thermodynamic characterization of viral procapsid expansion into a functional capsid shell

doi:10.1016/j.jmb.2012.02.020 J. Mol. Biol. (2012) 418, 167–180

Contents lists available at www.sciencedirect.com

Journal of Molecular Biologyj ourna l homepage: ht tp : / /ees .e lsev ie r.com. jmb

Thermodynamic Characterization of Viral ProcapsidExpansion into a Functional Capsid Shell

Elizabeth Medina†, Eri Nakatani†, Shannon Kruseand Carlos Enrique Catalano⁎Department of Medicinal Chemistry, University of Washington School of Pharmacy, H172 Health Science Building,Campus Box 357610, Seattle, WA, 98195-7610, USA

Received 20 December 2011;received in revised form14 February 2012;accepted 16 February 2012Available online23 February 2012

Edited by M. F. Summers

Keywords:virus assembly;herpesviruses;thermodynamics;macromolecular transitions;bacteriophage lambda

*Corresponding author. E-mail [email protected].† E.M. and E.N. contributed equaAbbreviations used: dsDNA, dou

β-ME, β-mercaptoethanol; EDTA,ethylenediaminetetraacetic acid; IHFfactor.

0022-2836/$ - see front matter © 2012 E

The assembly of “complex” DNA viruses such as the herpesviruses andmany tailed bacteriophages includes a DNA packaging step where the viralgenome is inserted into a preformed procapsid shell. Packaging triggers aremarkable capsid expansion transition that results in thinning of the shelland an increase in capsid volume to accept the full-length genome. Thistransition is considered irreversible; however, here we demonstrate that thephage λ procapsid can be expandedwith urea in vitro and that the transitionis fully reversible. This provides an unprecedented opportunity to evaluatethe thermodynamic features of this fascinating and essential step in virusassembly. We show that urea-triggered expansion is highly cooperative andstrongly temperature dependent. Thermodynamic analysis indicates thatthe free energy of expansion is influenced by magnesium concentration(3–13 kcal/mol in the presence of 0.2–10 mM Mg2+) and that significanthydrophobic surface area is exposed in the expanded shell. Conversely,Mg2+ drives the expanded shell back to the procapsid conformation in ahighly cooperative transition that is also temperature dependent andstrongly influenced by urea. We demonstrate that the gpD decorationprotein adds to the urea-expanded capsid, presumably at hydrophobicpatches exposed at the 3-fold axes of the expanded capsid lattice. Thedecorated capsid is biologically active and sponsors packaging of theviral genome in vitro. The roles of divalent metal and hydrophobicinteractions in controlling packaging-triggered expansion of the procapsidshell are discussed in relation to a general mechanism for DNA-triggeredprocapsid expansion in the complex double-stranded DNA viruses.

© 2012 Elsevier Ltd. All rights reserved.

Introduction

The pathways for the assembly of an infectious virusfrom macromolecular precursors are remarkably

ress:

lly to this work.ble-stranded DNA;

, integration host

lsevier Ltd. All rights reserve

similar in all of the complex double-stranded DNA(dsDNA) viruses, both eukaryotic and prokaryotic.1,2

In particular, the DNA replication, procapsid assem-bly, and genome packaging pathways are stronglyconserved in the herpesvirus groups and in manybacteriophages.3–7 In these cases, a terminase enzymespecifically recognizes viral DNA and the terminasemotor translocates the duplex into the interior of apreformed procapsid.3–6 DNA packaging triggers amajor reorganization of the proteins assembled intothe procapsid shell, which typically results in expan-sion of the shell into a thinner, more angularizedicosahedral structure.8–10 Bacteriophage lambda (λ)

d.

mailto:[email protected]

http://dx.doi.org/10.1016/j.jmb.2012.02.020

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168 Thermodynamic Characterization of Capsid Expansion

has been extensively characterized genetically, bio-chemically, and structurally and provides an idealsystem in which to define the molecular details ofgenome packaging.7,11,12

Assembly of the λ procapsid follows an orderedpathway that is generally conserved from phage tothe herpesviruses. Briefly, assembly initiates withself-association of the portal protein (gpB) into adodecameric ring structure.12–15 This nucleates poly-merization of the major capsid protein (gpE) into anicosahedral shell, chaperoned by co-polymerizationwith the scaffolding protein (gpNu3).16–19 A limitednumber of viral protease proteins (gpC) are alsoincorporated into the nascent procapsid interior,which auto digests, degrades the scaffold protein,and removes 20 residues from the N-terminus ofroughly half of the portal proteins.13,14 The proteol-ysis products exit the structure to afford the matureprocapsid composed of a portal ring situated at aunique vertex of the icosahedral shell; this portalvertex provides a hole through which viral DNA canenter during packaging and exit during infection.Genome packaging represents the intersection of the

DNAreplication andprocapsid assemblypathways.3,7

The terminase enzyme specifically recognizes viralDNA and then binds to the portal vertex of an emptyprocapsid (Fig. 1). This activates the terminase motor,which translocates DNA into the procapsid interior,fueled by ATP hydrolysis. Upon packaging ∼15 kbDNA, the procapsid undergoes an expansion process,which involves a significant reorganization of thecapsid proteins assembled into the shell (Fig. 1;discussed further below).7,11,20–23 Conventional wis-dom dictates that procapsid expansion represents “amajor, irreversible change in the assembled capsidproteins of the procapsid shell.”22,24,25

