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BIOLOGICAL SCIENCES
X-ray structure of the N and C-terminal domain of a coronavirus nucleocapsid
protein; structural basis of helical nucleocapsid formation
Hariharan Jayaram, Hui Fan&, Brian R. Bowman, Amy Ooi& ,Jyothi Jayaram, Ellen W.
Collison, Lescar Julian, B.V.Venkataram Prasad
Verna and Marrs McLean Department of Biochemistry and Molecular Biology; Baylor
College of Medicine; Houston, Texas, 77030; U.S.A , Department of Veterinary
Pathobiology; Texas A&M University; College Station, Texas ,77843;U.S.A; School of
Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore
637551
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Abstract (250 words allowed ..page 2-Current 202):
Coronaviruses cause a variety of respiratory and enteric diseases in animals and
humans including SARS, a disease with emerging global impact. Enveloped capsids of
these viruses enclose single stranded RNA genome that is associated tightly with a
nucleocapsid protein (N protein), a highly immunogenic protein that is implicated in
genome packaging, replication, and apoptosis in certain virus infected cells. Using
limited proteolysis we identified two stable domains of the nucleocapsid protein from
infectious bronchitis virus (IBV), a member of the Coronaviridae family. We present
here the crystal structures of the N and C-terminal domains (NTD & CTD) of IBV- N
protein determined to 1.3 Å and 2.2 Å resolution respectively. The NTD protein with
basic residues concentrated on two long tethers in the protein constitutes an RNA
interacting module. The CTD exist as intimate domain swapped dimers that tend to
organize into helical arrays. Inferring from crystal packing interactions observed at
different pHs for the NTD and CTD we hypothesize that the CTD is the key
determinant of helical nucleocapsid formation in the virus. Similarity between CTD
and the capsid forming domain of another related virus suggests that this fold
constitutes a new class of viral capsid folds that are employed in viruses with helical
nucleocapsids. The coronavirus nucleocapsid is thus made up of an N-terminal RNA
binding core connected to a C-terminal capsid forming domain that together organize
the helical nucleocapsid in the virus.
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Coronaviridae, a member of the order Nidovirales, is a family of viruses which are
significant causative agents of human upper respiratory infections including common
colds and other severe illnesses such as SARS (severe acute respiratory syndrome). The
coronaviruses are enveloped viruses with a diameter ranging from 80 to 160 nm . The
viral genome consists of a single 30 kb long segment of positive sense single-stranded
RNA (Siddell 1995). Upon infection the genomic RNA encodes a 3’ co-terminal set of
four or more subgenomic mRNAs with a common leader sequence at their 5’-ends. These
subgenomic RNA encode various viral structural and non structural proteins required to
replicate the virus and produce progeny virions. The enveloped capsid of the virus is
predominantly made up of the membrane glycoprotein (M) and another small
transmembrane protein (E) and an array of spikes composed of the spike protein
glycoprotein (S) which gives the roughly spherical particles a corona. A significant
protein component of the capsid is the nucleocapsid protein (N), which interacts with the
genomic RNA forming the central core of the virion.
Electron microscopic studies of detergent permeabilized transmissible gastroenteritis
virus capsids (TGEV a prototype coronavirus) revealed that the internal nucleocapsid is
helical and is composed of the ssRNA genome tightly associated with N (nucleocapsid)
protein (Risco, Anton et al. 1996; Risco, Muntion et al. 1998). The N protein is typically
a multifunctional basic phosphoprotein of molecular weight 50-60 kDa, which is
synthesized in large amounts along with its coding RNA during infection (Stohlman and
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Lai 1979; Lai and Cavanagh 1997). The highly basic N protein exhibits non-specific
ssRNA binding ability with an increased affinity for genomic RNA (Cologna and Hogue
1998) and bind consensus sequences at 5’ and 3’ termini of the genome. Biochemical
studies on some of the coronaviruses such as mouse hepatitis virus (MHV),infectious
bronchitis virus (IBV), and SARS virus have mapped the RNA binding function to a 55
residue segment in the N-terminal half of the N-protein and the dimerization function to a
the C-terminal half. (Nelson, Stohlman et al. 2000; Hui Fan 2005; Yu, Gustafson et al.
2005).
