Mechanics of bacteriophage maturation Wouter H. Roosa, Ilya Gertsmanb,c,1, Eric R. Mayd,1, Charles L. Brooks IIId, John E. Johnsonb, and Gijs J. L. Wuitea,2 a Natuur- en Sterrenkunde and LaserLab, VU University, 1081 HV, Amsterdam, The Netherlands; bDepartment of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037; cDepartment of Pediatrics, University of California, La Jolla, CA 92037; and dDepartment of Chemistry and Biophysics Program, University of Michigan, Ann Arbor, MI 48109
Edited by Charles M. Knobler, UCLA, Los Angeles, CA, and accepted by the Editorial Board December 20, 2011 (received for review June 27, 2011)
Capsid maturation with large-scale subunit reorganization occurs in virtually all viruses that use a motor to package nucleic acid into preformed particles. A variety of ensemble studies indicate that the particles gain greater stability during this process, however, it is unknown which material properties of the fragile procapsids change. Using Atomic Force Microscopy-based nano-indentation, we study the development of the mechanical properties during maturation of bacteriophage HK97, a λ-like phage of which the maturationinduced morphological changes are well described. We show that mechanical stabilization and strengthening occurs in three independent ways: (i) an increase of the Young’s modulus, (ii) a strong rise of the capsid’s ultimate strength, and (iii) a growth of the resistance against material fatigue. The Young’s modulus of immature and mature capsids, as determined from thin shell theory, fit with the values calculated using a new multiscale simulation approach. This multiscale calculation shows that the increase in Young’s modulus isn’t dependent on the crosslinking between capsomers. In contrast, the ultimate strength of the capsids does increase even when a limited number of cross-links are formed while full crosslinking appears to protect the shell against material fatigue. Compared to phage λ, the covalent crosslinking at the icosahedral and quasi threefold axes of HK97 yields a mechanically more robust particle than the addition of the gpD protein during maturation of phage λ. These results corroborate the expected increase in capsid stability and strength during maturation, however in an unexpected intricate way, underlining the complex structure of these self-assembling nanocontainers. AFM ∣ elastic network model ∣ nanoindentation ∣ normal mode analysis ∣ virus structural mechanics
H
K97 is a double stranded DNA bacteriophage infecting Escherichia coli. The capsid protein of HK97 has a subunit fold that is shared by phages λ, P22, T4, and phi29, among others, as well as the eukaryotic virus, Herpes (1). In addition, the maturation pathways of these viruses show similar morphological changes (2). HK97 provides a unique model system to study capsid maturation, as large quantities of isometric particles can be produced in an E. coli expression system. These particles are expressed without the portal and terminase proteins used for genome packaging, but can be isolated as procapsids and matured in vitro using chemical agents instead of DNA, and maturation can be trapped in various expansion states (3, 4). In vitro expansion can be effectively induced with a variety of chemical agents, including lowering the pH below 4. The particles expand from the Prohead II (P-II) form, which differs from Prohead I (P-I) by the absence of the 104AA N-terminal polypeptide (termed the Δ-domain) from each subunit (gp5). The Δ-domain is presumed to function in scaffolding capsid assembly (5, 6). The Δ-domain is cleaved by an internally packaged protease (gp4) upon assembly, allowing the particle transition to the maturation competent P-II particle. During maturation the procapsid, with an average outer diameter of approximately 50 nm, expands into a stable capsid of approximately 60 nm which possesses a twofold thinner shell (7, 8). In this process the skewed hexons of the P-II rearrange into the symmetrical configuration that characterizes the expansion intermediate (EI) (8, 9). Upon EI formation, cross-links form covalent isopeptide bonds between lysine 169 on one subunit 2342–2347 ∣ PNAS ∣ February 14, 2012 ∣ vol. 109 ∣ no. 7
and asparagine 356 of a different subunit at all threefold axes of the capsid. Each subunit takes part in such a reaction, ultimately resulting in an intertwined cross-link network in the mature Head II (H-II) particle that resembles chainmail. It was shown that upon approximately 60% cross-link formation, the EI particle expands further to the penultimate balloon state, and then finally to H-II. The maturation steps involve massive conformational changes between subunits (4, 8) (Fig. 1). A recent, crystallographic model of P-II revealed, with near atomic resolution, that twisting and bending of helix and β-sheet domains in the capsid protein occur during maturation (10). Whereas these results show that the structural basis of capsid maturation is increasingly well understood, the mechanics of this process remain elusive. It is, however, a mechanical model that has been put forward as the hypothesis to explain the need for capsid maturation: i.e., a stabilization of the fragile procapsid (2, 11). This idea has a long history but it has never been tested in a direct experiment. We examine, here, the mechanical consequences of maturation by Atomic Force Microscopy (AFM) nanoindentation (12–15). This technique has developed into a versatile tool to study particle mechanical properties (16). Such studies have successfully included Finite Element Methods (FEM) and/or full capsid Molecular Dynamics (MD) simulations to analyze the material properties of protein nanocontainers (13, 15, 17, 18). However, MD simulations of entire capsids are computationally expensive and FEM requires a priori knowledge of the elastic properties. In this work we employed a different method; a multiscale approach combining MD of only the asymmetric unit and normal mode analysis (NMA) of elastic network models (ENM) of the entire capsid (19, 20). This method allows for ab initio predictions of the mechanical properties, independent from the experimental data and in a computationally efficient way. Using this multiscale method we calculate the Young’s modulus of the HK97 particles and compare it to estimates obtained via thin shell continuum elastic theory. Combining these computational approaches with our experimental AFM data on stiffness, material fatigue, and ultimate strength we dissect the complex mechanics of viral capsid maturation and compare the properties of mature HK97 particles with the known mechanical properties of mature phage λ (21, 22). Results To assess the change in material properties of HK97 during maturation we looked at both the prohead states, an intermediate state, and the final matured particle. Before starting experiments we image the viral particles by AFM (23, 24). The morphologies of the particles in these images, as shown in Fig. 2, reveal subAuthor contributions: W.H.R., C.L.B., J.E.J., and G.J.L.W. designed research; W.H.R., I.G., and E.R.M. performed research; I.G., E.R.M., C.L.B., and J.E.J. contributed new reagents/ analytic tools; W.H.R., I.G., E.R.M., C.L.B., J.E.J., and G.J.L.W. analyzed data; and W.H.R., I.G., E.R.M., C.L.B., J.E.J., and G.J.L.W. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. C.M.K. is a guest editor invited by the Editorial Board. 1
I.G. and E.R.M. contributed equally to this work.
