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Molecular Dissection of the Forces Responsible for Viral Capsid Assembly and Stabilization by Decoration Proteins Shannon Lambert, Qin Yang, Rolando Wray De Angelis, Jenny R Chang, Marcos E Ortega, Christal Davis, and Carlos Enrique Catalano Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00705 • Publication Date (Web): 28 Dec 2016 Downloaded from http://pubs.acs.org on January 1, 2017

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Molecular Dissection of the Forces Responsible for Viral Capsid Assembly and Stabilization by Decoration Proteins Running Title: Viral Capsid Assembly and Stabilization

Authors: Shannon Lambert1,§, Qin Yang2,§, Rolando De Angeles2,†, Jenny R. Chang1,‡, Marcos Ortega3, Christal Davis4, and Carlos Enrique Catalano1,¶

Affiliations: 1Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Box 357610, Seattle, WA 98195; 2Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado, Anschutz Medical Campus, Mail Stop C238, Aurora, CO 80045; 3Department of Biology, Macalester College, St. Paul, MN, 55105; Program in Structural Biology and Biochemistry, University of Colorado, Anschutz Medical Campus, Mail Stop C290, Aurora, CO 80045. §

These authors contributed equally to this work.

†

Current Address: BioMarin Pharmaceutical Inc., 105 Digital Dr, Novato, CA 94949

‡

Current Address: Abenza, 360 George Patterson Blvd., Bristol; PA 19007

¶

Corresponding Author (Current Location): Carlos Enrique Catalano, Skaggs School of

Pharmacy and Pharmaceutical Sciences, University of Colorado, Anschutz Medical

ACS Paragon Plus Environment

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Campus, Mail Stop C238, 12850 E. Montview Blvd., Aurora, CO 80045-2605. (303) 724-0011 (office), (303) 724-7266 (fax), [email protected] Key Words Virus Assembly, Capsid Assembly, Capsid Stability, Viral Decoration Protein, Physical Virology

ToC Graphic

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Abstract Complex double-stranded DNA viruses utilize a terminase enzyme to package their genomes into a pre-assembled procapsid shell. DNA packaging triggers a major conformational change of the proteins assembled into the shell and most often subsequent addition of a decoration protein that is required to stabilize the structure. In bacteriophage lambda, DNA packaging drives a procapsid expansion transition to afford a larger, but fragile shell. The gpD decoration protein adds to the expanded shell as trimeric spikes at each of the 140 three-fold axes. The spikes provide mechanical strength to the shell such that it can withstand the tremendous internal forces generated by the packaged DNA, and in addition to environmental insult. Hydrophobic, electrostatic, and aromatic-proline non-covalent interactions have been proposed to mediate gpD trimer spike assembly at the expanded shell surface. Here, we directly examine each of these interactions and demonstrate that hydrophobic interactions play the dominant role. In the course of this study, we unexpectedly found that Trp308 in the lambda major capsid protein (gpE) plays a critical role in shell assembly. The gpEW308A mutation affords a soluble, natively folded protein that does not further assemble into a procapsid shell, despite the fact that it retains binding interactions with the scaffolding protein, the shell assembly chaparone protein. The data support a model in which the lambda procapsid shell assembles via cooperative interaction of monomeric capsid proteins, as is observed in the herpesviruses and phages such as P22. The significance of the results with respect to capsid assembly, maturation, and stability are discussed.

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An essential step in the assembly of complex double-stranded DNA (dsDNA) viruses, both prokaryotic and eukaryotic, is packaging of the viral genome into a protective procapsid shell (1-3). The packaging reaction is strongly conserved in these viruses and is catalyzed by a terminase enzyme (4-7). The terminase proteins specifically bind to viral DNA and to the portal vertex of a pre-assembled shell to afford the functional motor complex; the portal is a doughnut shaped structure that resides at a unique vertex of the icosahedron through which DNA is inserted during packaging and through which it exits during subsequent infection of a cell. The activated motor translocates the duplex into the shell interior, powered by ATP hydrolysis (4, 8). Icosahedral procapsid shells self-assemble by co-polymerization of major capsid proteins and a scaffolding protein (9, 10); the latter acts as a chaperone for high-fidelity shell assembly. In some cases such as phage HK97 the capsid proteins first assemble into capsomers (pentons and hexons) that subsequently assemble into icosahedrons (11)

. In other cases, such as the herpesviruses and phage P22, the scaffolding proteins

co-polymerize with capsid protein monomers that cooperatively assemble into the shells (12, 13)

. In all cases, DNA packaging into the pre-assembled procapsid triggers a major

conformational reorganization of the proteins assembled into the shell, which is often accompanied by shell expansion. This affords a capsid structure that can accept the full-length genome. A variety of strategies are used to stabilize the expanded shell for further DNA packaging (14-18) including (i) increased interactions between adjacent capsid proteins in the shell (13, 19, 20), (ii) autocatalytic chemical cross-linking of the major capsid proteins (21) and (iii) addition of "decoration" proteins (a.k.a., cementing proteins) to the shell surface (22-26). In all cases, the terminase motor continues to package DNA

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until the entirety of the genome has been inserted into the capsid. Finishing proteins and additional structures (tail, envelope, etc.) are finally added to afford an infectious virus particle. Bacteriophage lambda is a prototypic complex dsDNA virus and has served as a model system for interrogation of genetic, biochemical and structural features of virus development. Specifically, our lab has utilized lambda as a platform to characterize the enzymology and biophysical features of the genome packaging reaction using rigorous biochemical approaches (27-34). The packaging pathway outlined in Figure 1A is representative of dsDNA viruses (4). Lambda terminase initiates DNA packaging from a concatemeric precursor compose of multiple genomes linked in a head to tail fashion. Packaging triggers expansion of the procapsid to afford a larger, thinner and more angularized shell. Here we use the term “procapsid” to refer to the contracted shell and “expanded shell” to denote the mature capsid that results from DNA packaging in vivo and urea exposure in vitro (see Figure 1A). The gpD decoration protein adds to the expanded shell in numbers equivalent to the major capsid protein (420) and terminase continues to translocate DNA into the capsid until the entire genome has been inserted. The DNA is packaged to liquid crystalline density, which generates over 20 atmospheres of internal pressure (35-37). Importantly, the expanded shell itself is fragile and ruptures during DNA packaging in the absence of gpD (27, 38, 39). Thus, the decoration protein provides mechanical strength to the shell such that it can withstand the tremendous internal forces generated by the packaged DNA and in addition to environmental insults (26, 40).

