X-ray structure of the C-terminal domain of a coronavirus nucleocapsid protein;
structural basis of helical nucleocapsid formation
Hariharan Jayaram, Jyothi Jayaram, Brian R. Bowman, Ellen W. Collison,
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
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 globular domains of the nucleocapsid
protein from infectious bronchitis virus. We present here the crystal structure of the Cterminal domain (CTD) of IBV- N protein. The CTD exist as intimate domain swapped
dimers that tend to organize into helical arrays. Inferring from interactions observed in
crystals at different pHs 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.
Coronaviridae, a member of the order Nidovirales, is a family of viruses with ssRNA
genomes which are a significant causative agent of common colds and other severe
respiratory illnesses such as SARS. The coronaviruses have enveloped, non-icosahedral,
pleiomorphic capsids with diameters ranging from 80 to 160 nm. The capsid encloses the
viral genome consisting of a single 30kb long segment of positive sense ssRNA. Upon
infection the genomic RNA encodes a 3’ co-terminal set of four or more subgenomic
mRNAs that code for both structural and non-structural proteins. The enveloped capsid of
the virus if 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). A significant protein component of the capsid is the nucleocapsid
protein (N), which interacts with the genomic ssRNA forming a helical nucleocapsid that
comprises the central core of the virion.
Electron microscopic studies of TGEV a prototype coronavirus revealed that the internal
nucleocapsid is possibly helical and is composed of the ssRNA genome tightly associated
with N-nucelocapsid protein(Risco, Anton et al. 1996). The coronavirus N (nucelocapsid)
protein is typically a protein of molecular weight 50kDa to 60kDa.tag and is synthesized
in large amounts in an infected cell. The protein binds the genomic RNA as well as
subgenomic RNAs that are synthesized during a virus infection. Interactions with
conserved sequences in genomic RNA are hypothesized to mediate incorporation of RNA
into nucleocapsid cores. Proper assembly of capsids in reverse genetic systems also
requires complementary interactions between N protein and the major membrane protein
M.
The N protein displays a non-specific affinity for ssRNA in coronavirus including the
ability to recognize with increased affinity the consensus packaging signal of MHV and
also interactions between SARS-N protein N-terminal and consensus leader sequence in
RNA. The N protein also has a role in modulating viral sub-genomic RNA transcription
and mRNA translation along with control of packaging of genomic RNA. These activities
have led to the suggestion that N protein function to coordinate the involvement of subgenomic and genomic RNA in various stages of the virus life cycle and ensure its
packaging into a nucleocapsid.
The full length N-protein is prone to disorder, aggregation and degradation in solution
and its instability is suggested to be important for its role in virus capsid
formation(Wang, Wu et al. 2004). Limited proteolysis conducted on the full length N
protein from infectious bronchitis virus demonstrated that it was predominantly
composed of two domains an N-terminal domain that consisted of monomers and a Cterminal domain that existed as dimers at a very low concentration in solution. Similarly
dimers of the N-protein were observed for the homologous region in SARS-N protein
both in the context of the domain by itself and the full length protein(Surjit, Liu et al.
2004; Tang, Wu et al. 2005; Yu, Gustafson et al. 2005).It is our observation that Nterminal domain and C-terminal domain of SARS do not interact with each other at
moderate salt concentrations as assayed by co-fractionation during gel chromatography
and affinity pull down experiments. The N-protein therefore constitutes two functional
domains, an RNA binding N-terminal domain (Tang, Wu et al. 2005; Yu, Gustafson et al.
2005) and a C-terminal dimerization domain.
(Janowski, Kozak et al. 2001)
Structure of The CTD dimer: The CTD exists in both crystal forms as an intimate
domain swapped dimer. The domain swapping is brought about by interaction between
beta strands of one monomer with surrounding helixes and loops from the other monomer
to form a reciprocated,closed domain swapped dimer akin to that seen in crystal
structures of cystatin A(Janowski, Kozak et al. 2001; Newcomer 2001).The major
interface between the two monomers is brought about by a beta sheet constituted by
strand 2 (295 and 307) . The base of the dimmer is thus made up a ~400 Å area concave
surface.with the topology b1B-b2B-b2A-b1A. The basic structure can be described as
floor of anti-parallel b strands surrounded by helices and loops. The helices 3 and 4
connected by loop region surround the beta strand floor flanked by the longest C-terminal
helix 6.
The dimerization interactions are very tight and bury a very large surface area. Neither
the serine rich domain or the disulfide bonding was important in either protein
oligomerization or crystal packing. The two cysteine residues do not mediate
dimerization and are not disulfide bonded to each other.. The crystals and protein prep
was performed I the absence of reducing agent so the non disulfide bond mediated
interaction seen here is probably identical to that seen in the nucleocapsid .
Interestingly a DALI search of the PDB revealed a very striking similarity to the 12X
amino acid capsid forming domain of PRRSV a corona like virus which is a member of
the nidovirales family. This match had a similarity Z-score of XXX 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 . The
capsid forming domain 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 similarity
between PRRSV and CTD here clearly indicates that these viruses are3 more similar than
previously thought and hints at this architecture being a characteristic fold adopted by
helical nucleocapsid viruses.
Crystal packing interactions in CTD insights into stability of helical packing
interactions: The buried surface area of ~5000 Å2 in the dimer clearly indicates that the
dimer is very intimate and is almost likely to be the dimer found in the capsid. Further
insight into the nature of the CTD in the capsid or context of the virus can be got from
looking at the crystal packing interactions in both spacegroups.
