The Skaggs Institute
for Chemical Biology

Scientific Report 2006

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

In collaboration 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.

The 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 final state.

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 be invisible.

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 molecular machines.

Publications

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, 2006.

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.