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GpD (11.4 kDa) remains a monomer in solution up to 1.3 mM and does not bind to the unexpanded procapsid shell (17, 22, 41). In contrast, gpD proteins assemble as trimeric spikes at each of the 140 three-fold axes of the expanded shell (Figures 1A, 1B). Highresolution structural data reveal that gpD possesses a remarkably low content of regular secondary structure and is predominantly random coil (Figure 1C) (41-43). The N-terminal 14 residues of the folded protein are disordered in solution, but high-resolution cryoelectron microscopy studies suggest that they become ordered upon binding to the expanded shell surface (44). Specifically, the N-termini are proposed to directly interact with three β-strands donated by adjacent capsid proteins positioned at the three-fold axes of the expanded shell. This affords a four-stranded β-sheet structure that is recapitulated 420 times across the capsid surface to provide structural stability to the shell. Additional interactions between the gpD trimer and major capsid proteins assembled into the shell have been proposed, as follows. First, the crystal structure of the gpD trimer reveals that the spike base, which interacts with the capsid surface, presents a large hydrophobic surface area (Figure 2A) (42, 43). This observation led to the hypothesis that hydrophobic interactions play an important role in gpD assembly at the shell surface (42, 43). Within this context, we performed a thermodynamic analysis of the procapsid expansion reaction which revealed that the transition exposes significant hydrophobic surface area (17). Based on these observations we proposed that the exposed hydrophobic residues reside at the three-fold axes of the expanded shell to provide a nucleation site for spike assembly at this site.

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Unfortunately, high-resolution structural data is unavailable for the lambda gpE major capsid protein, which precludes identification of potential interacting hydrophobic residues in the expanded shell lattice. To address this issue, we recently constructed a pseudo-atomic model for gpE assembled into the procapsid shell, based on a computational model for gpE and the high-resolution cryoEM structure of the lambda procapsid (45). Highlighting hydrophobic residues assembled at the three-fold axes reveals hydrophobic spines, which coincide with those presented in the gpD spike (see Figures 2A and 2B). We propose that these residues become highly solvent exposed in the shell expansion transition to provide a hydrophobic nucleation site for spike assembly. In addition to extensive hydrophobic surface area, the crystal structure further reveals alternating rings of three prolines and three histidines that are presented at the spike base (Figure 2C); the authors suggested that these residues may be important in spike assembly at the expanded shell surface (42, 43). Presumably, these residues directly interact with specific residues in gpE assembled at the three-fold axes to promote cooperative trimer assembly and/or stabilization of the shell structure. Close inspection of the three-fold axes in the lambda procapsid shell model reveals a ring of three Asp residues that are poised to interact with the three His residues displayed in the gpD trimer spike, as depicted in Figure 2D. Additionally, an alternating ring of three Trp residues in the shell align with the three Pro residues presented in the gpD trimer spike; aromatic–proline interactions are quite strong and can provide up to 7 kcal/mol binding energy (46, 47). Based on these observations, we previously proposed that the Asp-His electrostatic and Trp-Pro interactions are important for gpD spike assembly

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and/or stabilization of the expanded shell, in addition to the hydrophobic interactions described above (45). In the present study, we employ a mutagenesis approach to directly test our hypotheses and examine the roles that hydrophobic, electrostatic, and aromatic-proline non-covalent bonding interactions play in gpD trimer spike assembly at the expanded shell surface and stabilization of the structure. In the course of this work, we identified mutations in the gpE major capsid protein that strongly affect procapsid assembly. Specifically, the gpD-W308A mutation abrogates procapsid shell assembly while retaining the ability to bind to the scaffolding protein. A robust capsid shell is essential to virus assembly and the forces mediating particle assembly and stability are fundamental to virus development and viability. The results presented here thus have broad biological implications. Experimental Procedures Construction of Mutant gpD Expression Vectors. We previously constructed a vector that expresses the wild-type gpD protein to high yields (48). The QuikChange II sitedirected mutagenesis kit (Agilent) kit was used to change gpD codons 17 (CCC) and 19 (CAT) to alanine (GCG and GCT, respectively) to afford plasmids pD-17A and pD-19A, vectors that express the mutant gpD-P17A and gpD-H19A proteins, respectively. The sequence of each vector was verified to ensure that the desired mutation was present in an otherwise wild-type gene. The plasmids were transfected into E. coli BL21 cells to afford BL21[pD-17A] and BL21[pD-19H] cell lines, respectively.

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Expression and Purification of Wild-Type and Mutant gpD Proteins. One liter cultures were grown in Terrific Broth media at 37˚C to achieve OD600 ~ 0.6 and protein expression was induced with the addition of 1 mM IPTG. Incubation was continued for an additional two hours and the cells were harvested by centrifugation. Purification of the wild-type and mutant gpD proteins was performed as previously described (39). The purity of each preparation was at least 95% as determined by SDS-PAGE (Figure S1A). Construction of Mutant Procapsid Expression Vectors. We previously constructed pT7Cap Dam7am43, a vector that expresses all of the lambda capsid proteins (but not gpD) and from which wild-type procapsids can be isolated in high yield (48). The QuikChange II site-directed mutagenesis kit (Agilent) was used to individually change E gene codon 292 (GAC) to (GCC), and E gene codon 308 (TGG) to (TTT) and (GCG) to afford the plasmids pT7Cap Dam7am43 ED292A, pT7Cap Dam7am43 EW308F, and pT7Cap Dam7am43 EW308A, respectively. The sequence of each vector was verified to ensure that the desired mutation was present in an otherwise wild-type sequence. The plasmids were transfected into E. coli BL21 cells to afford BL21[pT7Cap Dam7am43 ED292A], BL21[pT7Cap Dam7am43 EW308F] and BL21[pT7Cap Dam7am43 EW308A], respectively. Expression and Purification of Wild-Type and Mutant Lambda Procapsids. One liter cultures of each cell line were grown in Terrific Broth media at 37˚C to achieve OD600 ~ 0.6, and protein expression was induced with the addition of 1 mM IPTG. Incubation was continued for an additional two hours and the cells were harvested by centrifugation. The cell pellets were taken into 50 ml of TMS buffer (50 mM Tris, pH 8.0 at 4ºC, containing 10 mM MgCl and 100 mM NaCl) and the cells were lysed by 9


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sonication. The clarified cell lysates possess similar amounts of wild-type and mutant gpE proteins (not shown), indicating that expression of the major capsid protein is not significantly affected by the introduced mutations. Procapsid shells were isolated from the clarified lysate as previously described (49). The purity of the procapsid preparations was at least 95% as determined by SDS-PAGE (not shown) and electron microscopy (see Figure 4). Purification of Soluble gpE-W308A Mutant Protein. A four liter culture of BL21[pT7Cap Dam7am43 EW308A] was grown in Terrific Broth media at 37˚C to achieve OD600 ~ 0.8, and protein expression was induced with the addition of 0.4 mM IPTG. Incubation was continued for an additional two and one half hours and the cells were harvested by centrifugation. The cell pellets were taken into 100 ml of lysis buffer (20 mM Tris, pH 8.0 at 4ºC, buffer containing 500 mM NaCl, 1 mM EDTA and 10% glycerol), lysozyme was added to 0.4 mg/ml and the mixture was incubated on ice for 30 minutes prior to cell lysis by sonication. Insoluble material was removed by centrifugation (27,000 x g x 45 minutes), ammonium sulfate was added to the supernatant to 80% saturation, the mixture was incubated on ice for 20 minutes and the precipitated protein was harvested by centrifugation (27,000 x g x 30 minutes). The ammonium sulfate pellet was taken into 20 ml lysis buffer and dialyzed overnight against one liter dialysis buffer (20 mM Tris, pH 8 at 4˚C, buffer containing 100 mM NaCl and 1 mM EDTA). The dialysate was applied to a DEAE column (60 ml) equilibrated and washed with dialysis buffer and protein was eluted with a five column gradient of dialysis buffer containing 1 M NaCl. Fractions were examined by SDS-PAGE and gpE-W308A containing fractions were pooled and dialyzed against one liter 20 mM

