-stabilizing roles. Despite the strong conservation of the sucrose-recognition site observed in LsdA, Bs levansucrase and GH32 family Thermotoga maritima invertase, structural differences appear around residues involved in the transfructosylation reaction.