ProcapsidTerminase

ATPA

gpDAddition

DNA

DNAPackag

ATPA

DNAPackag

Fig. 1. Phage λ assembly pathway. A multi-genome concatsubstrate in vivo. Details of the packaging pathway are provid

In a number of viral systems, this irreversibletransition can be artificially triggered in vitro usingtemperature, pH, or denaturants.26–31 In this study,we examine urea-triggered expansion of the λprocapsid and report the surprising observationthat the transition is fully reversible. The equilibriumis strongly affected by urea concentration, magne-sium concentration, salt, and temperature. Wepresent physical, biochemical, and structural studiesthat characterize this transition and confirm that theurea-expanded structures faithfully recapitulatethose generated by DNA packaging in vivo. Therelevance of these studies with respect to a generalmechanism for DNA-triggered procapsid expansionin the complex dsDNA viruses is discussed.

Results

Urea triggers expansion of the λ procapsid

Expansion of the λ procapsid in vivo is triggeredupon packaging of∼15 kb duplexDNA (Fig. 1).20,21,23We examined a variety of approaches to artificiallyexpand procapsids to study this transition in vitro. Incontrast to other viral systems, neither pH in the rangeof 3–9 nor heat in the range of 4 °C to 50 °C areeffective in promoting expansion (SupplementalTable S1). Prior work has demonstrated that the λprocapsid can be artificially expanded by incubationwith 4 M urea for 30 min on ice.28 Here, werecapitulate this result and demonstrate that incuba-tion of our purified procapsids in 2.5M urea on ice for15 min triggers procapsid expansion (Fig. 2a). Due tothe time required for analysis by agarose gel, anaccurate quantitation of the reaction rate is not

DP

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Fig. 2. Expansion of the λ procapsid in vitro. (a) Urea and magnesium stabilize the procapsid and expanded capsidshells, respectively. Procapsids were expanded in 2.5 M urea, which affords the expanded capsid shell (urea). Theexpanded shells were then buffer exchanged into either high magnesium (15 mM, TMB) or low magnesium (1 mM,TMSO) buffer, as indicated. The migration of the procapsid (●) and expanded capsid ( ) in the agarose gel is indicated onthe right. (b) Urea-triggered expansion is strongly temperature dependent. Procapsids were incubated in 2.5 M urea for15 min at the indicated temperature and then analyzed by 0.8% agarose gel. (c) Urea-triggered expansion is inhibited bysalt. Procapsids were expanded as described in Materials and Methods except that NaCl was added to the reactionmixture as indicated. (d) Procapsid expansion is fully reversible. Procapsids were expanded in 2.5 M urea for 15 min at4 °C (urea) and then buffer exchanged into TMB buffer. The identical sample was again expanded and buffer exchangedas indicated.

169Thermodynamic Characterization of Capsid Expansion

possible using this assay. We note, however, thatprocapsid expansion is relatively rapid and essentiallycomplete in ∼1 min by gel assay (data not shown).Interestingly, urea-triggered procapsid expansion

is temperature dependent. While procapsids expandrapidly and completely on ice, the transition isstrongly inhibited by elevated temperature (Fig. 2b).We considered that this might reflect a kinetic effect;however, urea-triggered expansion was not ob-served even after 24 h at 25 °C (SupplementalTable S1). Urea-triggered expansion is also inhibitedby NaCl in a concentration-dependent manner (Fig.2c). Salt inhibition is likely responsible for theobservation that the λ procapsid does not expandin the presence of 4 M guanidine hydrochloride(Supplemental Table S1). For the remainder of thiswork, we will use the term procapsid to describe thecontracted shell (●) and the term capsid to describethe expanded, angularized structure ( ; see Fig. 2a).

Expansion of the λ procapsid is reversible

Previous studies reported that dialysis of ureafrom the reaction mixture affords a preparation ofλ capsids that remain in the expanded state.28 Incontrast, we observe that the structures contractback to the procapsid state when urea is removedby buffer exchange into TMB buffer [50 mM Trisbuffer, pH 8, containing 15 mM MgCl2 and 7 mMβ-mercaptoethanol (β-ME)] (Fig. 2a). Close inspec-tion of the published data reveals that the primarydifference between our studies and previous workis the buffer used to remove urea from the sample.This was investigated and we show that thecapsids remain in the expanded state whenexchanged into the TMSO buffer used in theprior studies [10 mM Tris buffer, pH 8, containing1 mM MgSO4 and 10 mM NaN3]. In contrast, theexpanded shells contract back to the procapsid


conformation when exchanged into our standardTMB buffer (Fig. 2a). It is generally accepted thatprocapsid expansion is an irreversible process invirus development and this surprising observationwas more fully explored.Each of the components in the two buffers was

individually examined, which reveals that theincreased concentration of Mg2+ in our TMB bufferis responsible for driving the expanded structureback to the procapsid state; none of the othercomponents has any effect (Supplemental TableS2). We next evaluated other metals, and the datapresented in Table 1 demonstrate that capsidcontraction is driven by all of the divalent metalsexamined. In contrast, none of the monovalent saltshave any effect at similar concentrations. Finally, weexamined the extent to which the transition isreversible. Procapsids that had been previouslyexpanded with urea and then contracted by bufferexchange into TMB buffer (15 mMMg2+) were againexpanded in urea and again buffer exchanged intoTMB buffer. The data presented in Fig. 2d demon-strate that the λ procapsid can be repeatedlyexpanded and contracted in solution by urea andMg2+, respectively.