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Tris, pH 8, buffer containing 500 mM NaCl, 1 mM EDTA, and 5% glycerol at 4˚C overnight. The dialysate was concentrated to a volume of 2 ml by centrifugal concentration (Sartorius Vivaspin-20) and the sample was applied to a Sephacryl S300 column SEC equilibrated and developed with dialysis buffer; gpE-W308A eluted as a single peak that was collected and concentrated by centrifugal concentration. The purity of the preparation was at least 95% as determined by SDS-PAGE (Figure S-1B) and the protein concentration was determined spectrally using an ε(280)= 33,350 M-1 cm-1 determined from the known sequence of the protein. Circular Dichroism Spectroscopy. Far UV CD spectra were collected on an Applied Photophysics Chirascan Plus instrument in 20 mM Tris buffer, pH 8.0 at 4˚C, containing 20 mM NaCl and the indicated concentration of protein. The sample was placed in a 0.5 mm quartz cuvette at 20˚C and spectra were recorded at 1 nm intervals with a bandwidth of 1 nm and a 1 second dwell time. Appropriate blank spectra were collected similarly. Ellipticity values were recorded by the instrument as millidegrees and converted to Mean Residue Ellipticity (MRE, θ) as previously described (50). Protein thermal denaturation was performed as above except that data were collected at 222 nm between 4°C and 95°C in 1°C increments. The raw data were converted to Fraction unfolded and fit to single or double sigmoidal curves, as appropriate, to obtain the TM for the transition(s), as described previously (51). Sedimention Velocity Analytical Ultracentrifugation. Sedimentation velocity experiments were performed using a Beckman Coulter XLA analytical ultracentrifuge in 50 mM Tris, pH 8.0 at 4˚C, buffer containing 20 mM MgCl2 and 100 mM NaCl; protein was included as indicated in each individual experiment. The samples were loaded into 11


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two sector Epon-filled charcoal centerpieces and placed in the centrifuge and equilibrated at 4 °C for at least 90 minutes prior to initiation of the experiment. The experiments were performed at 4 °C and at a rotor speed of 50,000 RPM. Absorbance data (280 nm) were collected at 0.003 cm intervals, with 2 replicates per position in the continuous mode. Buffer density and viscosity were calculated based on buffer components and the partial specific volume of each protein were calculated based on their known protein sequences using SEDNTERP (Biomolecular Interactions Technology Center, University of New Hampshire). The SV-AUC data were analyzed using SEDFIT (52). Characterization of gpE-W308A and gpNu3 Binding Interactions. Purified gpEW308A (15 µM) was incubated with the indicated concentration of gpNu3 scaffolding protein in a total volume of 2.5 mL at 4 °C for four hours. The proteins were then concentrated five fold in an Amicon Ultra-0.5 filter (MWCO = 10 kDa) prior to loading onto a Superose 6 10/300 (GE Healthcare) gel filtration column equilibrated with 50 mM Tris buffer, pH 8 at 4 °C, containing 100 mM NaCl and 20 mM MgCl2. The column was developed with the same buffer and absorbance (280 nm) of the eluate was monitored. Fractions (0.5 ml) were collected and analyzed by SDS-PAGE for protein content. Procapsid Expansion and Decoration. Purified procapsids (wild-type or mutant, as indicated) were expanded in vitro as previously described (17). Briefly, purified procapsids (20 nM) were incubated for one hour at 4˚C in 10 mM Tris buffer, pH 8.0 at 4°C, containing 2.5 M urea. The samples were then diluted five fold with 10 mM Tris buffer, pH 8.0 at 4°C, containing 1 mM MgCl2 and the expanded shells concentrated using an Amicon Ultra 100 kDa cut off filter. Thermodynamic characterization of urea12


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triggered procapsid expansion was performed and analyzed as previously described to afford [urea]1/2 (concentration of urea require to expand half the procapsid shells), ∆G(H2O) (the free energy of expansion) and the denaturant m-value. (17) Unless otherwise indicated, shell decoration was performed as previously described (17, 48)

. Briefly, the decoration protein at the indicated concentration was incubated with

expanded procapsids (20 nM, 20 µl) for 60 minutes at room temperature to afford decorated shells. Shell decoration was confirmed by agarose gel assay as previously described (17, 48). Electron Microscopy. Samples for negative stain were prepared by applying 3 µL of procapsids (mutant or wild-type, as indicated) to glow-discharged 300 mesh carbon grids (Electron Microscopy Sciences). The grids were washed thrice with TM buffer (10 mM Tris buffer, pH 8 at 4˚C, containing 15 mM MgCl2) and stained with 2% methylamine tungstate. Transmission electron microscopy was performed at the Electron Microscopy Center in the University of Colorado School of Medicine. DNA Packaging Assay. Packaging activity assays were performed as described previously (29, 49) with slight modification. Full-length λ genomic DNA (1.5 nM) was incubated with 200 nM terminase, 62.5 nM IHF, 20 nM wild-type or gpE-W308F mutant procapsids, as indicated, in 50 mM Tris buffer, pH 8.0 at 4°C, containing 10 mM MgCl2 and 1 mM ATP. Wild-type or mutant gpD protein, as indicated, was included to 10 µM and the reaction was allowed to proceed for 30 minutes at 22°C. DNAse I (0.2 mg/ml) was then added to digest unpackaged DNA and allowed to proceed for five minutes at

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25°C before quenching the reaction with EDTA. Packaged DNA was quantified by gel electrophoresis as previously described. Results Capsid decoration proteins are common in the dsDNA viruses and are required to stabilize the shell from both internal stress and environmental insult. Based on biochemical and structural data, we have proposed multiple non-covalent interactions that might play a role in gpD assembly at the expanded shell surface and/or stabilization of the structure. Here we directly test these hypotheses and define the extent that each plays in these fundamental processes. GpD Assembly at the Expanded Shell: Role of Hydrophobic Interactions. The crystal structure of the gpD trimer reveals a high concentration of hydrophobic residues exposed at the spike base and we have proposed that these residues interact with hydrophobic residues that reside at the three-fold axes of the expanded shell surface (see Figures 2A, 2B). According to this hypothesis, conditions that strengthen hydrophobic interactions will promote gpD decoration of the shell. Here we rely on the observation that the strength of the hydrophobic effect increases with temperature in the range of 4°C to 20°C (53, 54) and we examined the effect of temperature on the decoration reaction. Expanded shells (20 nM) were incubated with increasing concentrations of gpD as described in Experimental Procedures except that the incubation temperature was varied. Representative experiments performed at 22˚C and 4˚C are shown in Figures 3A and 3B, respectively. Consistent with our prior studies, the expanded shell is fully decorated in the presence of one molar equivalent of

