We have crystallized and solved the structure of the N-RNA binding domain of Mokola virus P via expression of full-length P which was subsequently truncated during the crystallization process. The MOKV P structure shows a high degree of structural similarity with the homologous domain of RABV P, consistent with the high degree of sequence identity (68%). Due to the longer sequence of MOKV P compared to RABV P, the C-terminal alpha helix (α6) is six residues longer than that of RABV P. Whether the MOKV P α1 in the intact protein is longer than observed here remains to be established, as the sequence prior to residue 197 is poorly conserved between the RABV and MOKV P proteins and secondary structure prediction methods do not predict the presence of a helix even for RABV P in this region.
Analysis of the crystal packing interactions revealed that a positively charged region of P, previously shown to be involved in the interaction with N, interacts with four acidic C-terminal amino acids in a symmetry-related P molecule that may resemble part of the putative P binding region in N. The interaction is electrostatically driven and, together with the small buried surface area, suggests that the interacting regions in P and/or N are likely to be larger than can be ascertained on the basis of the MOKV P crystal structure alone, consistent with the recent structure of VSV N-RNA in complex with P indicating that the opposing face of the domain in P, involving helices α4 and α5, is involved in the interaction with N (18
). The involvement of the positively charged region in P in binding N was further confirmed by a combination of site-directed mutagenesis and yeast two-hybrid analysis to study the effects of the mutations on the interaction of MOKV P with MOKV N. The analysis revealed that out of 10 amino acids in P potentially involved in binding N, five negatively affected the P-N interaction. As previously demonstrated (23
), we confirmed that K212, K215, and perhaps Y214 are involved in P-N binding. We further show that a positive charge at positions 212 and 215 is crucial for P-N interaction. Two other positions not previously described, L225 and R261, were also shown to be necessary for the P-N interaction to occur. However, our work suggests that the role of amino acids 210, 211, and 213, recognized as potentially involved in P-N binding, is less significant (or at least they contribute less to the interaction with N), as their mutation did not alter the interaction. This further reinforces the hypothesis of potential core packing destabilization (30
) of some mutations when generated at random since, only a small number of the residues of P identified show here a clear effect on N binding in the yeast two-hybrid screen when tested in isolation. The high degree of conservation of these positions in lyssaviruses despite the high variability of this region of the P further supports their importance in the interaction with N. The observation that at all sites alanine substitutions are tolerated in the interaction with N indicates that the acidic tail-positive cluster interactions are probably weak and therefore mutations in this region may be compensated for by the presence of other interacting regions in P and/or N.
The region of N interacting with P probably involves residues in the vicinity of S389, as its phosphorylation state may be important for the interaction with P (45
) and trypsin removal of RABV N residues 376 to 450 abolishes P binding (16
). S389 is followed by four acidic residues with a high degree of similarity with the acidic C-terminal extension of MOKV P (DEED in MOKV N versus EEAE in MOKV P). However, mutation of all four acidic residues in MOKV N (D390A, E391A, E392A, and D393A) did not affect binding of N to PΔ176 in a yeast two-hybrid screen (not shown). Another acidic region in N is also highly conserved among N proteins (ELEE in MOKV N, positions 371 to 374). Here again mutations E373A and E374A did not alter P-N binding in yeast two-hybrid experiments (not shown). One or both of these sites may still interact with P, since it has recently been shown that measles virus (MeV) N has two boxes of amino acid residues that are involved in P-N binding (5
). However, MOKV N mutated at both sites (residues 373 to 374 and 390 to 393) still binds to MOKV PΔ176 (not shown). This lack of effect on binding of P by mutating individually or in combination the two acidic regions of N may reflect the fact that these regions are simply not involved in P-N binding or that the P binding region involves more than just the acidic residues mutated in this study, as suggested by the structure of the VSV N-RNA P complex (18
). Given that, in our model, much of the molecular interaction of the negatively charged tail involves main-chain atoms, it remains possible that replacement of even the entire acidic region with alanine is not sufficient to destabilize the interaction with P, as a significant number of main-chain contacts with P are likely to remain in place, as shown for VSV (18
None of the MOKV P residues identified in this study as being important for the interaction with MOKV N appear to be conserved in the VSV P structure, suggesting that VSV P interacts differently with N than the lyssavirus P. In addition, biochemical data also indicate that P-N and P-L interactions are likely to be different (41
). This has been confirmed by the structure of VSV N-RNA complexed with the C-terminal domain of VSV P, which reveals that those residues of VSV P that are involved in binding lie close to but do not map directly onto the residues that we predict are involved in MOKV N-P binding (18
). This lack of agreement is probably due primarily to differences in the sizes of the P proteins (the C-terminal domain of MOKV P is 50% larger than the equivalent C-terminal domain of VSV).
Alongside the structures of the N-RNA binding domains from P proteins of members of the Rhabdoviridae
(MOKV and RABV for the Lyssavirus
genus and VSV P for the Vesiculovirus
genus), the structures of functionally equivalent domains of the P proteins from other members of the order Mononegavirales
have been solved. The structures of the N-RNA binding domains of Sendai virus (SeV) and measles virus (MeV) P proteins consist of an antiparallel triple-helix bundle, and, as for the Rhabdoviridae
, the N-RNA binding domains among paramyxoviruses are also structurally conserved in spite of low sequence conservation (5
). Careful analysis of the structures reveals a structural relationship between the N-RNA binding domains of the P proteins of the Rhabdoviridae
and the Paramyxoviridae
, where the three-helix bundles of the SeV and MeV P proteins align with helices α3, α4, and α6 of MOKV (and RABV) (Fig. ). Structural superposition using SHP (40
) shows that 43 and 40 residues of MOKV P (out of 107) can be aligned with SeV P and MeV P, with RMSDs of 3.3 Å and 3.2 Å, respectively, over the aligned residues, despite 14% and 0% sequence identity. The similarity between VSV P and the paramyxovirus and lyssavirus P proteins is much poorer due to a shorter C-terminal helix α4 and the absence of a helix equivalent to MOKV α3 in VSV P, although the alignment suggests that at least VSV P α3 (α4 in MOKV P) is conserved. This low but detectable degree of structural similarity would suggest that the P proteins of Rhabdoviridae
originated from a common ancestor in spite of the high degree of amino acid divergence.
FIG. 4. Comparison of the structures of Rhabdovirus and Paramyxovirus N-RNA binding domains of P. (A) Superposition of the conserved elements of MOKV P (orange), VSV P (yellow) (PDB accession no. 2K47), and SeV (magenta) (PDB accession no. 1R4G). Helix numbering (more ...)
Finally, it is of interest to note that the MOKV P positively charged cluster (K212, K213, and K215) in combination with R261 was recently identified as a functional nuclear localization signal (NLS) (34
). Although the possible role of P nuclear localization remains to be established, the structures of the RABV and MOKV P proteins show that these regions do not conform to the classic NLS structure (39
), as they are not part of a disordered or flexible loop but rather are part of an ordered region that includes beta sheet β1, the (ordered) loop separating α1 and β1, and helix α4. As noted by Pasdeloup et al. (34
), the overlap with the NLS suggests that the N-RNA binding region in P may additionally act to mask the NLS upon binding to N. If verified, this would constitute another example of the modulation of the biological activities achieved by a lyssavirus protein by sequestering a biologically active protein interface during morphogenesis and/or the viral life cycle, as already postulated for the matrix protein (17