NTD dimer mediates linear array formation
The dimer in the ASU of the present structure is formed by the interactions between the protruding basic arms of the U shaped monomer with the acidic base of the other U shaped monomer. These two monomers are rotated with respect to each other by about 90 degrees. This dimeric interaction has a buried surface area of 2168 A2 thereby indicating a strong interaction between the dimers. In the crystal the dimers from the neighboring unit cell related by translation interact with each other using the same inter-monomeric interactions as seen in the dimer to form a linear array (Figure 1d and 1e). This linear array has patches of basic residues owing to the conserved basic residues sequences from the internal arm (Figure 1e). In contrast the previous structure of NTD by Hui et. al. also consisted of a dimer in the asymmetric unit in which the “U” shaped monomers interact with each other using the bases of the globular core such that the arms of the U shaped monomers now face away from each other (Figure 1 b).Such a dimeric interaction therefore does not result in a linear array formation in that spacegroup.
The dramatic difference in packing by the NTD dimers in these two crystal forms possibly results from presence of magnesium ion and differences in the pH between them.
Packing of NTD dimers:
The swiveling asymmetry of A-B dimer formation and the head to tail interaction within and between A-B dimers results in a linear array of NTD monomers mediated primarily by the N-terminal loop from residues B22 to B29 ( figure 1c ) extending to the neighboring ASU and interacting with the A monomer therein (Figure 1c left black arrow).

The other internal arm is alternately ordered and disordered and participates in A interacting with B (cyan arrow) or is disordered in the B molecules . This arm in the B molecule is free to possibly participate in interactions with RNA (Figure 1c right dotted line). These basic arms therefore constitute the oligomerization and RNA binding regions of the NTD. The differences in pH of this high resolution structure and the one previously reported points to possible oliomerization and conformational behaviors mediated by these loop. The pH dependent dynamics of these basic loops may play an important role in nucleocapsid assembly and dis-assembly and the RNA binding behavior observed for the N-terminal domain.
CTD forms a domain-swapped dimer
The CTD in all three crystal forms exists as an intimate domain swapped dimer ( Figure
2 ). The domain swapping is brought about by interaction between β-strands of one monomer with surrounding helices and loops from the other monomer to form a reciprocated, closed domain swapped dimer akin to that seen in crystal structures of cystatin A and RNAseA (Janowski, Kozak et al. 2001; Newcomer 2001). Accordingly a
12 residue long β-strand β2 (295 and 307) constitutes the interface between the two monomers ( Figure 2 bottom ). The overall topology of the dimer of IBV-N can be said to be a concave β-stranded floor of ~400Å 2
area with the topology β 1Bβ 2Bβ 2Aβ 1A surrounded by helices and loops. The helices

3 and

4 connected by loop region arch over this floor and constitute the roof of the dimer. A 12 residue long α-helix α5 located at the extreme C-terminus of CTD forms an angled wall that flanks either side of the dimer and is held in place by a tight turn made up residues 307 to 310 (purple boxed residues Figure 2 and Figure 5 ).

The integrity of the dimer observed in solution is apparent when one considers the ~5000
Å 2 buried surface area involved in the dimerization. The dimeric structure observed at pH 4.5 is almost identical in all four dimers observed at pH 8.5 in the asymmetric unit and the dimer observed in the ASU for crystal form III with the rmsd. for Cα-atoms in the core region (233 to 328) being ~0.3 Å. This observation is in concordance with several biochemical studies which mapped the dimerization domain to this stretch in several homologs (Surjit, Liu et al. 2004; Yu, Gustafson et al. 2005). pH-dependent crystal packing interactions in CTD. The presence of one dimer in the
ASU in two crystal forms (CTD1 and CTD3) and 4 dimers in the ASU in one other crystal form (CTD2) allowed the analysis of dimer-dimer interactions not only at different pHs and crystallization conditions but also in the presence and absence of any constraints imposed by crystal packing.
The three dimeric structures presented here result in five kinds of inter-dimer interactions. Crystal packing in crystal I (CTD1) is brought about by dimer-dimer interactions with the nth dimer interacting with n-1 dimer and n+1 dimer from neighboring ASU ( Figure 3b
) burying a surface area of 1182 Å 2
. In crystal II with 4 dimers in the ASU, inter-dimer interactions are responsible for keeping the four dimers in the ASU together as well as mediating crystal packing ( Figure 4a ). Accordingly this gives rise to four classes of dimer-dimer interactions. Two of them (termed class I dimerdimer interactions) i.e AB-CD, CD-EF and the crystal packing interaction wherein GH dimer from one ASU interacts with the AB dimer from the neighboring ASU (i.e
GH:AB n+1
) belong to the same class as seen in the pH 4.5 crystal form and bury an almost similar surface area of 1122 Å 2
.

