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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):
Coronaviridae cause a variety of respiratory and enteric diseases in animals and man
including SARS, a disease with emerging global impact. Enveloped capsids of the
virus enclose the single stranded genome associated with the nucleocapsid protein ( N
protein). Using limited proteolysis we identified two stable domains of the
nucleocapsid protein from infectious bronchitis virus. We present here the crystal
structure of the N and C-terminal domains (NTD & CTD) of IBV- N protein. 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 a related virus family reveals 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 with ssRNA
genomes which are a significant causative agent of human upper respiratory infections
such as common colds and other severe illnesses such as SARS (severe acute respiratory
syndrome).
The coronaviruses are a family of enveloped positive strand RNA viruses. Their capsids
range in diameter from 80 to 160 nm and enclose a single 30kb long segment of positive
sense ssRNA(Siddell 1995). Upon infection and cell entry 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 the various viral structural and non
structural proteins required to replicate the virus and produce progeny virion capsids.
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 spherical particles a
corona. A significant protein component of the capsid is the nucleocapsid protein (N),
which interacts with the genomic ssRNA 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-nucelocapsid
protein(Risco, Anton et al. 1996; Risco, Muntion et al. 1998).
The N protein is typically a multifunctional basic phosphoprotein of molecular weight
50kDa to 60kDa.and its coding RNA and protein is synthesized in large amounts during
an infection(Stohlman and Lai 1979; Lai and Cavanagh 1997).
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The highly basic N protein was shown to have a general RNA binding ability with an
increased affinity for corresponding viral RNA(Cologna and Hogue 1998) and bind
consensus sequences at their 5’ and 3’ termini. 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 very important for proper genome encapsidation and nucleocapsid
formation.
In addition to this the N protein also plays a role in controlling mRNA transcription, and
translation and replication(Lai and Cavanagh 1997; Tahara, Dietlin et al. 1998; Schelle,
Karl et al. 2005).
The abundance of N produced during an infection results in N playing an important role
in host modulation during coronavirus infection. 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 major immunogen and an important diagnostic marker for coronavirus
disease(Leung, Tam et al. 2004) and can help improve the efficacy of avian coronavirus
vaccines(Cavanagh 2003; Zhao, Cao et al. 2005).
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Materials and Methods
Purification of full length nucleocapsid protein and identification of tryptically
stable fragments: Full length nucleocapsid protein was expressed as before. The purified
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 gel-electrophoresis and blotting onto PVDF. 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
nucleocapsid protein: All proteins were cloned and expressed as GST fusion proteins
using the pet41 EkLIC vector (Novagen) using the LIC methodology. The expressed
protein was purified using affinity on glutathione S sepharose (Pharmacia) followed by
on-bead cleavage with enterokinase (EK-Max 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 N-terminal and C-terminal domains was concentrated to 5-8
mg/ml and used for crystallization trials using Crystal Screen I (Hampton Research).
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followed by the Index screens 2 and 3 (Jena Biosciences) which were used to design
optimization strategy.
Data Collection and phasing: Data was collected at various beamlines as indicated in
Table I. For each crystal 180 or 360 oscillation images with 1 oscillation angle were
collected using the inverse beam approach with a wedge size of 30 in the case of MAD
data sets and a continuous wedge of 180 for the native data sets. For the NTD the data
were phased using molecular replacement in PHASER with the NTD coordinates (Hui
Fan et. al.). Following molecular replacement, further model building and refinement
were performed in a similar manner to the C-term below.
For the CTD the native and selenomethionine data were integrated and scaled using the
HKL2000 suite.From the 2 wavelength multiwavelength anomalous dispersion dataset
obtained for selenomethionine substituted protein ( pH 4.5 crystal form, Table I) four
methionine positions were located using the SHAKE and BAKE program. The initial
solution was then refined, phases calculated and density modified using SHARP. The
model was built using COOT and refined using a combination of CNS which was used
for initial rounds of simulated annealing refinement. followed by refinement using
REFMAC5. The structure of CTD in other crystal forms were phased using molecular
replacement from above model as implemented inn the program PHASER. Model bias
was reduced by using the prime and switch methodology implemented in
SOLVE/RESOLVE. All figures were generated using Pymol and Espript (figure 5) and
annotated in Adobe Illustrator.
