Journal of Molecular Biology
Volume 352, Issue 3, 23 September 2005, Pages 723-735
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Cooperative Reorganization of a 420 Subunit Virus Capsid

https://doi.org/10.1016/j.jmb.2005.07.024Get rights and content

The complex protein capsids of many viruses exhibit dramatic reorganizations at critical stages in their life-cycle. Here, time-resolved solution X-ray scattering was used to study a dynamic, large-scale conformational maturation of the 420 subunit, 13 MDa, icosahedrally symmetric HK97 bacteriophage capsid. Isoscattering points in the time-resolved scattering patterns and singular value decomposition revealed that the expansion occurs as a cooperative, two-state reaction. The analysis demonstrates that the population shift from Prohead-II to Expansion Intermediate I, EI-I (60 Å larger than Prohead-II) occurs in minutes, but does not reveal the time required for individual transitions that occur stochastically. Any intermediate forms that may be traversed during this conversion are unstable and do not constitute an appreciable population of the ensemble of particles. In an energetic landscape view, particles must undergo an energy barrier-crossing event in order to successfully convert from Prohead-II to EI-I. This implies that the particles “hop” over the energy barrier stochastically as they individually attain an expansion-active state. Interestingly, systematic deviations from single-exponential kinetics were observed for the population shift. This may indicate that in undergoing the irreversible conversion from Prohead-II to EI-I, particles are subject to a complex energy landscape that links the initial and final particle forms.

Introduction

Assemblies composed of tens to hundreds of subunits drive many essential processes in biological systems by transducing physicochemical signals from their environment into directed conformational change. Examples include flagella, cilia, cytoskeletal assemblies, protein-folding chaperones, nucleopore complexes, and proteosomes. As they perform their functions, these assemblies often exhibit significant structural changes, with the multitude of subunits moving in concert. Little is understood about how these massive assemblies function in their stochastic microscopic environment, how efficiently conformational transitions are effected, and the extent to which subunits within the assemblies behave cooperatively.

The protective protein capsids of many viruses exhibit some of the most dramatic conformational changes among large macromolecular assemblies. These changes take place at critical stages in their life-cycles, such as during assembly, maturation, host cell recognition and genome expulsion and have been documented in viruses including retroviruses, papillomaviruses, herpesviruses, flaviviruses, picornaviruses, and bacteriophages. Here, we ask whether such assemblies composed of hundreds of subunits behave as tightly integrated units or as loose assemblages of parts (Figure 1). At one extreme, each subunit in a particle may switch conformations independently of neighboring subunits. Alternatively, subunits may manifest some degree of coordination, such that their conformational switching occurs in a concerted fashion. Depending on the energetic landscape that underlies the conformational change, one may observe a gradual, progressive change; consistent with a barrierless, downhill energetic slope linking unexpanded and expanded particles. Or the transition may occur as an abrupt, two-state event with the two particle forms being separated by an energy barrier.

The bacteriophage HK97 head assembly is an excellent model system for studies of viral capsid dynamics. Many aspects of the changes that occur in HK97 are believed to be common to most double-stranded DNA phages as well as herpesviruses.1, 2, 3, 4, 5, 6 In double-stranded DNA bacteriophages, the capsid assembles first as a complete precursor shell (prohead), then DNA is actively pumped into the shell by an ATP-powered portal-terminase complex. DNA packaging induces the hundreds of capsid subunits to rearrange dramatically, bolstering inter-subunit interactions and expanding the capsid shell.7, 8, 9, 10, 11 This process is termed maturation or maturational expansion.

We have chosen to use HK97 particles that lack portals and have the experimental advantage of complete icosahedral symmetry to study the maturation process in vitro.12, 13 In all other respects, these particles are believed to follow the same pathway of particle maturation that occurs when DNA is packaged in vivo to make viable phage particles.7, 8, 9, 10, 11, 12, 13 The head assembly, lacking portal, is composed of 420 copies of one type of capsid protein. The subunits are arrayed into a thin, single-layer shell. Particles can be purified in the Prohead-II form that is metastable at neutral pH and has a molecular mass of 13 MDa. Conformational maturation of Prohead-II can be effected in vitro by exposing it to moderate concentrations of denaturant or by acidifying the solution.14 Acidified particles undergo a dramatic metamorphosis through a series of forms, termed Expansion Intermediate-I (EI-I) through EI-IV, that have been characterized biochemically, spectroscopically, and by cryo-electron microscopy (EM) and image reconstruction.10, 15, 16, 17 The fully mature Head-II state is attained by reneutralizing these acidified particles. Head-II is 25% larger in diameter than Prohead-II and exhibits a chainmail structure consisting of catenated rings of five and six covalently crosslinked subunits.16, 18, 19 We recently demonstrated that crosslink accumulation is a gradual process that begins once particles have attained the EI-II form.10, 16