This high pH crystal form CTD2 also displays a new class of “dimer-dimer” interactions and it involves the interaction between the GH dimer with an interface formed by the
CD-EF dimer ( Figure 4a ). This tri-dimeric interaction buries a surface area of 1385 Å
2
The uniformity of all but the last kind of dimer-dimer interactions observed in two crystals is apparent from a superposition of all four types of dimer-dimer interactions observed between the two crystals whereby the dimers all superpose with a minimum of
0.3 Å rmsd and a maximum of 0.8 Å rmsd (yellow dimer in Figure 3b ). When the three dimers (D n+1
-D-D n-1
) from three neighboring ASUs from crystal I are superposed from the three dimers from within the ASU of crystal II the rmsd between them is ~1.0 Å.
These interactions primarily involve residues between 308 and 328 which constitute a type II turn ( TT in Figure 5 ) and

5 and the terminal loop in CTD ( Figure 1 lilac boxes ). Apart from the class I dimer interactions the crystal packing interactions in crystal II (dimer GH interacting with dimer AB n+1
) bury only a surface area of 600 Å 2 and are brought about by a swiveling away of the GH dimer prompted possibly by its strong interaction with CD-EF dimers from within the ASU. This clearly indicates that the dimers tend to swivel only slightly w.r.t each other and constitute a subtle module that is well suited to interacting with itself ( arrow figure 3b middle panel ).
Although there is not significant surface complementarily between the two molecules the predominant interaction between dimers is a salt bridge between Arg-308 from one dimer and Asp-314 from a neighboring dimer ( Figure 3 b ). The salt bridge and the orientation of the dimers remain almost identical between the structures at pH 4.5 and pH 8.5. The inter dimer interactions other than for the salt bridge are strictly Vanderwaal interactions.
The multimerization interaction in addition to the dimerization interactions seen in CTD

very well maintained over this wide range of pHs. The ionic strength of the two crystal conditions is also different thereby providing further evidence as to the stability of dimerdimer packing interactions. Interestingly the structure of crystal form II of the CTD which is of the IBV-Beaudette strain has a cysteine residue instead of the Arg 308 in this position. Although no inter-dimer disulfide bond is seen in the structure interaction of cysteine residues across this dimeric interface may facilitate disulfide bond formation that replaces the salt bridge in this strain of IBV-N protein.
Discussion: (good place to start discussion).
One of the primary of functions of the N protein in coronaviruses is in the formation of the nucleocapsid through its interactions with the genomic RNA. From the biochemical characterization of the IBV N protein presented here and from similar characterization of the N protein from other coronoviruses it is apparent that this protein has two major protease-resistant domains. Our X-ray crystallographic structural characterization of these two domains provides some insights into how the two domain organization of the N protein may facilitate nucleocapsid assembly.
NTD as a an RNA binding module:
Several biochemical studies have shown that determinants for RNA binding reside in the
N-terminal region with the minimal region being mapped to residues 136 to 190 in IBV
(by analogy with residues 177 to 231 in MHV). Electrophoretic mobility shift assays on the analogous construct for IBV-Beaudette strain by Hui. Et al clearly showed that out
NTD construct binds RNA. A similar region in IBV-N was also shown to specifically interact with the 3’ termini of IBV RNA(Zhou, Williams et al. 1996).