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Results and Discussion:
The full length N protein from infectious bronchitis virus had been purified and
characterized previously(Zhou and Collisson 2000). The N protein has strong interactions
with 5’and 3’ conserved sequences of IBV RNA and also undergoes phosphorylation in
infected cells to generate multiple isoforms . Our structural characterization of full length
N protein was impeded by its aggregation and degradation on storage under a variety of
conditions. Purified full length N protein was also extremely polydisperse in solution as
characterized by dynamic light scattering analysis and not amenable to detailed structural
characterization using protein X-ray crystallography.
We employed the divide and conquer approach to study the protein structurally. Using
limited proteolysis we sought to identify regions of the protein that represented stable
domains that were resistant to proteolysis under limiting amounts of proteases trypsin
(that cleaves after basic residues Arg and Lys) and V8 protease (cleaves after acidic
residues Glu and Asp). The digestion pattern with v8 protease was not very distinct and
yielded several diffuse bands( data not shown). Trypsin proteolysed the full length
protein to a single ~17 kD band on a 17% denaturing SDS-PAGE gel within 15 minutes
of trypsinization. The “single” band thus observed was resistant to further degradation
even upon typsinization for several hours and represented a stable region(s) of the
protein. Using N-terminal sequencing of the cleavage fragment we identified four tryptic
fragments: two major cleavage sites that corresponded to cleavage at residues19 and 219
and two secondary cleavage sites at residues 27 and 226 (Figure 1a). The optimized
domain constructs termed NTD (N terminal domain) and CTD (C-terminal domain) were
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then cloned, expressed and purified to homogeneity. The N-terminal domain thus
identified was monomeric at moderate concentrations concentrations while the Cterminal domain protein was a dimer even at very low concentrations as assayed by gelfiltraion chromatography. The NTD and CTD proteins tended to aggregate during
purification and thus was purified at very low concentrations and concentrated only prior
to crystallization screening. The NTD and CTD proteins also 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 represent independent
domains of the full length protein and were suitable for structure determination
separately.
Crystals of both the NTD and CTD were obtained in a variety of conditions. For NTD
initial phasing attempts were carried out with several mutagenized proteins with
incorporated methionines since the wild type IBV-GRAY sequence in this region
(residues119-162) did not possess any Cys or Met residues. These mutant protein crystals
failed to yield a structure owing to pseudo-body centering and diffraction to moderate
resolution (~3.5 Å) that seriously limited the quality of the anomalous signal and made
phasing impossible.The crystal structure of a similar construct of IBV-N (Beaudette
strain) was then solved by some of the co-authors and was therefore used as a model to
phase the high resolution 1.3 Å data obtained for wild-type N-protein using molecular
replacement. The CTD phases were successfully obtained using anomalous data collected
at two wavelengths. The four methionine position identified yielded d an excellent map
with an initial FOM of 0.65. to 2.2 Å. Although almost 80% of the model could be traced
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using automated tracing as implemented in ARP-WARP, manual building yieleded a
refined model which was used to phase other native data for CTD using molecular
replacement. In all we have solved the structure of NTD in 2 pacegroups and CT in three
spacegroups in this study. The multiple packing arrangements seen for NTD and CTD in
the structures presented here suggest possible modes of interaction for these domains of
the nucleocapsid protein and suggest a possible model for nucleocapsid organization in
coronaviruses.
High resolution structure of the N-terminal domain: The secondary structure and fold
of the N-terminal domain solved at 1.3 Å is almost identical to the structure of the IBV
N-protein Beaudette reported before with the exception of five additional residues
discernible at the N-terminus.Briefly the structure is composed of a relatively acidic
globular core made up a twisted anti-parallel β-sheet center surrounded by a number of
loop regions. Prominent among the loop regions are two long loops corresponding to the
N-terminal 12 amino acids of this domain (residues 22 to 34) and a looped loop region
from residues 74 to 86 that extended outward like long tethers from this globular base
and a monomer forming a U shaped monomer.