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gpD:gpE at 22˚C (Figure 3A, filled arrow) (17, 48, 55). In contrast, gel data from a decoration reaction performed at 4˚C reveals that the shells are only partially decorated at the lower temperature (Figure 3B, open arrow). This result was unanticipated and we therefore performed a more detailed interrogation of shell decoration as a function of temperature, as follows. Expanded shells (20 nM, 8.3 µM gpE) were incubated with a saturating concentration of gpD (10.4 µM) for 60 minutes at the indicated temperature. The fraction of shells that were fully decorated (i.e., 420 copies of gpD) was then quantified by gel assay. The data presented in Figure 3C clearly demonstrate that while the expanded shell can be partially decorated at lower temperature (open arrow), complete decoration (filled arrow) is an endothermic process and strongly temperature dependent between 10˚C and 20˚C (Figure 3D). This result is consistent with our hypothesis that shell decoration is mediated, at least in part, by hydrophobic interactions. GpD Assembly at the Expanded Shell: Roles of Specific Residues. Inspection of the pseudo-atomic shell model reveals a juxtaposition of His-Asp and Pro-Trp residues in the trimer spike and the gpE subunits assembled at the three-fold axes of the shell, respectively (Figures 2C, 2D). We proposed that these ionic and aromatic-proline interactions play a significant role in gpD assembly and/or stability of the decorated shell and we here test this hypothesis using a mutagenesis approach. Biochemical Characterization of Mutant gpD Decoration Proteins. The mutant gpDP17A and gpD-H19A proteins were expressed and purified to homogeneity as described in Experimental Procedures. Yields for the two mutant proteins were equal to that of wild type grown in concert (data not shown), indicating that the mutations do not 15


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result in any gross structural aberrations. The far-UV CD spectra for the two mutant proteins are indistinguishable from that of gpD-WT (Figure S-2A) and are consistent with high-resolution structural data indicating that gpD is composed of primarily random structures (41, 42). Of note, all three spectra possess a strong positive band in the 230 region, which is likely associated with a Trp49-Trp87 exciton interaction in the folded protein (see Figure 1C) (56). This suggests that the Trp49-Trp87 interaction remains intact in the mutant proteins, an indication that their tertiary structures are not perturbed by the mutations. As a further confirmation, we performed thermal stability studies which demonstrate that introduction of the H19A mutation has little effect on the thermal stability of the protein (Figure S-2B, Table 1). Interestingly, however, mutation of Pro17 to Ala results in the appearance of a second, high-temperature transition that is not observed with either gpD-WT or the gpD-H19A mutant protein (Figure S2-B, Table 1). The basis for this subtle observation remains unclear at present. The wild-type gpD protein is a monomer in solution (41, 42) and to confirm that the mutations have not affected quaternary interactions we examined the hydrodynamic properties of the proteins using sedimentation velocity analytical ultracentrifugation. The data presented in Table 2 confirms that gpD-WT and both mutant proteins are monomers in the concentration range of 10 µM to 100 µM. In sum, the data indicate that the mutations cause little to no changes in secondary, tertiary or quaternary structure of the proteins. Expression of Mutant gpE Major Capsid Proteins. We next constructed gpE proteins that harbor mutations complementary to the gpD-P17A and gpD-H19A mutations described above; gpE-W308A, gpE-W308F, and gpE-D292A (see Figure 2D). The 16


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proteins were expressed, insoluble material was removed by low-speed centrifugation and assembled shells were harvested from the clarified supernatant by high-speed centrifugation (Experimental Procedures). The majority of the gpE-WT, gpE-W308F and gpE-D292A proteins were found in the high-speed pellet as anticipated for an assembled particle (17, 49). Quite unexpectedly, however, virtually all of the gpE-W308A capsid protein was located in the supernatant fraction of the high-speed centrifugation (not shown). This suggests that gpE-W308A does not assemble into procapsid shells and this surprising result is examined further below. Characterization of Mutant gpE-D292A Procapsid Shells. The majority of the expressed gpE-D292A mutant protein was found in the pellet fraction of the high-speed centrifugation spin as expected of an assembled particle. The protein purified in a similar fashion and with similar yield to wild type gpE procapsids (data not shown). The purified particles were examined by electron microscopy (EM), which revealed that while a fraction of gpE-D292A assembles into shells with wild-type morphology the majority of the protein assembles into a variety of malformed and aberrant structures (compare Figures 4A and 4B). Further, the gpE-D292A particles have markedly decreased solubility compared to wild type procapsids and are highly prone to aggregation. This feature precludes the use of mutant gpE-D292A shells in biochemical studies and the particles were not investigated further. Nevertheless, it is clear that the gpE-D292A mutation, which resides at the three-fold axes of the assembled shell, affords a major capsid protein that has a significant defect in high fidelity procapsid assembly.

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Characterization of Mutant gpE-W308F Procapsid Shells. The conservative gpEW308F mutation affords particles that purify in a similar fashion and with similar yield to wild type gpE procapsids (data not shown). This indicates that the mutation does not have a major effect on particle assembly. To confirm this presumption purified particles were examined by EM, which revealed morphology indistinguishable from that of wildtype shells at this resolution (compare Figures 4A, Figure 4C). We note, however, one important difference. Wild-type lambda procapsids are isolated as a mixture of contracted procapsids and expanded shells in proportions that vary with each preparation (17); a similar observation has been made with purified bacteriophage T4 capsids (57, 58). In contrast, the gpE-W308F mutant protein assembles shells that are resistant to expansion and the preparations contain almost exclusively contracted procapsids (data not shown). We interpret this observation to indicate that the gpEW308F mutation stabilizes the procapsid conformation. We next examined the mutant procapsids for their capacity to undergo ureatriggered expansion, a measure of shell functionality (17). The data presented in Figure 4D demonstrate that the gpE-W308F procapsids can indeed be expanded in vitro and indicate that the shells are functional in this respect; however, several features of the expansion curve are noteworthy. The first is the sloping pre-transition baseline for the wild-type procapsids. This has been noted previously and suggests that there is some heterogeneity in the wild-type procapsid preparation with a fraction (~ 15%) expanding in a stochastic manner (17). In contrast, the pre-transition baseline for the mutant gpEW308F procapsids is flat. This is consistent with our hypothesis that the mutation stabilizes the procapsid conformation (vide supra), which affords a homogenous