During the virus life-cycle multiple copies of the N protein interacts extensively with the
genomic as well as the subgenomic RNA that are synthesized (Baric, Nelson et al. 1988;
Narayanan, Kim et al. 2003) and possibly participates in genome packaging which is
initiated by recognition of a packaging signal by the M-protein. The M and N protein also
interact closely via their C termini, an interaction which is important for genome
encapsidation and nucleocapsid formation (Kuo and Masters 2002). In addition, N
protein is also shown to play a role in controlling mRNA transcription, translation and
replication (Lai and Cavanagh 1997; Tahara, Dietlin et al. 1998; Schelle, Karl et al.
2005).
The abundance of N protein produced during an infection results in this protein playing
an important role in host modulation. Accordingly the N protein has been shown to
interact with cycophilin, an immuno-modulator, activate the AP1 pathway involved in
cell cycle control, enter the nucleus as well as induce apoptosis in certain cell types (He,
Leeson et al. 2003; Luo, Luo et al. 2004; Surjit, Liu et al. 2004). The N protein is also a
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major immunogen and an important diagnostic marker for coronavirus disease (Leung,
Tam et al. 2004) and is shown to help improve the efficacy of avian coronavirus vaccines
(Cavanagh 2003; Zhao, Cao et al. 2005).
We present in this paper, structural analysis of the N-protein of infectious bronchitis virus
(IBV), a member of the Coronaviridae family. The recombinant N protein of
coronavirus is highly susceptible to proteolysis making the structural analysis of the full
length protein difficult. To date, there is only limited structural information on the
coronavirus N protein which includes an NMR structural analysis of the N-terminal
domain of SARS nucleocapsid protein (Huang, Yu et al. 2004)and our previously
published crystallographic studies on the N-terminal domain of the IBV nucleocapsid
protein at 2.8 A resolution (Hui Fan 2005). As yet there is no X-ray crystallographic
structure of the C-terminal domain of the coronavirus N protein. In the present study,
using limited proteolysis we have been able to identify two stable domains of the N
protein which represent N and C-terminal domains respectively (NTD and CTD) and
determine their crystal structures. Each of these domains crystallized in multiple crystal
forms allowing us to study their packing interactions and gain structural insights into
nucleocapsid formation. With one of the crystal forms of the NTD, we have been able to
determine the structure of the NTD to 1.3 Å resolution, significantly higher resolution
than our previous studies, and the structure of CTD, which represents the first crystal
structure in this region of the coronavirus N protein, to 2.2 Å resolution.
Materials and Methods
Purification of full length nucleocapsid protein and limited proteolysis: Full length
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nucleocapsid protein was expressed as before (Zhou, Williams et al. 1996). The protein
was further purified by heparin affinity chromatography, concentrated to 1-2 mg/ml and
was checked for monodispersity by dynamic light scattering (Dynapro) and negative stain
electron microscopy. Limited proteolytic cleavage of full length N protein (1-2 mg/ml)
was carried out with 2% (wt trypsin /wt protein) sequencing grade trypsin (Roche) to
identify tryptically stable domains. The identity of the amino termini of the proteolytic
product(s) was ascertained by N-terminal amino acid sequencing of band following gelelectrophoresis and blotting onto a polyvinylidene fluoride membrane (PVDFImmobilon-PSQ, Millipore). For construct optimization the carboxy termini were
estimated based on predicted secondary structure in terminal region and mass
spectrometric characterization of proteolyzed protein.
Cloning, expression, purification and crystallization of the tryptic fragments of N
protein: All proteins were cloned and expressed as GST fusion proteins using the pet41
Ek-LIC vector (Novagen). The expressed protein was purified using glutathione S
sepharose (Pharmacia) columns followed by on-bead cleavage with enterokinase (EKMax, Invitrogen). The cleavage reaction was performed by suspending 1 ml of beads in
40 ml of cutting buffer (250 mM NaCl, 50 mM Tris-HCl pH 8.0) with 10 units of
protease. Following proteolysis, the dilute supernatant was purified further by gel
filtration chromatography on a Superdex 75 16/60 column (Pharmacia). The purified Nand C-terminal domains were concentrated to 5-8 mg/ml and used for crystallization.