Thermodynamic analysis of urea-triggeredprocapsid expansion

The data presented above demonstrate thaturea-triggered expansion of the λ procapsid isreversible. Moreover, close inspection of the gelsfails to reveal any evidence of intermediate statesbetween the contracted procapsid and expandedcapsid conformations (see Fig. 2). These observa-tions suggest that the expansion reaction may bemodeled as a reversible, two-state transition and

Table 1. Divalent metals drive contraction to theprocapsid conformation

Salt (15 mM) Capsid state

MgCl2 ContractedMgSO4 ContractedCaCl2 ContractedBaCl2 ContractedMnCl2 ContractedNi(NO3)2 ContractedZnCl2 ContractedNaCl ExpandedNa phosphate ExpandedKCl ExpandedK glutamate Expanded100 mM NaCl Expanded500 mM NaCl Contracted

Procapsids were expanded in 2.5 M urea on ice for 15 min andthen exchanged into buffer containing 1 mM MgCl2 and theindicated salt at 15 mM, unless otherwise indicated. In all cases,the preparations contained either exclusively expanded capsid orcontracted procapsid structures, as indicated.

we adapted analytical tools developed to charac-terize two-state protein unfolding reactions.32

Procapsids were expanded as described in Mate-rials and Methods except that the concentration ofMgCl2 and urea was varied as indicated. In thepresence of 0.2 mM MgCl2, procapsid expansionis urea concentration dependent and stronglycooperative (Fig. 3a). Analysis of the dataaccording to a reversible, two-state transition[Eq. (1)]32 affords a free energy of expansion,ΔG(H2O)∼3 kcal/mol‡.As described above,Mg2+ drives the expanded shell

back to the contracted procapsid conformation. Weinterpreted the data to indicate that urea and Mg2+

have antagonistic effects on the conformation of theλ capsid and we directly tested this hypothesis. Theabove urea-expansion study was repeated exceptthat MgCl2 was included in the reaction mixture at3 mM or 10 mM. The data clearly indicate that Mg2+

antagonizes urea-triggered procapsid expansion in aconcentration-dependent manner (Fig. 3a) and thatthe concentration of urea required to expand halfof the procapsids ([urea]1/2) is strongly affected byMg2+ (Fig. 3b). Analysis of the expansion dataaccording to Eq. (1) affords a ΔG(H2O) of 10.0±3.0 kcal/mol and 13.4±2.4 kcal/mol in the presenceof 3 mM and 10 mMMg2+, respectively (Fig. 3c). Forcomparison, “typical” protein unfolding reactionsreport [urea]1/2 between 3 and 6 M and a ΔG(H2O)that ranges between 5 and 15 kcal/mol.32–35 Finally,we note that the denaturant “m” value for thetransition is relatively large and insensitive to theconcentration of Mg2+ in the reaction mixture(Fig. 3c). This is discussed further below.

Mg2+-driven capsid contraction

To further explore the antagonist effects of urea andMg2+, we prepared by buffer exchange expandedcapsids in buffer lacking both urea and Mg2+. Theexpanded capsids were then incubated in thepresence of increasing concentrations of MgCl2, andthe fraction of structures that had contracted back tothe procapsid state was quantified by gel assay. Thedata presented in Fig. 4 demonstrate that Mg2+

drives the expanded shell back to the procapsidconformation in a concentration-dependent andstrongly cooperative manner; analysis of the dataaccording to Eq. (3) affords a [Mg2+]1/2 =1.1±0.1 mM. We next repeated the experimentas described above except that 2.5 M urea wasincluded in the reaction buffer. While Mg2+ can

‡We note that the expansion transition under theseconditions is extremely facile and occurs with essentially nopre-transition baseline. The error associated with thisanalysis is thus rather large; ΔG(H2O) = 3.3 ± 4.4kcal/mol.

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Fig. 4. Magnesium drives contraction of the expandedcapsid to the procapsid state. MgCl2 at the indicatedconcentration was added to expanded capsids in theabsence (○) or presence (●) of 2.5 M urea as described inMaterials and Methods. The fraction of contracted capsidswas quantified by agarose gel assay as described. Eachdata point represents the average of at least three separatemeasurements with standard deviations indicated withbars. The continuous lines represent the best fit of the dataaccording to Eq. (3).