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preparation and a thus a single expansion transition with a flat pre-transition baseline. Second, higher concentrations of urea are required to expand the mutant shells. This observation is also consistent with our hypothesis that the gpE-W308F mutation stabilizes the procapsid conformation. Third, thermodynamic analysis of the transition reveals that the free energy of expansion [∆G(H2O)] and the denaturant "m-value" for the mutant procapsids are significantly less that those observed for the wild-type procapsids (Table 3). This indicates that less hydrophobic surface area is exposed during expansion of the mutant shells (59). These features are discussed further below. GpD Assembly at the Expanded Shell: Effect of Site Specific Mutations. We next test our hypotheses that Asp-His electrostatic and Trp-Pro interactions are important for gpD spike assembly and/or stabilization of the expanded shell. The data presented in Figure 5A demonstrate gpD-P17A and gpD-H19A each bind to wild-type expanded shells in a manner similar to that observed with the wild-type decoration protein. Consistently, gpDWT binds to the mutant gpE-W308F expanded shell in a manner similar to that observed with the wild-type expanded shell (compare Figures 5A and 5B). Finally, the data presented in Figure 5B demonstrate that gpD-P17A and gpD-H19A bind similarly to the gpE-W308F mutant shell. Thus, contrary to our prediction none of the mutations, alone or in combination have a significant effect on gpD trimer assembly at the expanded shell surface. Genome Packaging Into the Decorated Shells: Effect of Site Specific Mutations. A more rigorous measure of shell function is their capacity to accept a full-length genome. Packaging of a lambda genome is strictly dependent on efficient decoration of the capsid and stabilization of the expanded shell with gpD. In the absence of gpD, no 19


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packaging is observed, presumably due to rupture of the shell at high filling densities (39)

. The data presented in Figure 5C show that both the gpD-P17A and gpD-H19A

mutant proteins support packaging of a lambda genome into wild-type capsids, with only modest defects relative to wild-type gpD. The gpE-W308F mutant capsids also sponsor packaging when gpD-WT is used to decorate the shells, albeit at a slightly lower efficiency (Figure 5D). A more severe defect is observed when either mutant gpD protein is used to decorate the mutant gpE-W308F capsids, with a 25% reduction in packaging efficiency observed relative to wild type. The gpE-W308A Major Capsid Protein is a Soluble and Folded Monomer in Solution. In contrast to the gpE-D292A and gpE-W308F mutations described above, the mutant gpE-W308A capsid protein does not assemble into shells when expressed in E. coli and we here we further characterize this unusual capsid protein. The far-UV CD spectrum of gpE-W308A indicates that that the protein possesses significant secondary structures (Figure 6A). Comparison of the CD spectrum of gpE-W308A in solution with that of wild type gpE assembled into shells shows that the mutant protein possesses greater helical character (~10%, Table 4). While the structural basis for this is not known, we speculate that this results from the formation of β-strands (at the expense of helical character) when soluble gpE assembles into shells. This hypothesis is based on the observation that an intermolecular three-strand β-sheet is formed between major capsid subunits assembled at the three-fold axes of the HK97 shell (44); this may be a general feature of icosahedral shell assembly but that is abrogate by the W308A mutation.

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The CD data suggest that the mutant gpE-W308A protein is folded in solution and to further demonstrate this point we examined thermal denaturation of the protein. The data presented in Figure 6B reveals a relatively broad, but cooperative unfolding transition (TM= 54.4 ± 0.5 ˚C); unfortunately, unfolding is irreversible which precludes a rigorous thermodynamic analysis of the transition. Thermal unfolding of gpE-WT assembled into shells indicates that the protein is much more stable (TM= 80.8 ± 0.1 ˚C). This strongly cooperative transition likely represents concomitant disassembly of the shell and irreversible unfolding of the wild-type protein. Indeed, gpE-WT shows a strong tendency to aggregate in the absence of denaturant (51), not unexpected given its role in assembling a capsid shell. In contrast, gpE-W308A remains a soluble monomer in the concentration range of 5 µM to 80 µM, as determined by size exclusion chromatography (Figure 6C). As anticipated for a soluble monomeric protein of this size, no discernible higher order structures are observed in electron micrographs (data not shown). In sum, purified gpE-W308A is a soluble, natively folded, and stable monomer in solution that unlike the wild-type protein shows no tendency to self-associate. gpE-W308A Binds to the Scaffolding Protein. That gpE-W308A remains a soluble monomer at elevated concentrations is surprising and indicates that the mutation specifically introduces a severe defect in self-association required for shell assembly. The lambda scaffolding protein (gpNu3) "chaperones" assembly of the major capsid protein into an icosahedral shell and we next asked whether these interactions have been affected by the mutation. Purified gpNu3 and gpE-W308 proteins were mixed and incubated at 4˚C for four hours as described in Experimental Procedures. The mixture was analyzed by size exclusion chromatography and relevant chromatograms are

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presented in Figure 6D. The data clearly show that incubation of the mutant protein with gpNu3 affords a complex of the two. Importantly, while gpNu3 binding to wild-type capsid protein results in co-polymerization into icosahedral shells, scaffold binding to gpE-W308A only affords a small complex, likely composed of only one or two capsid proteins. Thus, the mutant protein retains the capacity to bind to the scaffolding protein but nevertheless remains defective in higher-order interactions required to assemble a shell. Discussion Viral capsids provide a protein coat that is essential to protection of the viral genome in the environment. In complex dsDNA viruses, the capsid is an icosahedral shell composed of one or a few proteins into which viral DNA is packaged by a terminase motor complex (1, 3, 4). These motors are extremely powerful and can package DNA to liquid crystalline density, which can generate over 20 atmospheres of internal pressure (35, 36)

. Thus, the shell must be quite robust in order to withstand these packaging forces

and a variety of mechanisms have evolved to ensure shell integrity (see Introduction). In the herpesviruses and several bacteriophages, a decoration protein adds to the exterior surface of the expanded shell to stabilize the structure (22, 26). The lambda gpD decoration protein is essential for packaging the viral genome and in its absence only sub genomic lengths of DNA can be packaged without loss of shell integrity (39). In addition, the capsid must survive a multitude of sub-lethal insults in the environment. Within this context, we recently reported that gpD provides significant mechanical reinforcement to the shell such that it can withstand sub-lethal collisions that resemble environmental insults (40). 22


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Forces Mediating gpD Addition to the Expanded Shell. While it is clear that decoration proteins are essential to capsid integrity, the non-covalent forces mediating structural stability are less well defined. Based on structural studies, Johnson and coworkers proposed that the N-terminal 14 residues of gpD interact with three β-strands donated by adjacent capsid proteins at the three-fold axes of the lambda capsid (44). This affords a four-stranded β-sheet structure that is recapitulated 420 times across the capsid surface and is predicted to provide a significant stabilizing force to the shell. Importantly, this model is consistent with genetic studies demonstrating that these residues are required for virus viability in vivo (38); however, we suggest that this is only part of the story and the studies reported here provide insight into additional stabilizing forces. DNA packaging triggers a procapsid expansion transition at which point gpD cooperatively adds to the shell surface (Figure 1A) (39). Thermodynamic analysis of the expansion transition reveals that hydrophobic residues sequestered in the procapsid are exposed during shell expansion (17). We hypothesized that these exposed residues reside at the three-fold axes of the shell and that this provides a nucleation site for gpD trimer spike assembly. The data presented here demonstrate that gpD addition is endothermic and strongly affected by temperature in the 10˚C - 20˚C range, consistent with a hydrophobic decoration reaction. At lower temperatures the shell is only partially decorated despite the fact that gpD is present at saturating concentrations. The nature of the partially decorated shells is unclear. One possibility centers on the observation that while the icosahedral shell contains 140 three-fold axes, only 20 of these are true icosahedral three-folds with the rest exhibiting local "pseudo-" or "quasi-" three-fold