X-ray crystallography and cryo-EM provide valuable information about conformational change in the form of detailed but static structures. However, in order to better understand how the assemblies function as machinery, it is necessary also to characterize the real-time conformational dynamics, energetic landscapes, and structural flux that underlie the static snapshots. In this study, we have applied time-resolved, solution small-angle X-ray scattering (SAXS) to investigate the early stage of the acid-induced expansion of HK97 capsids. This maturation stage converts Prohead-II into the significantly larger EI-I particles (∼60 Å greater diameter) via rotational and translational reorientation of subunits and remodeling of interfaces (Figure 1). The data reported here advance limited SAXS measurements of HK97 maturation reported recently by covering the previously inaccessible first 2.5 min (during which most of the changes take place), with the first measurements now made only a few seconds after the transition is initiated.15 Current measurements are made at 5 s intervals, increasing time-resolution more than tenfold over the previous study. Taken together, these advances have enabled a rigorous signal processing method, singular value decomposition (SVD), to be applied in order to assess objectively the number of particle states present during the transition. We demonstrate that this conformational event occurs as an abrupt, global, two-state transition. No evidence of particles intermediate in size between the initial and final states in these experiments was detected, implying that intermediates between Prohead-II and EI-I do not exist as a stable state. Particles undergo this conversion in an all-or-nothing fashion; once a transition begins within a particle, it goes rapidly to completion. Such concerted movement among the 420 subunits within each particle reflects the striking associational and conformational dexterity of the subunit components and the icosahedral lattice. These types of cooperative transformations in viruses are likely to be ubiquitous, due to general features of icosahedral virus construction.

Section snippets

Time-resolved small-angle X-ray scattering for acid-induced HK97 maturation

Acidifying particles to around pH 4 by rapid syringe mixing induced HK97 Prohead-II maturational expansion. A steep pH-dependence of the initial expansion rate centered around pH 4 was observed previously.10, 15 In order to test whether the pH of acidification affects the number of conformational states populated and the kinetic order, we examined two pH conditions, 3.8 and 4.1 (accuracy of pH measurement, ±0.05 unit). Figure 2 shows the scattering curves collected over a 5 min experiment at pH

Large-scale reorganization of Prohead-II to EI-I occurs as a two-state reaction

This study demonstrates that the early phase of the HK97 capsid maturation involves only two distinct structural states, unexpanded Prohead-II and a particle that appears to correspond to the previously observed EI-I form which is ∼13% (∼60 Å) greater in diameter. No intermediately sized particles were found to exist as stable states, including asymmetric particles in which only a fraction of the subunits have switched conformations (Figure 1(a), Scheme I) and spherically-symmetrical, partially

Conclusions

Capsid maturation is a complex process that requires the efficient conformational reorganization of hundreds of subunits. As in the case of protein folding, in which proteins fold efficiently and cooperatively despite the astronomical number of configurations that can be sampled by the polypeptide chain, conformational change in virus capsids can theoretically involve a vast number of possible configurations. Even if each subunit can adopt only two conformations, in a 420 subunit capsid there

Purification of HK97 capsid particles

HK97 Prohead-II was produced using the expression vector pT7-Hd2.9, which co-expresses the HK97 gp4 protease and gp5 capsid protein. Particles were purified by precipitation with PEG, sedimentation by ultracentrifugation, and ion-exchange chromatography as described.39 The purified material appeared homogeneous by all applicable assays. These particles lack the portal complex that is not essential for HK97 in vitro morphogenesis. Prohead-II particles were dialyzed into 10 mM Hepes (pH 7), 500 mM

Acknowledgements

We gratefully acknowledge Dr Peter Doerschuk (Purdue University), Dr Doug Barrick (Johns Hopkins University), Lu Gan (TSRI), and Dr Alasdair Steven (NIH) for valuable discussions, and Dr Kazuki Ito (SSRL) for processing some of the SAXS patterns. This work was supported by NIH grants to R.W.H. & J.E.J.; K.K.L. is supported by a postdoctoral fellowship from the NIH. Experiments were carried out at SSRL, a national facility operated by Stanford University on behalf of the US Department of Energy,

References (44)

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    Indeed, the ability to arrest maturation at the EI-1 stage constituted a unique opportunity to demonstrate that in the absence of crosslinks or such non-covalent E-loop interactions the capsid still resides in a stressed conformation harboring distorted coat subunits despite the formation of ∼6-fold symmetric hexamers. The observation that expansion can be induced by various physico-chemical stimuli (such as pH change or iso-butanol) and the characteristic two-state transition between Prohead-2 and EI-1 evidenced by SAXS measurements indicate that the latter has a lower energy than its precursor (Gertsman et al., 2010b; Lee et al., 2005). However, our results indicate that EI-1 is still storing energy in its structure, probably to ensure that, in combination with formation of the first crosslinks, the maturation moves forward (Figure 1) and reaches the EI-2 particle form (with numerous crosslinks and coat subunits with canonical tertiary structure).

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