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Fig. 3. Thermodynamic characterization of urea-trig-gered procapsid expansion. (a) Procapsids in the absence(○) or in the presence of 3mM(●) or 10mM (◊)MgCl2wereincubated on ice in the presence of increasing concentra-tions of urea, as indicated, and the fraction of expandedcapsids was quantified by gel assay. Each data pointrepresents the average of at least three separate measure-ments with standard deviations indicated with bars. Thecontinuous line represents the best fit of the data to Eq. (1).(b) The data presented in (a)were analyzed according to Eq.(2) to afford the concentration of urea required to expandhalf of the procapsid shells ([urea]1/2). (c) The free energy ofcapsid expansion (●) and the denaturant m values (○)derived from (a) are plotted as a function of MgCl2.


still drive the contraction transition, significantlyhigher concentrations of divalent metal are re-quired; [Mg2+]1/2=6.5±0.4 mM (Fig. 4).

Biological activity of the expanded λ capsids

We have demonstrated that the λ procapsid canbe reversibly expanded and contracted in vitro,which has allowed a rigorous thermodynamiccharacterization of the transition (vide supra). Eval-uation of these data with specific reference to virusassembly requires that urea-triggered expansionmimic the natural pathway that is triggered byDNA packaging in vivo. The three most essentialfunctions required of procapsids during DNApackaging are (i) the ability to bind the terminasemotor and sponsor DNA packaging, (ii) the abilityto bind the gpD decoration protein to the expandedcapsid lattice, and (iii) the capacity of the decoratedshell to physically withstand the internal forcesgenerated by the packaged λ genome.Our laboratory has developed an in vitro DNA

packaging assay where viral DNA is packaged intopurified procapsids in a defined biochemical assaysystem.36,37 These studies have demonstrated thatthe packaging reaction is magnesium dependentand that the optimal MgCl2 concentration is∼5mM.This presents a problemwhen trying to package intothe expanded capsids since they contract in thepresence of N1 mM Mg2+ (Fig. 4). Therefore, as afirst step towards demonstrating the biologicalactivity of the urea-expanded capsids, we examinedgpD binding to the capsid shell. An expandedcapsid preparation in 1 mM Mg2+ (no urea) wasprepared and purified gpD was added to themixture. Figure 5a clearly shows that the gpDdecoration protein efficiently adds to the expandedcapsid lattice. Several features of this interaction are

image of Fig. 4


noteworthy. First, while gpD binds to the expandedcapsid, no interaction is observed with unexpandedprocapsid shell (Fig. 5a); this feature is similarlyobserved in vivo.7,39 Second, the gpD-decoratedshell migrates faster in the gel than does theexpanded capsid alone. This indicates that gpDaddition affords a more negatively charged capsidshell. Third, gpD stabilizes the expanded shell andthe decorated capsids no longer contract in thepresence of elevated Mg2+ concentrations (E.M.,S.K., and C.E.C., unpublished results40). Finally,electron micrographs demonstrate that the gpD-decorated structures show thinning of the capsidshell and increased angularization of the icosahedralstructure (Fig. 5b), as observed in vivo.23 We notethat the expanded shells are fragile and easilydamaged during preparation for electron microscopy

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analysis (see Fig. 5b). In contrast, the gpD-decoratedshells are robust and intact structures are evident inthe micrographs.To further demonstrate biological activity, we

examined the genome packaging activity of thegpD-decorated capsids. Terminase-mediated pack-aging of the λ genome into a capsid renders theduplex resistant to DNase and the packagingproducts are visualized on an agarose gel.36 Ourstandard DNA packaging assay was modified bypre-incubating the expanded capsids with gpD andthen adding the other reaction components requiredfor genome packaging. The data presented in Fig. 5cdemonstrate that the expanded, gpD-coated capsidsare fully competent for DNA packaging. Important-ly, the only packaging product is the full-length λgenome (inset). This indicates that packaging intothe urea-expanded capsids is highly processive andthat DNA-filled particles can withstand the tremen-dous internal forces generated by the tightlypackaged, highly pressurized DNA genome.20,38,41

These features mirror the observations with bona fideλ capsids both in vitro20,38 and in vivo.7,14,39

Discussion

The packaging of a viral genome into a preformedprocapsid structure is an essential step in theassembly of complex dsDNA viruses. A universalfeature is that DNA packaging triggers maturationof the procapsid, often resulting in expansion andthinning of the shell to afford a larger, moreangularized structure. Dogma contends that this

Fig. 5. Urea-expanded capsids are biologically func-tional. (a) Expanded capsids bind the gpD decorationprotein. Procapsids and expanded capsids (in the absenceof urea) were incubated with gpD as described inMaterials and Methods, and the products were analyzedby 0.8% agarose gel. Note that the gpD-decorated shellmigrates faster than both the procapsid and the expandedcapsid shells. (b) The gpD protein stabilizes the expandedcapsid shell. Micrographs of negatively stained procapsids(i), expanded capsids (ii), expanded capsids that have beenre-contracted with 15 mM MgCl2 (iii), and gpD-decoratedexpanded shells (iv). Note that the expanded shells arefragile and deteriorate during preparation for electronmicroscopy analysis. Expanded capsids that have been re-contracted with magnesium or stabilized with gpD arestructurally sound. (c) Expanded capsids are biologicallyactive. Procapsids and gpD-decorated expanded procap-sids (capsids) were used in an in vitro DNA packagingassay. The data represent the average of three separateexperiments with standard deviations indicated with bars.Inset: DNase-resistant (packaged) viral DNA was ana-lyzed by gel assay. Note that only full-length genomicDNA (48.5 kb, arrow) is rendered DNase resistant. Thisindicates that both capsid preparations package DNA in aprocessive manner.38