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symmetry. Thus, partial decoration at low temperature may reflect differential trimer assembly at selective three-fold axes, leaving the others unoccupied. Alternatively, it is feasible that that spike assembly is uniformly stalled at the dimer stage at low temperature and that addition of the third subunit is strongly temperature dependent. Within this context, we note that the trimer subunit interfaces are also hydrophobic (42). Thus, an assembled dimer provides an expansive hydrophobic platform for addition of the third subunit, which is consistent with the strong temperature effects observed. Structural studies are underway to characterize these partially decorated intermediates. We further postulated that specific ionic (His19-Asp292) and proline-aromatic (Pro17-W308) interactions between the gpD trimer and gpE assembled into the capsid are important; however, a mutational analysis of these interactions indicates that they play a modest role in gpD spike assembly. A greater effect is observed when genome packaging is examined, but the effect is similarly modest. It is feasible that these interactions play a more prominent role in subsequent assembly steps leading to an infectious virus particle and/or survivability of the virus in the environment. Notwithstanding, the current data force us to accept the null hypothesis that neither of these residues play a major role in gpD assembly at the expanded shell nor in stabilization of the structure. Polymerization Pathway for Procapsid Assembly. An unexpected outcome of these studies is the observation that the gpE-D292A and gpE-W308A mutations, which reside at the three-fold axes afford major capsid proteins that are defective in shell assembly. Most strikingly, the gpE-W308A mutation affords a soluble protein that remains a monomer in solution up to 80 µM. The mutant protein is folded, stable and retains the 24


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Biochemistry

capacity to bind to the gpNu3 scaffolding protein, but nevertheless fails to assemble into shells. Thus, the gpE-W308A mutation fails to assemble beyond the monomer state despite the fact that gpNu3 "chaperone" interactions remain intact. This indicates that Trp308 plays a major role in higher-order interactions required to assemble a native, icosahedral capsid shell. Trp308 resides at a position distant from the gpE-gpE interface in the capsomers (pentamers and hexamers) that make up the icosahedron (Figure 7) and this peripheral mutation would not be expected to affect capsomer assembly. That gpE-W308A capsomers are not observed in solution nor in micrographs indicates that these intermediates do not play a major role in the procapsid assembly pathway. This in turn suggests that lambda procapsids assemble via the stepwise interaction of monomeric proteins to the nascent shell, as is observed in the herpesviruses and phages such as P22 (13). Trp308 lies at the center of the three-fold axis of the assembled shell (Figure 7) and we speculate that gpE trimers, the assembly of which is abrogated by the W308A mutation, play an important role in shell assembly. Consistent with this hypothesis, the gpE-D292A mutation, which also lies at the center of the three-fold axis, assembles heterogeneous, aberrantly shaped shells. The conservative gpE-W308F mutation further demonstrates that residues residing at the three fold axes affect shell assembly and stability. While this mutant protein assembles shells that are structurally indistinguishable from wild type procapsids, subtle differences are observed and the ensemble of data suggest that this mutation stabilizes the procapsid conformation of the shell. We previously postulated that (i) hydrophobic interactions between capsid proteins assembled into the shell stabilize the procapsid 25


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conformation and that (ii) the expansion transition exposes these hydrophobic residues for interaction with gpD (17). This predicts that increasing the strength of this putative interaction will favor the procapsid conformation and thus increase the concentration of urea required to trigger shell expansion. Phenylalanine is more hydrophobic than tryptophan (60) and consistently both of these predictions are observed with the gpEW308F shells. We note that the free energy of expansion and the denaturant m-value for the mutant procapsids are less than those observed with the wild-type shells. On the surface this may seem counter intuitive; however, this simply indicates that less hydrophobic surface area is exposed in the expansion transition of the mutant shells compared to wild type. This may similarly reflect stronger hydrophobic subunit interactions with the gpE-W308F mutant that interfere with complete expansion of the shells in isolation. Presumably gpD binding to the mutant shells drives expansion to completion to allow complete decoration and stabilization of the capsids. Conclusions. The data presented here indicate that the three fold axes of the lambda capsid are central to shell assembly, maturation and decoration by gpD. This decoration protein provides capsid stabilization by numerous and redundant non-covalent proteinprotein interactions. It is likely that this “polyvalent” stabilization approach is a common feature of all virus particles, whether this be via a decoration protein, inherent interactions between the proteins in the expanded shell, and even in those viruses that employ physical cross-linking of their capsid proteins. These redundant interactions may allow viruses to explore mutational space to avoid cellular antiviral systems with minimal effects on virus viability, since attenuating one or a few interactions may not significantly affect particle stability. All viruses utilized a protein coat to protect their genomes in the

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environment and we suggest that similar redundant binding interactions are generally utilized to ensure long-term virus survival.

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Associated Content Supporting Information SDS-PAGE image showing purity of our protein preparations. CD spectra and thermal denaturation data for the mutant decoration proteins. This material is available free of charge via the Internet at http://pubs.acs.org.

Author Information Corresponding Author. Carlos Enrique Catalano, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado, Anschutz Medical Campus, Mail Stop C238, 12850 E. Montview Blvd., Aurora, CO 80045-2605. (303) 724-0011 (office), (303) 724-7266 (fax), [email protected]

Funding. This work was supported by National Science Foundation grant # 1550993.

Notes. The authors declare no competing financial interest.

