CTD forms a domain-swapped dimer
The CTD in all the three crystal forms exists as an intimate domain swapped dimer
(Figure 2) formed by monomers related by non-crystallographic symmetry in the
asymmetric unit of these crystals. Although the structure of the dimer remains invariant,
the number of dimers in the crystallographic asymmetric unit varies from one to eight
between the crystal forms. The domain swapping in the CTD dimers is brought about by
exchange of beta strands from one monomer to the other. 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 CTD can be described as a concave floor of ~400Å2 area consisting of
anttiparaaleell beta sheet area with the topology β1B-β2B-β2A-β1A 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
Å2 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 Å2. 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:ABn+1) belong to the same class as seen in the pH 4.5 crystal form and bury an
almost similar surface area of 1122 Å2 .
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 Å2
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 inFigure 3b ). When the three
dimers (Dn+1-D-Dn-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 ABn+1) bury only a surface area of 600 Å2
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 Å2)as the primary crystalpacking (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 1-23 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 SARSN 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.