step is irreversible so that unidirectional assemblyof the viral particle is ensured. This presumption isconsistent with a number of in vitro studies whereprocapsid expansion has been artificially triggeredby using pH, heat, or denaturants;26–28,30,42–44however, there has been some indication that strictirreversibility may not necessarily be the case. Forinstance, pH-induced expansion of the HK97procapsid proceeds through a number of interme-diates and there is evidence that at least onetransition may be partially (10%) reversible.45 Thatsaid, the fully expanded capsid structure is stableand does not contract back to the procapsidconformation. Thus, our demonstration that urea-triggered expansion of the λ procapsid is fullyreversible and that the expansion–contractiontransition can be repeated for multiple cycles isunexpected and quite remarkable.

Energetic features of procapsid expansion

We have demonstrated that Mg2+ and ureastabilize the contracted and the expanded confor-mations of the λ capsid, respectively. We havefurther shown that interconversion between the twostructures is highly cooperative and fully reversible.These features allow a rigorous thermodynamiccharacterization of the expansion reaction, whichhas not been possible in any other system. Our dataindicate that the free energy of urea-triggeredprocapsid expansion, ΔG(H2O), is ∼13 kcal/mol inthe presence of 10 mM MgCl2. This translatesdirectly to ∼90 piconewton ·nanometers (pN·nm)of work done to expand an individual capsid (seeSupplementary Data). Is this relevant to DNA-packaging-triggered procapsid expansion in vivo?It is generally presumed that packaging DNA intothe procapsid interior generates pressure thatultimately provides the energy, at least in part,required to trigger expansion.6–8,20 Previous single-molecule studies suggest that expansion is triggeredwhen the terminase motor inserts ∼15 kb duplexDNA into the procapsid§ (see Fig. 1).20 The motorgenerates ∼5 pN force at this point,46,47 whichreflects the energy required to condense the DNAinto the confines of the procapsid interior. To thefirst approximation, this force will generate apressure of 0.06 N/cm2 on the inside of the shell(see Supplementary Data). Expansion increases thediameter of the particle from 50 nm to 60 nm,23,41

which results in a volume increase of 4.5×10− 20 L.This means that the mechanical work performed bythe motor at the point of procapsid expansion is∼70 pN·nm per capsid (work=PΔV), which to the

§A magnesium concentration of 10 mMwas used in thelaser tweezer studies.

first approximation corresponds quite well to thework required to expand the structure with urea invitro (∼90 pN·nm, above). While admittedly quali-tative in nature, these simple calculations indicatethat the free energy of procapsid expansion by ureais commensurate with the work performed by themotor at the point of expansion during DNApackaging. This is discussed further below.

Magnesium stabilization of the procapsid:Possible biological role?

Divalent metals play an essential role in theassembly of many viruses. For example, Ca2+ playsan important role in the assembly and stabilization ofpolyoma virus48–50 and herpes virus51 particles.Similarly, early studies demonstrated an importantrole for Mg2+ in the assembly and stability of aninfectious λ virus.52,53 Chelation of Mg2+ withethylenediaminetetraacetic acid (EDTA) destroys λinfectivity, and it has been proposed that Mg2+

serves as a counterion to maintain DNA in thecondensed state within the nucleocapsid.54 Stabili-zation of the procapsid conformation by divalentmetals as demonstrated here indicates that there is ametal–capsid interaction in addition to the metal–DNA interaction. Indeed, recent structural studieshave identified putative metal-binding sites local-ized at the 3-fold axes on the interior surface of theHK97 procapsid.55 We suggest that this feature isrecapitulated in λ and that this metal–capsidinteraction is responsible, at least in part, forstabilizing the procapsid conformation. This modelhas some interesting predictions.In addition to the pressure generated by the

packaged DNA (vide supra), it is presumed thatduplex interactions with the inner surface of theprocapsid shell trigger the expansion transition;however, mechanistic details of these putativeinteractions remain obscure. We suggest the follow-ing. Condensation of DNAwithin the capsid interioris energetically unfavorable due, in part, to signif-icant charge repulsion by the closely packedphosphodiester backbone. Charge neutralizationby polyamines and divalent metals is required toensure efficient condensation and packaging of thegenome.54–57 The terminase motor translocates over600 bp/s into the capsid interior,20,58 and it istempting to speculate that the rapidly packagedduplex effectively strips the Mg2+ bound at theinterior capsid surface. This would serve to decreasethe free energy requirement for expansion of theshell, perhaps as much as 10 kcal/mol (Fig. 3), andto promote the transition. As shown here, expansionis accompanied by addition of gpD to the capsidsurface, which stabilizes the expanded shell andprevents contraction back to the procapsid state. Inthis situation, divalent metals are free to bind to and


stabilize the condensed DNA. Work currentlyunderway in our laboratory seeks to directlymeasure the duplex length requirement for procap-sid expansion in the presence of various divalentmetals to directly test this hypothesis.