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Biochemistry

51. Medina, M. M., Andrews, B. T., Nakatani, E., and Catalano, C. E. (2011) The Bacteriophage Lambda gpNu3 Scaffolding Protein is an Intrinsically Disordered and Biologically Functional Procapsid Assembly Catalyst, Journal of Molecular Biology 412, 723-736. 52. Lebowitz, J., Lewis, M. S., and Schuck, P. (2002) Modern Analytical Ultracentrifugation in Protein Science: A Tutorial Review, Protein Science 11, 20672079. 53. Baldwin, R. L. (1986) Temperature Dependence of the Hydrophobic Ineraction in Protein Folding, Proc Natl Acad Sci U S A 83, 8069-8072. 54. Schellman, J. A. (1997) Temperature, Stability, and the Hydrophobic Intereaction, Biophysical J. 73, 2960-2964. 55. Kruse, S. V. (2015) Thermodynamic and Directed Physical Characterization of Bacteriophage Lambda Capsid Maturation, In Medicinal Chemistry, p 110, University of Washington, Seattle, Washington. 56. Woody, R. W., and Dunker, A. K. (1996) Aromatic and Cystine Side-Chain Circular Dichroism in Proteins, In Circular Dichroism and the Conformational Analysis of Biomolecules (Fasman, G. D., Ed.), pp 109-151, Springer Science+Business Media, New York, N.Y. 57. Rao, V. B., and Black, L. W. (1985) DNA packaging of bacteriophage T4 proheads in vitro. Evidence that prohead expansion is not coupled to DNA packaging, J Mol Biol 185, 565-578. 58. Zhang, Z., Kottadiel, V. I., Vafabakhsh, R., Dai, L., Chemla, Y. R., Ha, T., and Rao, V. B. (2011) A Promiscuous DNA Packaging Machine from Bacteriophage T4, PLoS Biol 9, e1000592. 59. Bolen, D. W., and Santoro, M. M. (1988) Unfolding free energy changes determined by the linear extrapolation method. 2. Incorporation of delta G degrees N-U values in a thermodynamic cycle, Biochemistry 18, 8069-8074. 60. Rose, G. D., Geselowitz, A. H., and Zehfus, M. H. (1985) Hydrophobicity of Amino Acid ResiduesinGlobularProteins, Science 229, 834-838. 61. Louis-Jeune, C., Andrade-Navarro, M. A., and Perez-Iratxeta, C. (2012) Prediction of protein secondary structure from circular dichroism using theoretically derived spectra, Proteins: Structure, Function, and Bioinformatics 80, 374-381.

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Biochemistry

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Tables

Table 1. Thermal Stability of Wild-Type and Mutant gpD Proteins.

TM (˚C) gpD-WT

60.1 ± 0.1

gpD-H19A

60.1 ± 0.2

gpD-P17A

61.4 ± 0.4 78.6 ± 1.4

The thermal denaturation data presented in Figure S-2B were analyzed as described in Experimental Procedures. The data for gpE-WT and gpD-H19A were fit to a single transition model and the data for gpD-P17A were fit to a double transition model. The best-fit (TM ± standard deviation) is presented in the table.

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Biochemistry

Table 2. SV-AUC Analysis of Wild-Type and Mutant gpD Proteins.

Calculated Molecular Weight (Da) a

Construct

Experimental Molecular Weight (kDa)

10 µM

100 µM

gpD-WT

11,572

11.5 ± 0.3

11.0 ± 0.1

gpD-H19A

11,506

10.7 ± 0.4

11.5 ± 0.1

gpD-P17A

11,546

11.4 ± 0.2

11.6 ± 0.1

The purified gpD proteins at the indicated concentrations were examined by SV-AUC and the data analyzed using SEDFIT as described in Experimental Procedures. The (experimental molecular weight ± standard deviation) is presented in the table. a

Calculated based on the known protein sequence.

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Table 3. Urea-Triggered Procapsid Expansion

Procapsids

C1/2 a

∆G(H2O)

m

gpE-WT

1.5 molar

12.8 ± 1.0 kcal/mol

8.1 ± 0.6 kcal/mol•M

gpE-W308F

2.2 molar

6.0 ± 0.8 kcal/mol

3.3 ± 0.8 kcal/mol•M

The data presented in Figure 4D were analyzed as described in Experimental Procedures to afford the thermodynamic parameters presented in the table. The best fit of each data set are displayed as solid lines superimposed on the data in Figure 4D. a

C1/2 is the concentration of urea required to expand half of the procapsids.

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Biochemistry

Table 4. Secondary Structure of gpE-W308A and the Lambda Procapsid.

Protein

α Helix

β-Strand

"Other"

Soluble gpE-W308A a

36%

15%

49%

Lambda Procapsid (gpE-WT) b

24%

22%

54%

a

Calculated from the far-UV CD data presented in Figure 4A using K2D3 (61).

b

Calculated from the far-UV CD data presented in Figure 4A using K2D3 (61).

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Biochemistry

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Figure Legends Figure 1. Lambda DNA Packaging Pathway and Model for Capsid Shell Decoration. Panel A. DNA packaging triggers procapsid expansion, which exposes hydrophobic surface area in the shell (depicted as blue ovals). This provides a nucleation site for cooperatively assembly of gpD monomers as trimer spikes at the 140 three-fold axes of the expanded shell. Panel B. Cryo-EM structure of gpD trimer spikes assembled at the three-fold axes of the lambda procapsid (Electron Microscopy Data Bank #EMD-5012). The major capsid protein has been deleted from the structure for clarity. Note that while the N-terminus of the gpD monomer is disordered in solution, density extending from the trimer spikes assembled on the capsid shell is attributed to ordering of the gpD N-terminus. Panel C. High Resolution NMR Structure of gpD. Trp49 and Trp87 are shown as red spheres. Figure 2. Proposed Molecular Interactions between the gpD Trimer Spike and the gpE Major Capsid Protein Assembled at a Shell Three-Fold Axis. Panel A. Crystal structure of the gpD trimer spike (PDB IC5E) shown in surface representation, viewed from the spike base outwards from the capsid surface. Hydrophobic residues are colored yellow. Panel B. Structural model for gpE trimers residing at the three-fold axes of the lambda procapsid shell, viewed from the shell exterior (45). Hydrophobic residues, colored yellow, are sterically hindered in the procapsid conformation but presumably become solvent exposed in the expanded capsid shell. This provides a complementary hydrophobic nucleation site for assembly of the gpD trimer spike. Panel C. Crystal structure of the gpD trimer spike (PDB IC5E) in the same orientation as in Panel A. Proline 17 and Histidine 19 are colored yellow and purple-blue, respectively. Panel D. 38


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Biochemistry

Superposition of gpD-Pro17 (yellow) and gpD-His19 (purple-blue) in the gpD trimer spike with gpE-Trp308 (blue) and gpEAsp292 (red) trimers assembled at the three-fold shell axis, respectively, viewed from the external capsid surface. Figure 3. Hydrophobic Interactions Mediate gpD Trimer Spike Assembly. Panel A. Agarose gel showing the concentration dependence of gpD addition to expanded shells (20 nM capsids, 8.4 µM gpE) at 22˚C. GpD was added at the molar ratios indicated at the top of each lane. We note that a gpD trimer assembles at each of the 140 three-fold axes of the shell, or 420 copies per capsid. A lambda capsid contains 415 copies of the major capsid protein, gpE. Thus, a capsid concentration of 20 nM contains 8.3 µM major capsid protein (gpE) and requires 8.4 µM gpD to fully decorate the expanded shell. The solid arrow at right of the gel shows the migration of a fully decorated shell (420 copies of gpD assembled into 140 trimer spikes). Panel B. Agarose gel showing the concentration dependence of gpD addition to expanded shells as described in Panel A except that the reaction was performed at 4˚C. The lane labeled 1.25* indicates a binding reaction that was performed at 22˚C for comparison. Note that at a gpD:gpE ratio of 1.25, the shells are completely decorated at 22˚C (solid arrow) but are only partially decorated at 4˚C (open arrow). Panel C. Expanded shells were decorated at a gpD:gpE ratio of 1.25 as described in Panels A and B except that the reaction was performed at the indicated temperature. Expanded, undecorated shells are indicated with a dashed arrow at left. Migration of the partially decorated shells is indicated with an open arrow (left) while migration of the fully decorated shells is indicated with a solid arrow (right). Panel D. The representative data presented in Panel C were quantified in triplicate and the fraction of fully-decorated plotted as a function of