The previous structure NTD by Hui et. al. consisted of two dimers in the asymmetric unit
in which the “U” shaped monomers were arranged in a side to side fashion (Figure 1 b).
The NTD in this study crystallized as an asymmetric homodimer with two interlocking
monomers arranged in a head to tail fashion in the crystal and asymmetric unit. The
interlocking of the U shaped monomers is aided by the predominantly basic N-terminal
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residues from 22 through 29 which extend outward (figure 4a) and occupy an acidic
groove situated on one phase of the base of the U. The extended conformation of this Nterminal “tether” arm interacting with the neighboring monomer in the ASU is in contrast
to its random loop conformation in the structure by Hui et. al.. Besides the N-terminal
region the other basic protruding loop segment consisting of highly basic residues from
residues 74 to 88 that wrap around the other phase of the NTD monomer.The region from
79 to 83 is disordered in this monomer as is the entire loop from residues 75 to 87 in the
neighboring monomer . The natural twist in the asymmetric dimeric interaction results in
this loop being alternatively interacting with the NCS related monomer and exposed and
disordered. This flexible loop which is full of conserved basic residues therefore
represents a mobile region which is prone to interacting with other NTD monomers and
free to interact with other ligands like RNA. The buried surface area in this NTD dimer is
2168 Å2 and represnts a faily tight dimeric interaction.
The dramtic difference in packing by the NTD dimers observed here possibly results
from the presence of magnesium ions and pH which are different between the two
oberveered crystal forms. The resukting propagated head to tail interaction resulting in a
linear array made up of NTD monomers which have overhanging basic loops from
alternating monomers (figure 4a) and possibly represent the RNA interacting regions in
the NTD which was clearly shown to bind RNA (Hui et al)T.hese basic loops with their
dynamic and pH dependent protein-protein interactions may play an important role in
nucleocapsid assembly and dis-assembly.
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Structure of the CTD:
The CTD had to be purified under extremely dilute conditions and migrated as a dimer
during gel-filtration. The concentrated protein crystallized readily as needles, rods or flat
sheets indicating a strong tendency of the protein to organize itself in two dimensions.
AT a slightly lower pH as crystal form I (Table I) hexagonal crystals were obtained
which were un-usually three dimensional . These bipyramidal crystals were however
poorly packed and diffracted extremely anisotropically to 3.5 Å resolution and gave an
almost helical diffraction pattern when diffracted along the long axis of the crystal.This
behavior is also characteristic of strong tenedency of the protein to organize along two
dimensions.
The CTD was successfully phased from two wavelength anomalous data obtained for
selenomethionine substituted protein from rod shaped crystals which were very rarely
obtained in crystal condition I. The phases for CTD in two other crystal forms were
obtained by molecular replacement.
Structure of The CTD dimer: The CTD exists in all three crystal forms 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
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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 ~400Å2 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 αhelix 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 was almost identical to 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).
The presence of a dimer in the ASU in two crystal forms and 4 dimers in the asu in the
other crystal form 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.
Crystal packing interactions in CTD insights into stability of helical packing
interactions: The two dimeric structures presented here result in five kinds of inter-dimer
interactions. Crystal packing in crystal I is brought about by dimer-dimer interactions
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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 dimer-dimer
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 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
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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.
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 crystal-packing (or fiber
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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.
Electrostatic surface, conservation of surface residues and interaction with other
other capsid components: The Grasp surface of the N-term crystal clearly shows a
predominantly basic exposed patches and th heat to tail linear array formed by NTD
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dimers interlocking with each other aided by the N-terminal basic loop protrusion. The
NTD fiber is quite loose and is possible more flexible to allow for interactions with RNA.