Procapsid expansion exposes hydrophobicsurface area

We propose that the procapsid conformation isstabilized not only by divalent metal but also byhydrophobic interactions between the capsid pro-teins in the contracted shell (see Scheme 1). Theexpansion transition requires disruption of theseinteractions and exposure of hydrophobic patcheson the expanded capsid surface. This hypothesis isbased in part on the observation that urea triggersprocapsid expansion; while the mechanism re-mains controversial, it is generally accepted thaturea disrupts hydrophobic interactions and effective-ly “solvates” hydrophobic residues in water.34,59,60

Thus, in analogy to protein unfolding, urea stabilizesthe expanded capsid conformation and shifts theequilibrium towards the expanded state. The modelis also consistent with the observation that procap-sid expansion is strongly inhibited by salt andincreased temperature, both of which increase thestrength of hydrophobic interactions. For instance,expansion occurs efficiently at 4 °C but not at 25 °C,a temperature range in which the strength ofhydrophobic interactions increase and are maximalat ∼20 °C.59,61 In sum, our data are consistent witha transition that requires overcoming hydrophobicinteractions.The model proposed in Scheme 1 is further

consistent with the large denaturant “m” valueobtained in our thermodynamic analysis of urea-triggered expansion. This value reflects the depen-dence of ΔG on denaturant concentration, which isrelated to the heat capacity change (ΔCp). This inturn is related to the change in hydrophobic surfacearea; the larger the m, the greater hydrophobicsurface area is exposed in the transition.33 The mvalue obtained here (N4 kcal/mol·M) is relativelylarge compared to most protein unfolding reactions(typically 1–3 kcal/mol·M32 , 34,35,62). This indicatesthat a large hydrophobic surface area is exposedupon capsid expansion. Importantly, while theΔG(H2O) for expansion increases with increasingmagnesium concentration, the denaturant m valueremains relatively constant. This indicates that whilemagnesium stabilizes the procapsid state, it does notaffect the change in hydrophobic surface areaexposed upon transition to the expanded conforma-tion. In other words, the conformation of theexpanded state is the same regardless of the Mg2+

concentration, as would be expected of a simpletwo-state transition.

Mechanism for addition of gpD to the expandedcapsid lattice

The terminase motor packages DNA to liquidcrystalline density within the capsid, which gener-ates over 20 atm of internal pressure.20,63–65 Viraldecoration proteins add to the surface of theexpanded capsid structure to stabilize the shellagainst the tremendous internal forces generated bythe tightly packaged DNA.20,38,41 Specifically, the λgpD protein assembles as a trimeric spike at the 3-fold axes of the expanded capsid lattice.23,41 WhilegpD is dispensable for packaging of duplexes up to∼40 kb, packaging of larger duplexes requires gpDto maintain capsid integrity.38

Structural studies indicate that the base of thegpD trimer is hydrophobic, and it has beenproposed that the spike interacts with hydrophobicpatches on the capsid surface;41,66,67 however,biochemical evidence for this model is lacking.Our data indicate that procapsid expansion isassociated with exposure of hydrophobic patcheson the capsid surface. We propose that thesepatches, which are buried in the procapsid confor-mation, are exposed at the icosahedral 3-fold axesof the expanded shell and provide a nucleation sitefor assembly of the gpD trimer. Importantly, gpDadds to the surface of the expanded capsidefficiently at room temperature but only poorly at4 °C (data not shown; see Ref. 40); this temperaturedependence is consistent with an increase inhydrophobic binding energy within this tempera-ture range (vide supra).59,61

Although our data show that expansion of the λprocapsid is fully reversible, gpD addition effectivelylocks the capsid shell into the expanded state. Indeed,large-scale conformational changes accompany DNApackaging in all of the complex dsDNA viruses andthis is intrinsically irreversible. In vivo, these transi-tions are further made irreversible by proteolyticcleavage events, capsid protein cross-linking events,or the addition of decoration proteins as is observed inλ. Subsequently, “finishing” proteins add, in anordered, stepwise sequence, interactions that arenucleated by addition of the previous protein. Inthis manner, the particle progresses to the next step ofassembly to minimize “off-pathway” intermediatesand ensures fidelity in the assembly process.

Why do procapsids expand?

Procapsid expansion is a common feature in thepackaging of viral DNA, but it is not clear why this isnecessary. One possibility is that the assembly of aprocapsid shell provides a mechanism for theterminase packaging motor to select only packag-ing-competent shells. This presumes that pre-ex-panded capsids represent defective, off-pathway

Scheme 1. Model for reversible procapsid expansion and gpD addition. (Upper pathway) Procapsid expansion is triggered by DNA packaging in vivo. Mg2+

strongly stabilizes the procapsid conformation. Packaged DNA binds Mg2+ at the interior procapsid surface, lowering the free energy required for the expansiontransition. gpD trimer spikes assemble at hydrophobic patches exposed at the icosahedral 3-fold axes of the expanded shell lattice and stabilize the particle. (Lowerpathway) Procapsid expansion is triggered by urea in vitro. Mg2+ strongly stabilizes the procapsid conformation and higher concentrations of urea are required totrigger the transition. gpD trimer spikes assemble at hydrophobic patches exposed at the icosahedral 3-fold axes of the expanded shell lattice. The decorated particlesare structurally robust and biologically active.