Data Collection and phasing: Data were collected at various synchrotron beam lines as
indicated in Table I (supplementary material). For each crystal, the diffraction data were
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collected with 1 degree oscillation angle and integrated and scaled using HKL2000
(reference). For the NTD, the diffraction data to 1.3 A were phased using molecular
replacement procedures in PHASER (Storoni, McCoy et al. 2004) with the previously
published NTD structure at 2.8 A resolution (Hui Fan et. al.). Following molecular
replacement, further model building and refinement was performed in a similar manner to
the CTD as described below.
The CTD crystallized in three crystal forms (Table I , supplementary material). The
structure of the CTD was determined from selenomthionne substituted protein (pH 4.5
Crystal from Table I) to 2.0 A resolution using MAD (multi ewav wefgweg) datasets
collected at two different wavelengths (Se-peak , 0.9734; Se-inflection, 0.9748).
Positions of the four selenium atoms were located using the SnB program (Weeks and
Miller 1999). The initial solution with an figure of merit of 0.65 was then refined and a
electron density map was then calculated following density procedure using SHARP
(Bricogne 1997). An initial model was built using ARP/WARP (Lamzin 2001) followed
by manual model building using COOT (Emsley and Cowtan 2004). Refinement was
performed using a combination of CNS (Brunger, Adams et al. 1998) which was used in
the initial rounds of simulated annealing refinement followed by refinement using
REFMAC5 (Pannu, Murshudov et al. 1998). The structure of CTD in the two other
crystal forms were phased using molecular replacement procedures implemented in
PHASER. Model bias in both NTD and CTD structures was reduced by using the prime
and switch methodology implemented in SOLVE/RESOLVE (Terwilliger and Berendzen
1999). The stereochemistry of the structures was checked by PROCHECK(Roman A
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Laskowski 1993) during the course of model building and refinement. All figures were
generated using Pymol (DeLano 2002) and Espript (Gouet, Courcelle et al. 1999).
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Results and Discussion:
Limited proteolysis yields two stable independent domains. Our structural
characterization of purified full length N protein was impeded by its aggregation and
degradation on storage under a variety of conditions. The full length N protein was also
extremely polydisperse in solution as characterized by dynamic light scattering analysis
and not amenable to X-ray crystallographic analysis.
Using limited proteolysis we sought to identify regions of the protein that represented
stable domains that were resistant to proteolysis under limiting amounts of trypsin and
V8 protease. The digestion pattern with V8 protease was not very distinct and yielded
several diffuse bands . However with trypsin the full length protein was cleaved to a
“single”, stable ~17 kD band within 15 minutes of trypsinization. N-terminal sequencing
identified this band to be composed of four tryptic fragments with two major cleavage
sites at residues19 and 219 and two secondary cleavage sites at residues 27 and 226
(Figure 1a).
The optimized domain constructs termed NTD (residues 19 -162) and CTD (residues
219-349) were then cloned, expressed and purified to homogeneity. The NTD was
monomeric at moderate concentrations, whereas the CTD was a dimer even at very low
concentrations, as assayed by gel-filtration chromatography. The NTD and CTD proteins
tended to aggregate during purification and thus were purified at very low concentrations
and concentrated only prior to crystallization screening. The NTD and CTD proteins
failed to interact at a variety of salt and protein concentrations as assayed by gel-filtration
co-fractionation and pull down experiments (data not shown). NTD and CTD therefore
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represent independent non-interacting domains of the full length protein and were
suitable for X-ray crystallographic analysis separately.
NTD and CTD crystallized in multiple crystal forms: In contrast to the recently
reported structure of NTD which corresponded to the Beaudette strain and crystallized in
the P1 spacegroup, this structural analysis employs the IBV-Gray strain which
crystallized in a different spacegroup and diffracted to 1.3 A resolution. The CTD also
crystallized in different forms as needles, rods, flat sheets or hexagonal crystals under
different conditions(Table I crystal forms I II and III). Rod shaped CTD1 crystals of SeMet substituted protein that diffracted to 2.0 A in the P212121 spacegroup was used for
structure determination. Structure of CTD in two other crystal forms (P21212 at 2.2 A
resolution and P43 at 2.6 A resolution) were determined by molecular replacement.