2
To whom correspondence should be addressed. E-mail:
[email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/ doi:10.1073/pnas.1109590109/-/DCSupplemental.
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Fig. 1. Expansion diagram of the HK97 maturation pathway. P-I: Prohead I, P-II: Prohead II, EI: Expansion Intermediate, H-II: Head II.
Fig. 3. Force-indentation curves. The thin lines show the individual indentation curves, together with their average (thick, red line). The relative indentation is plotted on the x-axis, using the measured dimension for every particle. For clarity only the linear parts of the curves are shown (i.e., until breakage occurs). Error is SEM. (A) Prohead II, (B) Head II.
Fig. 4 demonstrates that at the EI state a relatively small number of cross-links have been formed (9, 25). Nevertheless, the critical force is already at the level of the H-II particle as can be seen in Fig. S3. However, there is a clear difference in proneness to fatigue, when EI particles are compared to H-II ones. During imaging, a small force is exerted to the particles with the AFM cantilever tip, which for some particles induces material fatigue. We observed, that during high resolution imaging, considerably more EI particles break (>80%) than H-II particles (57 nm. It was however not possible to deduce to what degree the particles had expanded and this data was discarded. See Fig. S1 for a distribution of the heights of P-II and H-II particles. Molecular graphics images (Fig. 2 B and D) were made with Chimera software (see Fig. S1).
normalized based on the ratio of the sum of the spherical harmonic coefficients between the MD and ENM simulations. This renormalization ensures that our ENM spectrum is scaled to the appropriate thermal scale. The initial configurations of the asymmetric units for the MD simulations were taken from the crystal structures in the VIPER database (47) (P-II:3e8k; H-II:1ohg, H-I:2fs3). These structures were energy minimized and then equilibrated at 300 K, using the GBSW implicit solvent model (48) under icosahedral boundary conditions (49). The CHARMM simulation package and CHARMM19 force field were utilized to perform the MD simulations (50). To include the effects of crosslinking in H-II, adjacent protein units in the structure were cross-linked by introducing bonds, angle, and torsional terms between the side chains of the appropriate residues (K169 and N356). These interactions occur between the asymmetric unit which is being explicitly simulated and neighboring (image) proteins, and the force field terms were modeled after the terms describing the peptide backbone. All systems were equilibrated for 10 ns, production simulations were conducted for at least 9 ns for all systems. The magnitude of the equilibrium fluctuations were calculated by performing block averaging over 4 ns blocks of data; the blocks were offset by 1 ns. There were seven blocks used for P-II and H-I and six blocks for H-II, for which we had slightly less production simulation data. The ENM models were constructed from the average structure of the production MD simulations. The icosahedral symmetry operators were imposed on the asymmetric unit to construct the full capsid structure. The ENM models consisted of only heavy (nonhydrogen) atoms and the rotation-translation of blocks method (51) was used to determine the normal modes of the system. In this method, segments of the structure are treated as rigid units, we chose to make two consecutive residues a rigid unit in our analysis. The networks were propagated along the 999 lowest frequency modes (excluding the first six modes which correspond to rigid body motions), to construct a trajectory long enough to observe approximately 20 oscillations of the slowest modes.
Multiscale Modeling Approach. To generate the equilibrium capsid dynamics representative of a particular temperature, we employ a multiscale approach (20), in which we construct an ENM, propagate the network along the lowest frequency normal modes, and project the renormalized equilibrium thermal fluctuations onto a spherical harmonic basis. To renormalize the fluctuations in the ENM dynamics, we compute a MD trajectory of the capsid asymmetric unit under icosahedral boundary conditions using all atom force field based molecular models, and project these fluctuations onto the same spherical harmonic basis. The spherical harmonic spectrum from the ENM is then re-
ACKNOWLEDGMENTS. R. Hendrix and R. Duda (University of Pittsburgh) are thanked for discussions, I. Ivanovska (Vrije Universiteit) is thanked for technical support and David Veesler (Scripps) is thanked for purifying the P-I particles. G.J.L.W. acknowledges the support by the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) for a CW-ECHO and Vici grant and the Stichting voor Fundamenteel Onderzoek der Materie (FOM) for support under the “Physics of the Genome” research program. W.H.R. is supported by NWO through a Rubicon grant. I.G. and J.E.J. were supported by National Institutes of Health (NIH) Grant R01 AI040101. E.R.M. is supported by a National Science Foundation (NSF) postdoctoral fellowship. C.L.B. is supported through the NIH (RR012255) and NSF (PHY0216576).
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