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Structural studies of HK97 bacteriophage assembly and maturation

Final Report Summary - HK97 MATURATION (Structural studies of HK97 bacteriophage assembly and maturation)

We have used an HK97 subunit mutation that prevents formation of crosslink or comparable non-covalent interactions and an expression system that produces virus-like particles indistinguishable from authentic proheads but with the portal replaced by a twelfth coat subunit penton. The mutation stops maturation at the Expansion Intermediate-I intermediate generating a homogeneous population of these particles without E-loop “chainmail” interactions. Comparing these particles with mature Head-2 allowed the mechanical role of the Brownian ratchet in maturation to be identified. We determined the subnanometer structure of the crosslink-free Expansion Intermediate-I particle with electron cryoEM employing single particle protocols (Figure 1). The reconstruction, unexpectedly, reveals that coat subunit monomers exhibit distortions comparable to those observed in the prohead forms although the hexamers are approximately 6-fold symmetric (Figure 1). The observed coat subunit conformations suggest that release of their structural strain adds an energetic assist to crosslinking, driving capsid maturation forward with multiple energetic components. In addition, the structure suggests that the exothermic nature of capsid maturation is a consequence of enhanced quaternary interactions that stabilize the downstream intermediates.

Figure 1. Subnanometer CryoEM Reconstruction of the HK97 Expansion Intermediate-1. (Left) Icosahedrally averaged reconstruction at 9.3 Å. A pseudo-atomic model was obtained by rigid-body fitting the Prohead-II coat subunits in the map. (Right) A Prohead-II coat subunit is shown in the corresponding density extracted from the Expansion Intermediate-1 reconstruction.

Employing a mutant protease in the expression system, without enzymatic activity but otherwise normal, we obtained subnanometer cryoEM reconstructions of the protease-free as well as the protease-loaded Prohead-1 and used these as a basis to analyze the influence of the protease on the assembly (Fig. 2 A-C). The virion interior exhibits extended densities intruding nearly radially relative to the capsid shell (with a high-degree of apparent disorder) and into which we modeled the N-terminal region of the coat subunits as well as an elongated helical segment corresponding to the scaffolding domain C-terminal region (Fig. 2 B-D). A difference map computed by subtracting the protease-free to the protease-containing Prohead-1 reconstructions revealed that addition of the protease strengthens the scaffolding domain region that extends further toward the capsid center. The effect of the viral protease on the reconstruction suggested that it is incorporated into the procapsid shell in one of the following ways: (i) the protease molecules may be interacting with the scaffolding domains in a transient manner through moderate affinity contacts; (ii) or they remain anchored to the scaffolding domains (high-affinity) but are not organized with icosahedral symmetry (similarly to what was proposed for herpes viruses) as a result of scaffolding domain disorder and/or due to the non stoichiometric ratio of encapsidated protease to scaffold. The absence of discernable density attributable to the protease in our reconstruction is in agreement with either of these two hypotheses but do not allow their discrimination. Hydrogen/Deuterium exchange studies coupled to mass spectrometry (HDXMS) further confirmed these results and allowed us to accurately map the influence of the protease on the coat subunit structure, providing the first experimental evidence of a direct interaction between the viral protease and the scaffolding domain (Fig. 2 D). Comparison of the two reconstructions also suggested that the thermodynamic consequences of protease packaging are to shift the equilibrium between isolated coat subunit capsomers and procapsid in favor of the latter by stabilizing the assembled particle before making the process irreversible through proteolysis of the scaffolding domains.
We also estimated the number of protease molecules that are packaged into the procapsid based on native mass spectrometry of the two Prohead-1 particle forms used to perform three-dimensional reconstructions. A well-defined spectrum for the protease-free procapsid allowed resolution of individual charge states and yielded a mass estimate of 17.9 +/- 0.004 MDa (Fig. 2 E, top), in excellent agreement with the theoretical mass (17.7MDa). Analysis of the protease-containing particle resulted in an unresolved spectrum at a higher m/z than the empty Prohead-1 (Fig. 2 E, bottom). We thereby estimated the mass of the protease-filled Prohead-1 particle at approximately 21.4 ± 0.34 MDa under the assumption of normal charging, which corresponds to 144 ± 14 copies of the protease. The lack of resolution in the native mass spectrometry experiment is a clear indication of heterogeneity in the number of packaged protease molecules and supports the hypothesis put forward based on the cryoEM results. The observation of a broad distribution of protease copy number (instead of a fixed coat subunit-to-protease ratio) was unexpected and constitutes a change of the current paradigm for tailed phages. This study represents the most detailed analysis available to date of the interactions between a dsDNA viral capsid and the maturation protease and provides a basis to further our understanding of the first steps of virion assembly in Caudovirales phages and herpesviruses. From a methodological standpoint, this outcome is also a significant milestone as we obtained for the first time native mass spectra with charge-state resolution for a particle of such a size.

Figure 2. Recruitment of the maturation protease during assembly of the HK97 procapsid. (A) Surface rendering of the HK97 protease-containing Prohead-1 reconstruction low-pass filtered at 8.3 Å resolution and radially colored. The fit of a Prohead-1 coat subunit into the corresponding region of the reconstruction is shown on the right. The seven subunits of the icosahedral asymmetric unit are conformationally distorted at the level of the spine helix and P-domain β-sheet. (B) Cross-section of the reconstruction showing the scaffolding domains extending toward the procapsid center. (C) Difference map (low-pass filtered to 20 Å resolution) resulting from the subtraction of the protease-free Prohead-1 to the protease-containing one after scaling of the two reconstructions. (D) View of the coat subunit hexon from the procapsid interior. The regions featuring the highest solvent protection in presence of the protease, as revealed by hydrogen/deuterium exchange coupled to mass-spectrometry, are colored individually for each monomer whereas the other parts of the coat subunits are colored gray. (E) Native mass-spectrometry analysis of the protease-free and protease-containing Prohead-1 indicated masses of 17.9 and 21.4 MDa. (insets) Zoom-in of the indicated m/z region to reveal the fine structures of the signal.