In all we have here determined the structure of NTD in two space groups and CTD in
three spacegroups in this study. The different packing arrangements in these crystal forms
reveal multiple modes of self-interaction for these domains of the nucleocapsid protein
and help suggest a plausible model for nucleocapsid organization in coronaviruses.
High resolution structure of NTD
The NTD in this study crystallized as an asymmetric dimer of two interlocking
monomers arranged in a head to tail fashion in the crystallographic asymmetric unit
(Figure 1a).The secondary structure and fold of the NTD (IBV-Gray strain) is almost
identical to the structure of the NTD-IBV N-protein Beaudette strain reported previously
(Hui Fan 2005),with the exception of five additional residues discernible at the N1
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terminus in the present structure. Briefly, the structure is composed of a relatively
acidic globular core of twisted anti-parallel β-sheet that is surrounded by a number of
loop regions. Prominent among the loop regions are two long loops corresponding to the
N-terminal 12 amino acids (residues 22 to 34) and a loop region from residues 74 to 86
that constitutes an internal arm. These loops extend outward like long tethers from the
globular core resulting in a U shaped monomer (cyan and black arrow, Figure 1b).
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 (molecule A) with the acidic base of the
other U shaped monomer (molecule B, figure 1B). These two monomers are rotated with
respect to each other by about 90 degrees. The main difference between these two
molecules related by non crystallographic symmetry is that in molecule B one of the arms
of “U” (internal arm) is disordered. The dimeric interaction has a buried surface area of
2168 Å2 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
only the N-terminal loop to form a linear array (Figure 1d and 1e). This linear array has
patches of basic residues from the internal arm (Figure 1e). These basic residues are
conserved across the Coronaviridae. In contrast to the dimers seen in the present
structure the previous structure of NTD by Hui et. al. also consisted of a dimer in the
asymmetric unit wherein the “U” shaped monomers interacted with each other using the
bases of the globular core such that the arms of the U shaped monomers faced away
from each other (Figure 1 b).Such a dimeric interaction therefore does not result in a
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linear array formation in this spacegroup.
The dramatic difference in packing by the NTD dimers in these two crystal forms
possibly results from differences in ionic strength and pH between the two crystallization
conditions.
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. The domain swapping in the CTD dimers is brought
about by exchange of beta strands from one monomer to the other. The overall topology
of the CTD dimer can be described as a concave floor of ~400Å2 area consisting of an
anti-parallel beta sheet (β1B-β2B-β2A-β1A ) surrounded by helices and loops (Figure 2
B). The helices 3 and 4 connected by a 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
forms an angled wall that flanks either side of the dimer and is held in place by a tight
turn made by residues 307 to 310 (Figure 2).The integrity of the dimer observed in the
crystal with a buried surface area of ~5000 Å2 is consistent with the observation that
CTD is a dimer in solution and several biochemical studies which map the dimerization
domain of the full length protein to the C-terminal domain.
Packing interactions in CTD crystals. Although the structure of the dimer remains
invariant, their molecular packing is considerably different in the three crystal form we
have studied. The presence of one dimer in the ASU in two crystal forms (CTD1 and
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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. Such an
analysis of these inter-dimeric interactions is of relevance, considering the primary role
of N-protein in nucleocapsid formation.
In two of the crystal forms CTD1 (at pH 4.5) and CTD2 (at pH 8.5) the dimers form a
linear array similar to that seen in the NTD. This “fibre” forming dimer-dimer
interaction (designated as Type I) takes place between dimers from neighboring unit cells
in CTD1 and between three non-crystallographic symmetry related dimers in CTD2
(figures 3a and 3b). Despite the differences in crystal forms and pH the inter-dimeric
interaction is well preserved between the two crystal forms with an overall rmsd of 1.0 Å
between the “fibres” and a similar buried surface area of ~1100 Å2. This “fibre” forming
interaction is mediated by the extreme C-terminal residues between 308 and 328 which
constitute a type II turn (TT in Figure 5) and the terminal α-helix 5 (Figure 5 lilac
boxes). The two dimers are held together by a network of water-bridged polar
interactions and a salt bridge between residues Arg 308 and Asp 314 (Figure 3b, right).