The electrostatic surface of the NTD crystal, as calculated using GRASP, clearly shows a predominantly basic exposed patches and the head to tail linear array formed by NTD dimers interlocking with each other. The loosely packed NTD fiber with exposed conserved basic residues therefore allows for interactions with RNA. The alternating disordered internal arms in B monomers may possible become ordered in the presence of
RNA which might interact with the flexible NTD linear array.
The looseness of this dimer might be in order to allow for interactions with RNA during nucleocapsid formation in addition to compensating for the relative rigidity of the CTD dimer as described below . Unlike the NTD dimer the CTD dimeric surface is predomiantnly acidic with a basic swath that runs in a helical fashion aroung the fibre axis. The CTD shows no Nterminal binding in vitro and therefore possibly serves predominantly as a dimerization and helical array forming module with the NTD specializing in conforming to the helical template provided for by the CTD in addition to mediating interactions with
RNA. Such a two domain organization is similar to the nucleocapsid protein of HIV where one of the domains is involved in dimerization with the other domain is free to interact with other capsid components(Gamble, Yoo et al. 1997)
Possible role of inter-dimeric CTD interactions in nucleocapsid assembly and disassembly : The additional dimer (GH) is clearly auxiliary (and not part of the primary fibre see below) and reveals a higher mode of interaction with CTD dimers. The interacting surface comprises residues from all over the dimers ( underlined residues
Figure 1, T2 dimer interface figure 3a ). Since this interaction involves three different molecules and yet the buried surface area is similar (~1200 Å 2
)as the primary crystal-

packing (or fiber forming interaction), we hypothesize that it is less likely and therefore secondary to the primary interaction seen for other dimers. Considering this dimer mediates crystal packing in this spacegroup by the same region on its other face, the tight salt bridge observed between R308 and D315 is preserved in only one of the cases and disrupted in the two fold related case. Despite this skewing the overall rmsd is only 0.8 Å indicating the extreme adaptability of the dimer with α5 and preceding loop mediating these interactions.
This additional interaction also leads to the possibility that the fibre-hexamer made up of dimer 1-2-3 with a dimer 4 appendage could circularize or form planar triangles under certain conditions with the GH dimer serving as a bridge to bring the otherwise rigid 1-2-
3 fibres together. Such bridging interactions may indeed be necessary for spherical particle formation driven by triangularization of three hexamers with the fourth dimer serving as the linker.
In addition the greater flexibility of various regions of the protein at alkaline pH as obtained from their large B factors coupled with the swiveling seen by dimer 4-dimer 1 crystal packing interaction in crystal II could represent a snapshot into the dis-assembly of dimer-dimer interactions considering how this may be important for nucleocapsid disaasembly and genome release.
The clear tendency of the dimer-dimer interaction to promote fibre formation is evident from superposition of three dimers from both spacegroups (Figure 4c). The relevance of this interaction is greater when one considers that it occurs as discussed above at both pHs and also occurs free of crystal packing induced forces at the alkaline pHs. The dimer induced fibre formation is even more striking when one puts it in context of the

relatedness of the protein to another capsid forming domain N protein from a related virus.
Plausible model for structural organization of the coronavirus genome. The NTD with its demonstrated RNA binding activity ( Hui et al ) and the clearly dimeric CTD are two highly adaptable modules on an otherwise largely flexible and possible disordered protein (Wang, Wu et al. 2004).The two basic tethers in the NTD possible are held alongside a C-term mediated fibre with the tethers grabbing onto and sequestering RNA.
This NTD_CTD_RNA superstructure possible then packs via secondary interactions made possible by both RNA interacting with CTD and the CTD-CTD class II dimerdimer interactions and also possibly the NTD-NTD dimeric interactions to form a highly compacted ribonucleoprotein complex. The C-terminus of N-protein is also known to interact with the CTD of M-protein which is predominantly basic. This may be possible by interactions of M with the acidic patches on the CTD mediated fibre. These interactions possible explain the rescue of unstable M-protein mutants by compensatory mutations in the CTD region of MHV-N (Kuo and Masters 2002)) indicating a strong interaction mediated in part by this region. Together This suggests a model for genome organization wherein the CTD domains form a helical template with extending NTD
RNA-grabbers that organize the genomic RNA that is brought along for the ride by interactions of consensus packaging signal with M protein which nucleates along the
CTD fibre by interacting with it. The CTD-fiber thus serves as a structural template for the NTD-RNA complex to wind around with intermittent interactions between M and
CTD. Once assembled this complex is not prone to disruption by treatment with RNAse
A as observed by Narayannan et al (Narayanan, Kim et al. 2003)