We were fortunate to crystallize CTD in three crystal forms. Two of which yielded
structures presented here. The third crystal form yields highly anisotropic data with most
of the data looking similar to helical packed arrays with layer lines and smeared spots.
This clearly indicates the tendency of the CTD to organize into helical arrays.
Majority of the crystal packing interactions in the two crystal forms primarily involve
residues between 308 and 328 which constitute the terminal loop and 6. A salt bridge
mediated by Arg90 from one dimer and Asp96 from the other dimer stabilized the ~1200
Å2 interaction area between the two dimers. Interestingly enough the mode of
interactions between asymmetric units in the crystal at pH 4.5 is conserved within the
asymmetric unit at pH 8.5. The salt bridge and the orientation of the dimers remain
almost identical such that the rmsd between three adjacent dimers from one spacegroup
and the three molecules in the assymetric unit from the other spacegroup is ~1.0 Å.
Although the surface complementarity does not exist between the two molecules and the
iteractions other than for the salt bridge are strictly vanderwaal interactions. The
multimerization interaction in addition to the dimerization interactions seen in CTD are
therefore 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
dimer-dimer packing interactions.
Auxilary interactions with fourth dimer in second spacegroup: The additional dimer
is clearly not part of the primary fibre and instead forms a weak secondary transverse
fibre. The primary residues involved in contact of this cond molecule with the three other
dimers are scattered over both molecules of the dimer. This interaction mode might
represent interactions necessary to yield a spherical structure in the context of fibre
formation. Since this interaction involved three different molecules and yet buried a
similar surface area as the primary crystalpacking or fribre forming interaction, we
hypothesize that it is energetically less likeky yet equally stabilizing.
Analysis of the GRASP surface of the octamer further reveals that the surface is primarily
acidic with a swath of basic residues running in an expectedly helical fashion throught the
fibre. Although the pimary interactions with RNA are conferred by the Nterminus
secondary interactions may be facilitated by this basic stretch which is clearly solvent
exposed.
Interactions with M protein:
This fact taken together with the interaction seen in the PRRSV crystal packing
interaction similarly mediated by helix helix vanderwaal stacking and a simlar salt-bridge
between ArgX aqnd ASpX 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.
Materials and methods:
Purification of full length nucleocapsid protein and identification of tryptically
stable fragments: Full length nucleocapsid protein was expressed as before. The
expressed protein was purified by Ni-NTA agarose affinity followed by Heparin affinity
to almost 95% purity ( as assessed by denaturing SDS-PAGE followed by coomassie
staining). The protein was checked for monodispersity by dynamic light scattering (
Dynapro ) and negative stain electron microscopy. Cleavage of full length N protein was
carried out at 1-2 mg/ml concentration with 2% (wt trypsin /wt protein) sequencing grade
trypsin (Roche) to identify tryptically stable fragments . Following trypsinization the
protein was run on a denaturing SDS-PAFGE gel and the protein band that resulted was
blotted onto a PVDF (polyvinyldine fluoride) membrane and subjected to N-terminal
amino acid sequencing. For construct optimization the carboxy termini were estimated
based on predicted secondary structure in terminal region and mass spectrometric
characterization of purified protein.
Cloning , expression purification and crystallization of the tryptic fragments of
nucleocapsid protein: The two major and minor bands identified were expressed as GST
fusion proteins using the pet41 EkLIC vector (Novagen) into the LIC site . 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 mls of cutting buffer ( 250 mM NaCl, 50
mM Tris-HCl ph 8.0) with 10 units of protease for 1ml of beads. Following proteolysis
the dilute supernatant was purified further by gel filtration chromatography on a superdex
75 16/60 column ( Pharmacia). The protein migrated as a dimer and was concentrated to
5-8 mg/ml and used for crystallization trials. Initial crystallization trials were carried our
using Crystal Screen I ( Hampton Research). Following several leads in conditions with
Peg 4000. The Index screens 2 and 3 ( Jena Biosciences) were used to design
optimization strategy. Crystals of the C-terminal dimer grew in three to ten days and were
mostly needle shaped ,thin plates or hexagonal three dimensional bipyramidal crystals
that grew around two base conditions: one with citrate i.e 100 mM pH 4.5-5.2 trisodium
citrate, 0.1M MgCl2, 25-30% PEG 4000 and the other had32% PEG 4000, 0.8 M LiSO4,
0.1 M Tris-HCl pH 8.5.
Data Collection and phasing: Data was collected at the beamlines as indicated in Table I.
180 or 360 oscillation images with 1 oscillation angle were collected using the inverse
beam approach with a wedge size of 30. The entire dataset was integrated and scaled
using the HKL200 suite and scalepack. Four methionine positions were located using
shake and bake. The solutions were then refined, phases calculated and density modified
using SHARP. The final FOM after structure solution and phasing inn SHARP was 0.65
which yielded maps of an excellent quality to 2.2 Å. Although almost 80% of the model
could be traced using automated tracing in ARP-wARP, manual building of the dimer in
the asymmetric unit was performed using the program COOT. Refinement was carried
out in CNS or refmac5 . Refined coordinates for the dimer were used to phase data
obtained in the P21212 spacegroup by molecular replacement in the program phaser.
Phaser was able to coorectly identify positions opf all 4 dimers. Model bias was avoided
during refinement by using the prime and switch methodology implemented in
SOLVE/RESOLVE. All figures were generated using espript in combination with Adobe
Illustrator or pymol.
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