175Therm

odynamic

Characterization

ofCapsid

Expansion


intermediates that may result from aborted packag-ing events; however, our data demonstrate that theexpanded λ capsid is catalytically competent andpackages DNA quite efficiently. Indeed, this hassimilarly been demonstrated in the bacteriophage T4system.68,69 Thus, while DNApackaging triggers thetransition in vivo, expansion per se does not influencethe capacity of the shell to accept the viral genome.This begs the question as to why viruses utilize a

procapsid structure at all. One possibility proposedby Gertsman et al. is that the extensive interactionsbetween the protein subunits assembled into themature capsid shell are unlikely to form in a singleassembly step.55 This model suggests that theprocapsid represents a metastable intermediate thatis required for fidelity in shell assembly and thatexpansion is a consequence of the transition to thestable mature shell. Such a transition impliesirreversibility; however, our data show that thisneed not be the case.

Conclusions

We have demonstrated that magnesium and ureastabilize the contracted and expanded conforma-tions of the λ capsid shell, respectively. The λsystem is unique in that expansion is fully reversible,which has allowed thermodynamic characterizationof the transition. Expansion of the shell is associatedwith exposed hydrophobic surface area to which thegpD decoration protein adds. This stabilizes theexpanded capsid lattice, abrogates contraction, andprovides structural integrity so that the genome canbe tightly packaged into the capsid interior. Thiswork further provides mechanistic insight into howDNA packaging triggers shell expansion that maybe generalizable to the complex dsDNA viurses,both prokaryotic and eukaryotic.

Materials and Methods

Materials

Tryptone, yeast extract, and agar were purchased fromDIFCO. Molecular biology enzymes and mature λ DNAwere purchased from New England Biolabs. Chromatog-raphymediawere purchased fromGEHealthcare, andureawas purchased from Fisher Scientific. All other materialswere of the highest quality available. Unless otherwisestated, Tris buffers were adjusted to the indicated pH at atemperature of 4 °C. Bacterial cultures were grown inshaker flasks utilizing an Innova 4430 Incubator Shaker. Allprotein purifications utilized the Amersham BiosciencesÄKTApurifier™ core 10 System from GE Healthcare. UV–VIS absorbance spectra were recorded on a Hewlett-Packard HP8452A spectrophotometer. Sucrose densitygradients were prepared using a Biocomp Model 107Gradient Master® and gradient centrifugation utilized aBeckman L-90K ultracentrifuge with an SW 28 rotor.

Protein purification

λ terminase and the Escherichia coli integration hostfactor (IHF) were purified as previously described.70,71

The gpD decoration protein was purified by our publishedprotocol,36 with modification. Briefly, gpD was expressedfrom the pT7Cap vector and ammonium sulfate wasadded to the cell lysis supernatant to 50% saturation. Themixture was gently stirred on ice for 50 min and insolubleprotein was removed by centrifugation (9000g, 20 min).The supernatant was adjusted to 90% ammonium sulfateand stirred on ice for 50 min, and insoluble protein washarvested by centrifugation (30,000g, 30 min). The pelletwas resuspended in 20 mM Tris buffer, pH 8, containing1 mM EDTA and 15 mM NaCl, and the sample wasapplied to a diethylaminoethyl column equilibrated withthe same buffer. The column flow-through fraction, whichcontained gpD, was applied to an S-300 gel-filtrationcolumn equilibrated and developed with 20 mM Trisbuffer, pH 8, containing 1 mM EDTA, and 100 mM NaCl.The gpD-containing fractions were pooled; dialyzed into20 mM Tris buffer, pH 8, containing 1 mM EDTA; andconcentrated to ∼500 μM using an Amicon® centrifugalfilter unit. All of our purified protein preparations werehomogenous as determined by SDS-PAGE (not shown).

Procapsid purification

λ procapsids were purified as described previously,36

but the purified preparations contained variable amountsof pre-expanded procapsids. To obtain a homogenouspreparation of unexpanded capsids, we fractionated thesample on a 0.8% agarose gel, which readily separatesexpanded capsids from unexpanded procapsids (see Fig.2). The lower band was excised with a sterile razor blade,and the unexpanded procapsids were eluted from the gelslice into Buffer A (25 mM Tris base, 192 mM glycine, and1 mM MgCl2) using a Bio-Rad Model 422 Electro-Eluter(100 V for 1 h). The sample was dialyzed into TMB bufferand concentrated using an Amicon® centrifugal filter unit.

Procapsid expansion and buffer exchange protocol

A freshly prepared stock solution of 8 M urea in waterwas used for all of the expansion experiments. Unlessotherwise specified, purified procapsids and 8 M ureawere mixed to afford a reaction mixture (20 μl) containing30 nM procapsids in 10 mM Tris buffer, pH 8, containing2.5 M urea and 3 mM MgCl2. Subsequent modification ofthe expansion reaction composition was accomplished bya buffer exchange protocol. Briefly, a sufficient volume ofbuffer was added to adjust the magnesium and ureaconcentrations as indicated in each experiment and themixture was then concentrated to its original volumeusing an Amicon® centrifugal filter unit. The expansionreaction mixtures were incubated on ice for 15 min, andprocapsid expansion was analyzed by agarose gel assay,as described below.

