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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
dimmers 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 illness 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 30km 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.
The coronavirus N (nucelocapsid) protein is typically a protein of molecular weight
50kDa to 60kDa.tag and is sythesized 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 gneomic 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 display 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 specific interaction between N protein and packaging
signal in RNA has only been demonstrated for MHV-N interaction with the packaging
signal.
Structure of The CTD: The cTD was purified to homogeneity as a dimer by affinity and
size exclusion. The CTD exists in both crystal forms as an intimate domain swapped
dimer. The major interface between the two monomers is brought about by a beta sheet
constituted by strands 1 (287 and 293) between and and 2 (296 and 308) from both
monomers constituting a sheet whose base has the topology b1B-b2B-b2A-b1A. such
that the final topology is. The basic structure can be described as floor of anti-parallel
beta strands surrounded by helices and loops. The XXX strands XX and xX interacting
with XxX and XXX strands from thee other chain constitute this floor which spans a
convex/concave floor of XXX square Å. The major helixes XXX and XXX surround the
beta strand floor and are attached to the beta strands via an interaction between residues
XXX and XXX on helix XXX and XXX with strands XXX and XXX on the floor. The
major helices XXX and XXX each XXX and XXx residues long form the basis of the
wings of the butterfly shaped dimer.
The dimerization interactions are very tight and bury a very large surface area. Unlike
expected from previous biochemical data neither the serine rich domain or the disulfide
bonding was important in either protein oligomerixation or crystal packing. The two
dislufide XXX and XXX ar located at XXX and are not in any position to mediate
oligomerization . The crystalsand protein prep was perfomed I the absence of redcing
agent so the non dislufide 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 capsid
forming domain of PRRSV a corona like virus. This match had a similarity score of XXX
with a corresponding RMS deviation of XXX Å. PRRSV a corona like virus is also a
XXX stranded 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 in the
presence of RNA . 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:
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.
The buried surface area of XXX Å 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.
Majority of the crystal packing interactions in the two crystal forms primarily involve
residues between XXX and XXX which constitute the terminal
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
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 dimmer grew in