Similarity to other coronaviral nucleocapsid proteins and evolutionary implications for viral architechture. A DALI search of the PDB revealed a very striking similarity to the 73 amino acid capsid forming domain of PRRSV a corona like virus which is a member of the nidovirales family. This match had a high similarity Z-score with a corresponding RMS deviation of 2.8 Å .PRRSV a corona like virus is also a + single stranded RNA-virus with a similarly large genome. PRRSV also forms a helical nucleocapsid and the full length N-protein was shown to form fibers in solution for the full length protein(Doan and Dokland 2003). Similar helical nucleocapsids have been observed in orthomyxovirus, paramyxovirus, flivovirus, rhabdovirus , bunyavirus and arenavirus families all of which contain genomic RNA associated with their respective nucleocapsid proteins(Narayanan, Kim et al. 2003).
The capsid forming domain in PRRSV also packed into helical arrays using crystal contacts in the crystal studied. The arrangements of CTD, PRRSV and MS2 coat protein all show a similar feature of an anti-parallel beta strand floor with flanking helixes and loops. The major difference between the two structures lie in the fact that the CTD floor is more concave while the PRRSV floor is perfectly flat. Besides this the number of surrounding loops and helical regions are greater for CTD considering that it is almost
120 residues long compared to the 90 residue length of PRRSV-capsid forming domain.
This fact taken together with the interaction seen in the PRRSV crystal packing interaction similarly mediated by helix helix Vanderwaal stacking and a similar saltbridge between Arg 65 and Asp43 in PRRSV suggests a common theme in helical fibre formation across the viruses in the Nidovirales family to which PRRSV and IBV both

belong. This strengthens the suggestion that this fold is commonly employed in viruses with helical nucleocapsids.
Similarity with SARS N protein. Despite the very low sequence homology between
IBV-N and SARS-N and (25%) the predicted secondary structure of SARS-N for the
CTD domain matches the observed secondary structure of IBV-N very closely ( Figure 5 black topology diagram top ). The NMR structure for the N-terminal domain for SARS-
N clearly shows that The N–terminal domain is largely composed of coiled structure and interacts with RNA in solution (Huang, Yu et al. 2004). A similar solution structure by
NMR of a part of the dimerization domain of SARS coronavirus reveals a similarity to the PRRSV capsid protein as reported in this publication but differ from this study in the arrangement of the C-terminal helix which is the key mediator of dimer-dimer – interactions which we hypothesize are the determinants of helical nuclocapsid formation(Chang, Sue et al. 2005). The helix corresponding to helix α5 in the SARS structure packs against the helix from the same dimer forming an asymmetric homodimer(Chang, Sue et al. 2005). The IBV and PRRSV dimer are very symmetric homodimers and have the same helix mediating dimer-dimer interactions which are thee main determinant of strand formation. It is quite likely that the absence of the residues Nterminal to the β-stranded floor might have easily allowed the C-terminal helix in SARS to move dramatically and interact with itself within the dimer. This interaction might not be possible in the context of the whole protein and the nucleopcapsid as seen in the structure of IBV-N protein and PRRSV N-protein.
Concluding pargraph

In summary, (expand a bit) The overall structural similarity between PRRSV and CTD here clearly indicates that these viruses within the Nidovirales order are more similar than previously thought and hints at this architecture being a characteristic fold adopted by helical nucleocapsid viruses.