Analysis of capsid expansion and contraction

The samples were applied to a 0.8% agarose gel, andelectrophoresis was performed at 110 V for 100 min. The


contracted procapsid (lower) and expanded capsid(upper) bands were visualized by staining with Coomas-sie brilliant blue. Video images of the destained gels werecaptured using an EpiChemi3 darkroom system with aHamamatsu camera (UVP Bioimaging Systems) and thevideo images were quantified using either the LabWorks4.6 (UVP Bioimaging Systems) or the ImageQuant(Molecular Dynamics) software packages.

Thermodynamic analysis of urea-triggeredprocapsid expansion

Procapsid expansion in urea is a two-state reversibletransition and we adapted analytical tools developed tocharacterize reversible protein unfolding transitions toanalyze the data, as follows. The fraction of procapsidsin the expanded state was quantified by agarose gelassay (above). The data were fit by nonlinear least-squares analysis using the linear extrapolation methodas outlined by Santoro and Bolen32 according to Eq. (1):

FE=mP4 U½ �+bPð Þ+ mE4 U½ �+bEð Þ4exp −

DGH2O

RT+mG4 U½ �RT

� ��

1+exp −DGH2O

RT+

mG4 U½ �RT

� �� ð1Þ

where FE is the fraction of capsids in the expanded stateas a function of the urea concentration ([U]). The valuesmP and mE represent the slopes, and bP and bE representthe y-intercepts of the pre- and post-transition baselines,respectively. R is the ideal gas constant and T is thetemperature (K). The free energy of procapsid expansion(ΔGH2O; kcal/mol) and the denaturant m value (mG;kcal/mol·M) represent the intercept and slope, respec-tively, of the linear dependence of the expansiontransition energy as a function of denaturant concentra-tion. Each data set was fit to Eq. (1) with mP, bP, mE, bE,ΔGH2O, and mG as parameters using the IGOR®graphics/analysis package (WaveMetrics, Lake Oswego,OR).The urea-expansion data were also fit according to

Eq. (2):

FE = Base +Max

1 + expurea½ �1=2 − urea½ �

mT

� �26664

37775 ð2Þ

where FE is the fraction of capsids in the expanded state asa function of the urea concentration ([urea]). Base andMaxrepresent the pre- and post-transition baselines, mT is thetransition slope, and [urea]1/2 is the concentration of urearequired to expand half of the procapsids to the expandedstate. Each data set was fit to Eq. (2) with Base, Max, C1/2,and mT as parameters using the IGOR® graphics/analysispackage (WaveMetrics).

Analysis of Mg2+-triggered capsid contraction

Expanded capsids were prepared as described above inthe absence and presence of urea at the indicated

concentration. The samples were incubated in thepresence of the indicated concentration of MgCl2 atroom temperature for 5 min and the fraction ofexpanded capsid structures quantified by agarose geland video densitometry as described above. The datawere fit by nonlinear least-squares analysis according toEq. (3):

FE = Base +Max

1 + expMg� �

1=2 − Mg� �

mT

!266664

377775 ð3Þ

where FE is the fraction of capsids in the expanded stateas a function of the Mg2+ concentration ([Mg]). Baseand Max represent the pre- and post-transition base-lines, mT is the transition slope, and [Mg]1/2 is theconcentration of Mg2+ required to contract half of theexpanded shells to the procapsid state. Each data setwas fit to Eq. (3) with Base, Max, [Mg]1/2, and mT asparameters using the IGOR® graphics/analysis package(WaveMetrics).

DNA packaging into expanded capsids

The in vitro DNA packaging reaction was performedas described previously,36 with modification. Briefly,purified procapsids (40 nM capsid; 16.6 μM gpE capsidprotein) were expanded as described above and thenbuffer exchanged into 10 mM Tris buffer, pH 8. PurifiedgpD was then added to a final concentration of 15 μM,and the mixture was incubated for 30 min at roomtemperature. The gpD-coated, expanded capsids wereadded to a reaction mixture containing 50 mM Trisbuffer, pH 7.4, containing 9 mM NaCl, 5 mM MgCl2,2 mM spermidine, 1.3 mM β-ME, 1 mM ATP, 100 nMIHF, and 2 nM mature λ DNA. The packaging reaction(20 μl) was initiated with the addition of terminaseholoenzyme to a final concentration of 100 nM andallowed to proceed for 30 min at room temperature.DNase (10 μg/ml) was then added to the reactionmixture and incubated at room temperature for 5 min.The DNase reaction was stopped with the addition ofphenol:chloroform (21 μl), and the aqueous layer wasremoved and loaded onto a 0.8% agarose gel. Packaged(DNase resistant) DNA was quantified by videodensitometry as previously described.36

Acknowledgements

This work was supported by National ScienceFoundationGrantMCB-0648617 and byWashingtonState Life Sciences Discovery Fund Grant # 2496490.The authorswish to thank Rishi Sanyal for providingthe IHF used in these studies. We are also indebtedto Dr. Tamir Gonen for his help with the electronmicroscopy studies and to Dr. Jenny Chang forhelpful discussions.


Supplementary Data

Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2012.02.020

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thermodynamic characterization of viral procapsid expansion into a functional capsid shell

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