Alternate modes of dimer-dimer interactions in CTD
The fourth dimer in the asymmetric unit in the CTD2 structure (molecule4, Figure 3a)
interacts via a series of predominantly vanderwaal contacts with two neighboring dimers
from the “fibre” burying a surface area of 1385 Å2.
A third crystal form CTD3 in the P43 spacegroup for the IBV-Beaudette strain was also
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obtained with one dimer in the ASU. Like the CTD2 crystal form, CTD3 also at pH
8.5, revealed two additional modes of dimer-dimer interactions mediated by salt bridges
on opposite ends of each dimer with dimers from the neighboring unit cell (Figure 5a).
Unlike the linear fibre forming interactions these interactions were weaker with a
combined buried surface area of ~1300Å2 and result in helical-columnar arrays (Figure
5b).
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 modular organization
of the N protein may coordinate nucleocapsid assembly.
NTD as 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 177 136 to 231 190
in MHV (corresponding to 136 to 190 in IBV). RNA binding ability for IBV NTD has
been shown by Hui et. al. using gel-shift assays. The N-terminal region of IBV-N was
also shown to specifically interact with the 3’ termini of IBV RNA(Zhou, Williams et al.
1996).
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Based on their structure of the NTD , Hui et. al. have proposed that the arms of the
“U” shaped monomer which are quite basic in nature are likely the regions of the Nprotein binding to RNA. This is also consistent with NMR –NOE analysis of NTD-RNA
interactions in the SARS-coronavirus N-protein. A novel finding in our crystal structure
analysis of the NTD is that NTD dimers can associate to form linear arrays with these
basic tethers exposed along the surface of such a fibre. This fibre with its exposed basic
tethers could provide for closely packed interactions of NTD with the genomic RNA.
CTD may provide a structural scaffold for helical nucleocapsid formation
In contrast to NTD which alone can bind RNA the CTD does not have the ability to bind
to RNA. Several biochemical studies have mapped the dimerization domain of IBV-N
protein to the CTD. Our crystal structure analysis, clearly indicate a tight dimer mediated
by domain swapped interaction. Although the NTD also forms a dimer the inter-dimeric
interactions are significantly stronger in CTD and thus is the primary force in the
formation the full length protein dimers. The inter-dimer NTD interactions are not very
rigid and mediated by an electrostatic association between the N-terminal arm and an
acidic groove. It is interesting to not that dis-assembly of nucleocapsid components was
achieved by high-salt conditions which destabilize electrostatic and ionic interactions.
The CTD inter-dimeric fibre forming interactions besides their relative rigidity and
slightly greater buried surface area also are preserved at a wide range of pH ( between
CTD1 at 4.5 and CTD2 at pH8.5). This rigidity of the dimer makes the CTD ideally
suited to serving as a structural scaffold around which the helical nucleocapsid is
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organized. The CTD fibre is also predominantly acidic wih a swath of basic
electrostatic potential that also follows a helical pattern. This region may be important in
making auxiliary contacts with RNA and the predominantly basic M protein with which
the C-terminal half of the N-protein is known to interact.
The additional interactions displayed by CTD also demonstate its ability to form higher
ordere dimer-dimer aggregates using more than one interface. The type II interactions
seen in CTD2 and the type IV and type V interactions seen in CTD3 suggest the
possibility that these interactions may be brought into play during spherical shell
formation after the linear fibre which provides the helicity to NTD interaction with RNA
is already put in place.The bridging nature of these non-fibre dimer-dimer interactions
may be important during compaction of the N-protein RNA complex into the
nucleocapsid.
The overall B-factor for one of the three dimers that participate in fibre formation is a lot
higher than other 3 dimers in the CTD2 ASU. This disorder seen at pH 8.5 together with
the observation of weaker interfaces and absence of linear arrays in CTD3 also at pH 8.5
might provide clues into the possible role of pH in nucleocapsid dis-assembly.
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
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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
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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 SARS1
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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.
Here we have shown that N protein of coronavirus is organized into two modular
domains with NTD serving as RNA binfd and the CTD forming scaffold. Such modular
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org is conducive for nucleocapsid formation with RNA biding to RNA and CTD
provodong as scaffold Overall struc similarity between coronavirus CTD amd PRRSV
Capsid forming somian indicates that these viruses within the nidovirales family are more
similar than previously thought.
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