The Skaggs Institute
for Chemical Biology
Nuclear Magnetic Resonance Studies of a Viral Capsid Maturation Intermediate
J.R. Williamson, F. Agnelli, A. Beck, C. Beuck, A. Bunner, A. Carmel, J. Chao, S. Edgcomb, L. Gan, I. Gertsman, M.
Hennig, E. Johnson, J. Johnson, D. Kerkow, E. Kompfner, S. Kwan, E. Menicelli, P.
Mikulecky, W. Ridgeway, H. Schultheisz, L.G. Scott, E. Sperling, B. Szymczyna
with J. Johnson, Scripps Research, we have characterized 2 maturation intermediates
of the capsid of the bacteriophage HK97. The capsid is an enormous particle with
an effective molecular weight of 13.1 MD and a diameter of nearly 600 Å. Characterization
of such a large molecule is a tremendous challenge for structural biology, particularly
for nuclear magnetic resonance (NMR) spectroscopy. NMR of large molecules is extremely
difficult because of the broad NMR resonances. Using NMR, we successfully observed
the N terminus of the HK97 capsid in 2 distinct maturation intermediates.
HK97 procapsid can be recombinantly expressed in Escherichia coli as an empty
procapsid precursor, known as the prohead II state. Maturation of prohead II can
be induced by shifting the pH to 4, to produce expansion intermediate III (EI-III),
which finally forms the mature head II conformation when returned to a neutral pH.
The capsid consists of 420 identical copies of the capsid protein assembled into
a spherical shell.
Both electron cryomicroscopy and x-ray
crystallography are being used in an extensive structural characterization of the
series of intermediates in the HK97 capsid expansion. The prohead II state is characterized
by a rough and corrugated surface (Fig. 1, inset at lower right in prohead II panel).
Upon expansion to EI-III, the surface becomes smoother as the radius of the maturing
capsid increases (Fig. 1, inset at lower right in EI-III panel). Finally, a very
smooth but thin capsid wall is achieved in the final conformational change to generate
head II. Remarkably, this maturation process is accompanied by an intermolecular
cross-linking reaction. Denaturing gel electropherograms are shown for each capsid
state at the lower left of each panel in Figure 1 to illustrate the cross-linking.
|Fig. 1. NMR
analysis of the HK97 maturation process. Upper left panel, In the 15N
heteronuclear single quantum correlation of the prohead II maturation intermediate,
each peak corresponds to an amide proton in the extreme N terminus of the capsid
protein, and only flexible residues are observed. Because of the symmetry of the
capsid, the spectra of the 420 copies of the capsid protein in the procapsid are
assumed to be identical. The denaturing gel electropherogram of the capsid protein
(inset at left) shows that the protein is monomeric, and the electron cryomicroscopy
reconstruction (inset at right) shows the rough capsid surface. Lower left panel,
Similar data for the EI-III intermediate, which is the next step in the maturation
process. Lower right panel, Similar data for the final mature head II capsid. Upper
right panel, A schematic model for the changes in the N terminus revealed by NMR
during capsid maturation. The capsid is shown as a quarter cross-section, and the
peptide location and mobility are indicated by the short lines. As the capsid matures,
the N terminus first becomes more flexible and then is completely ordered in the
x-ray structure of the final head II state shows experimental electron density for
all of the residues of the capsid protein. However, some residues are disordered
in the earlier intermediates. We wished to determine if these segments were mobile
enough to be visible in NMR spectra. Normally, no peaks would be visible in the
NMR spectrum of such a large molecule. If we could observe NMR resonances, we might
be able to learn about the role of the disordered residues not observed in the x-ray
or electron microscopy structures.
We produced a sample of prohead II labeled
with 15N, which permits application of heteronuclear NMR methods. A 2-dimensional
heteronuclear single quantum correlation NMR spectrum of prohead II is shown in
the upper left panel of Figure 1. Approximately 9 resolved peaks are attributable
to the amide protons on the backbone of the protein. Using heteronuclear NMR and
comparing the findings with the spectra of a short peptide, we determined that the
observed resonances are due to the extreme N-terminal region of the capsid protein.
Both the N terminus and an internal region termed the E-loop are disordered in the
x-ray structure, but only the N terminus gives rise to observable NMR resonances.
The prohead II state was converted to
the EI-III state by decreasing the pH to 4, and the resulting spectrum, shown in
the lower left panel of Figure 1, is quite different. The position and the shape
of the peaks have changed, indicating that both the structure and the dynamics of
this region have changed during the maturation process. Finally, the pH was returned
to normal to complete the maturation, and the resulting spectrum of head II, shown
in the lower right panel of Figure 1, is nearly empty. This result was expected
because all the residues in the mature capsid are highly ordered and should therefore
In summary, in the prohead II state,
the N terminus of the protein is not rigid, but undergoes transient interactions
with the procapsid shell. During the next step of maturation, a subset of N termini
becomes more flexible and disordered in the EI-III state. This finding is somewhat
counterintuitive, because one might expect increasing rigidification during maturation.
Finally, the N terminus becomes completely ordered in the mature head II state after
cross-linking has occurred. This macromolecular complex is the largest one for which
informative NMR spectra have been recorded. We are continuing to develop methods
and approaches for using NMR to study biologically important processes of large
Chao, J.A., Lee, J.H., Chapados, B.R.,
Debler, E.W., Schneemann, A., Williamson, J.R.
Dual modes of RNA-silencing suppression by Flock House virus protein B2. Nat. Struct.
Mol. Biol. 12:952, 2005.
Hennig, M., Munzarova, M.L., Bermel,
W., Scott, L.G., Sklenar, V., Williamson, J.R. Measurement
of long-range 1H-19F scalar coupling constants and their glycosidic
torsion dependence in 5-fluoropyrimidine-substituted RNA. J. Am. Chem. Soc. 128:5851,
Vallurupalli, P., Scott, L., Hennig,
M., Williamson, J.R., Kay, L.E.
New RNA labeling methods offer dramatic sensitivity enhancements in 2H
NMR relaxation spectra. J. Am. Chem. Soc. 128:9346, 2006.