Besides a significant part of the loop from residues 74 to 88 is disordered in every
alternating dimer.in the crystal (Figure 4a). The looseness of this dimer might be in order
to compensate for the relative rigidity of the NTD dimer.
Analysis of the GRASP surface of the CTD fibre as constructed from the pH 8.5 octamer
further reveals that the surface is primarily acidic with a swath of basic residues running
in an expectedly helical fashion throught the fibre (Figure 4b). Although the pimary
interactions with RNA are conferred by the N terminus secondary interactions may be
facilitated by this basic stretch which is clearly solvent exposed.
The clearly demonstrated role of the CTD in mediating dimerization and the
demonstrated affinity of the NTD construct for RNA therefore suggests a degree of
specialization and cooopertivity between the two domains, with the CDT mediating
dimerization and secondary interactions with RNA and the NTD also mediating23
oligomerization important for fibre formation and interactions with RNA. Such atwo
domain organization is similar to the nucleocapsid protein of HIV where one of the
domains (Gamble, Yoo et al. 1997)
Fibre formation : 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
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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.
Similarity to other 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 RNAvirus 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
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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.
Also despite the very low sequence homology between SARS-N and IBV-N (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).The 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 N-terminal to the β-stranded floor might
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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.
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.
Genome organization in coronaviruses as suggested from the structure of NTD and
CTD. 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 CTDCTD class II dimer-dimer 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
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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)
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2
2
Dataset
(crystal
condition)
CTD
PEG 4000,
28.75% to
29.5 %, pH
4.8 Citrate,
0.1 M
MgCl2
X-ray
source/Wavelength
Resolution Number of
reflections
Completeness Redundancy
23
SBC-CAT 19ID
Advanced Photon
Source (Argonne)
Space Group P2(1)2(1)2(1)
a=38.389 b=65.939 c=92.306 α=90.000 β=90.000 γ=90.000
Rsym
0.97937 Å
50-2 Å
(360º , 1º oscillation)
15139
96% (68.9%)
11(8.2)
0.072(0.28
0.97951 Å
50-2Å
(360º , 1º oscillation)
28037
97.9(85.5)
5.3(3.2)
0.076(0.35
CTD
BIOCARS-14ID
Space Group P2(1)2(1)2
30% PEG
Advanced Photon
a=108.99 b=128.534 c=71.435 α=90 β=90.00 γ=90.00
4000, 100
Source (Argonne)
mM Tris0.9000 Å
50-2.2 Å
97377
99.7(98.8)
3.4(3.1)
0.081(0.58
HCl pH 8.6, (180º , 1º oscillation)
800 mM
LiCl
NTD
BIOCARS-14ID
Space Group C 1 2 1
25 % PEG
Advanced Photon
a= 100.055 b=46.210 c=74.176 =90.00 =121.06 =90.00
4000,100
Source (Argonne)
mM MES
0.9000 Å
50-1.3 Å
204381
87.9(60.5)
3.3(2.6)
0.
Sodium Salt
(180º , 1º oscillation)
6.2, 200 mM
Magnesium
Chloride
Refinement Statistics
Parameters
PDB 1 (1778 atoms, 92
PDB 2 (7169 atoms 215 solvent
PDB
water molecules)
atoms)
atom
Resolution Range
50-2 Å
48-2.2 Å
50-1
Number of reflections
15110
48564
6075
Rcryst
0.238
0.236
0.21
Rfree
0.269
0.291
0.250
Mean Bond length deviation
0.005491
0.006096
0.008
Mean Bond angle deviation
1.30545
1.31765
1.235
Ramachandran statisics
94.2% most allowed, 5.8% 91.6% most allowed, 7.6%
886%
additional allowed
additional allowed , 0.6%
addit
generously allowed, 0.2%
0.5 %
disallowed (poor density)
Values in parenthesis are for outermost resolution shells
Rsym = hi|Il(h) - <I(h)|/hiIl(h)
Rvalue = (|Fobs|-k|Fcal|)/|Fobs|
Rfree is calculated based on 10% of reflections not used during the refinement
2
3
24
2
4
25
2
5