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Biochemistry

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temperature. The solid line represents the best fit of the data to a sigmoidal curve function and is meant only to guide the eye. Figure 4. Structural Characterization of gpE-D292A and gpE-W308F Particles. Panel A. Micrographs of gpE-WT procapsids. Panel B. Micrographs of gpE-D292A procapsids. Note the presence of small shells and numerous aberrant structures. Panel C. Micrographs of gpE-W308F procapsids show a phenotype indistinguishable from wild-type procapsids. Note the absence of pre-expanded shells in the mutant preparation. Panel D. Urea-triggered Procapsid Expansion. gpE-WT () and gpEW308F () procapsids were expanded with urea at the indicated concentration and the fraction of expanded capsids was quantified and is plotted. Each data point represents the average of at least 3 independent experiments with standard deviation indicated with error bars. The solid lines represent best fits of the data according to a linearextrapolation model for shell expansion, as described in Experimental Procedures. Thermodynamic parameters for this analysis are presented in Table 3. Figure 5. Biological Activity of the Mutant Decoration Proteins and gpE-W308F Procapsids. Panel A. Wild-type and mutant gpD constructs bind efficiently to gpE-WT procapsids. Each protein (18 µM) was incubated with 20 nM WT procapsids, as indicated, for 60 minutes at 22˚C and shell decoration examined by gel assay. Further addition of either gpD did not result in additional shift in migration of the decorated shells, indicating that all bound efficiently at one stoichiometric equivalent. Panel B. Wild-type and mutant gpD constructs bind efficiently to mutant gpE-W308F procapsids. Each protein (25 µM) was incubated with 20 nM procapsids, as indicated, for 60 minutes at 22˚C and shell decoration examined by gel assay. Note that particles 40


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Biochemistry

decorated with the mutant gpD proteins migrate slightly slower relative to gpD-WT. The mechanistic basis for this observation is unclear. Panel C. Genome packaging into gpEWT procapsids decorated with the indicated decoration protein. Panel D. Genome packaging into gpE-W308F procapsids decorated with the indicated decoration protein. The packaging reactions were preformed as described in Experimental Procedures. Each bar represents the average of results from three separate experiments and the error bars represent standard deviation. All data are normalized to WT procapsids decorated with WT gpD. Figure 6. Characterization of Soluble gpE-W308A. Panel A. Far-UV CD spectrum of soluble gpE-W308A (, 0.2 mg/ml) and gpE-WT assembled into procapsid shells (, 0.19 mg/ml). Spectra were recorded at 4˚C. Modeling of the CD data suggests that the soluble gpE-W308A protein possesses greater α-helical content than gpE-WT assembled into shells (Table 3). Panel B. Thermal denaturation of soluble gpE-W308A () and gpE-WT assembled into procapsids () monitored by far-UV CD (220 nm). The data were analyzed as described in Experimental Procedures, which yielded TM= 54.4 ± 0.5 ˚C and TM= 80.8 ± 0.1 ˚C for gpE-W308A and gpE-WT shells, respectively. Panel C. Chromatogram of gpE-W308A eluting from a Superose 6 10/30 column (SEC) at a concentration of 5 µM (dashed line) and 80 µM (solid line). Elution of albumin (66 kDa, 16.45 mL) and carbonic anhydrase (29 kDa, 18.01 mL) molecular weight standards is indicated with black arrowheads. Panel D. SEC chromatogram showing elution of gpEW308A (15 µM) from the column in the absence (dashed line) and in the presence (solid line) of gpNu3 (64 µM). Note that gpNu3 does not contain any aromatic residues and is not observed in the chromatogram. Inset. SDS-PAGE analysis of fractions eluting

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Biochemistry

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from the column; gel 1, gpE-W308 alone; gel 2, gpNu3 alone; gel 3, gpE-W308 plus gpNu3. GpE and gpNu3 are indicate with a “E” and “N”, respectively, at the right of each gel. The elution of gpE-W308 is shifted to a smaller volume indicating a direct interaction with the scaffolding protein. Figure 7. Structural model for gpE major capsid protein assembled into hexamers (left), pentamers (center), and at the three-fold axes, viewed from the shell interior. GpEW308 is depicted as red spheres.

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Biochemistry

Figure 1.

A

Expanded Shell

Procapsid Terminase DNA

Procapsid Expansion

DNA Packaging

ΔG(H2O)= 3 kcal/mol

ATP ADP

B

C


gpD Addition

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2.

A

B

C

D


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Figure 3.

A

gpD:gpE Ratio (22 deg) 0

0.1

0.2

0.3

0.4 0.5

0.6 0.75 0.9 1.25

C

B

D Temperature (C) 4

6

8

10

12

14

18

22

25

gpD:gpE Ratio (4 deg) 0

Fraction Decorated (F)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

0.1

0.2

0.3 0.45 0.6 0.75 0.9 1.25 1.25*

1.0 0.8 0.6 0.4 0.2 0.0 5

10

15

20

25

Temperature (˚C)


30

35

Biochemistry

Figure 4.

A

B

C

D

Fraction Expanded

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1.0 0.8 0.6 0.4 0.2 0.0 0


1 2 3 [Urea] (molar)

4

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Figure 5. gpD Addition and Genome Packaging

Wild Type Procapsids

B

gpE-W308F Procapsids

D

1.0

Relative Packaging Efficiency

C

0.8 0.6 0.4 0.2 0.0

no ne gp D -W gp T D -P gp 17A D -H 19 A

no ne gp D -W T gp D -P gp 17A D -H 19 A

A

Relative Packaging Efficiency

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

WT

P17A

H19A

1.0 0.8 0.6 0.4 0.2 0.0

gpE-WT Capsids


WT

P17A

H19A

gpE-W308F Capsids

Biochemistry

0 -5 -10 -15 200

220

240

Fraction Unfolded

-1

B

5

2

A

MRE (deg-cm -dmol )

Figure 6.

1.0

0.5

0.0 20 40 60 80 Temperature (˚C)

260

C

D

80 60 40 20 0

Absorbance (280 nm)

Wavelength (nm)

Absorbance (280 nM)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 48 of 50

600 14 15 16 17 18

500

1

E

400

2

N

300

E 3

200

N

100 0

14 16 18 20 22 Elution Volume (mL)


10

15

20

25

30

35

Elution Volume (mL)

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Biochemistry

Figure 7.


Biochemistry

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ToC Graphic


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Molecular Dissection of the Forces Responsible ... - ACS Publications

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