1. Volume 14, Issue 5, Pages 559-569 (June 2004)
Control of Crosslinking by Quaternary Structure Changes during Bacteriophage HK97 Maturation 
Lu Gan, James F Conway, Brian A Firek, Naiqian Cheng, Roger W Hendrix, Alasdair C Steven, John E Johnson, Robert L Duda 
Molecular Cell 
Volume 14, Issue 5, Pages (June 2004)
DOI: /j.molcel

2. Figure 1 Assembly and In Vitro Maturation of HK97 Capsids
(A) Assembly and processing. HK97 proheads assemble when capsid protein gp5 and protease gp4 are coexpressed in E. coli. Prohead I, a transient intermediate, consists of a shell formed from 60 hexamers and 12 pentamers of gp5 subunits with ∼60 copies of gp4 inside. (When present, a dodecamer of portal protein gp3 replaces one pentamer.) The gp4 protease digests the N-terminal 102 residues of each gp5 and itself into fragments that exit from the shell to create Prohead II. Prohead II can be induced to expand in vitro at pH 4 (via expansion intermediates EI-I, EI-II, etc.) and further mature upon neutralization.
(B) The in vitro pH-controlled sequential pathway. In this older model expansion can be induced at low pH, but crosslinking does not begin until the entire structure has undergone the transition to Head I, identical to Head II, but without crosslinks.
(C) The in vitro pH-controlled concurrent pathway. In the revised model of expansion, crosslinking begins early in the expansion process. EI-I can revert to Prohead II. The first crosslinks are found at the EI-II stage. The end product of either pathway is Head II, which has isopeptide bond crosslinks between pairs of subunits at residues K169 and N356.
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2
Expansion and Crosslinking Are Concurrent in HK97 Maturation at Low pH
(A) Crosslinking during low pH expansion. HK97 Prohead II was diluted 8-fold into pH 4.2 buffer to induce expansion; samples were taken at the times indicated, rapidly denatured with TCA, and analyzed by SDS-PAGE as described in the Experimental Procedures.
(B) HK97 particles present during low pH expansion. Types of particles observed by cryo-electron microscopy under identical conditions as in (A) and (C) summarized from a previous study (Lata et al., 2000). Increasing bar thickness indicates increasing relative proportions of particles: thin bar, >3%; medium bar, >10%; thick bar, >90%.
(C) Prohead II was induced to expand at pH 4.2 as in (A). Samples were neutralized by dilution into pH 7.5 buffer and run in an agarose gel as described in the Experimental Procedures.
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4. Figure 3 In Vitro Expansion and Purification of EI-III and EI-IV
(A) Survey of salt and temperature dependence of crosslinking at pH 4. Samples of Prohead II were treated for 4 days at 4°C, 22°C, or 37°C in 50 mM citrate (pH 4.0) buffer containing 0, 200 mM, 2 M, or 4 M KCl, as indicated in the figure. The samples were TCA precipitated and analyzed by SDS-PAGE.
(B) Crosslinking during expansion in citrate buffer. Prohead II was acidified by dilution 50-fold into EI buffer. Samples were taken at the indicated times, TCA precipitated, and analyzed by SDS-PAGE.
(C) Hydrophobic interaction chromatography of HK97 EI-III, EI-IV. Prohead II was incubated in EI buffer for 4 months and chromatographed on a column containing a phenyl ligand as described in the Experimental Procedures. The A280 elution profile and the relative conductivity are shown. The large peak eluting at ∼920 s is EI-III; the shoulder at ∼860 s was identified as EI-IV.
(D) Comparison of the gel crosslinking patterns of EI-III and EI-IV to Prohead II and Head II. Samples of Prohead II, EI-III, EI-IV, and HII were TCA precipitated, denatured, and run on an SDS-polyacrylamide gel. EI-III has monomers and oligomers from dimer up to hexamer as well as one prominent circular oligomer (circular pentamer, see Figure 4A). EI-IV has mostly hexameric oligomers and the same closed circular form that occurs in EI-III. In Head II most of the protein is crosslinked into covalently closed circles that are topologically entangled in complexes (chain mail, Figure 7C) so large that they cannot even enter the stacking gel (Duda, 1998). So, only a small fraction of total capsid protein is visualized by staining—a small amount stuck to the top of the stack and a small fraction in covalently crosslinked open hexamers and pentamers dislodged from the bigger complexes because they are not closed circles, but broken or incompletely formed.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4 Characterization of EI-IV
(A) Covalent circles in EI-III and EI-IV are pentamers. HII was denatured by boiling in 1% SDS and subsequently digested with 0.2, 0.4, 0.8, 1.0, 1.5, and 2.0 μg/ml V8 protease at room temperature. The samples were TCA precipitated, denatured, and run on a 7.5% low crosslinker SDS-polyacrylamide gel. Comparing the HII digest pattern with EI-III and EI-IV shows that the circular oligomer in the expansion intermediates is a closed pentamer circle or 5-circle.
(B) Separation of EI-III, EI-IV, and HII by hydrophobic interaction chromatography. Samples of EI-III, EI-IV, and HII were chromatographed separately as described in Figure 3C. A280 plots were overlaid and labeled in the figure. In the EI-III plot, the small peak is EI-IV.
(C) Enhancement of ANS fluorescence by EI-III, EI-IV, and Head II. Samples of each particle at ∼1 μM were incubated with 5 μM ANS in EI buffer for ≥4 hr to ensure that any structural perturbations were complete before each measurement. Excitation was at 360 nm, and fluorescence intensity was recorded from 420 to 550 nm. Background fluorescence from ANS alone was subtracted.
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5 Cryo-Micrograph of HK97 Expansion Intermediate IV
Field of EI-IV particles observed by cryo-EM. The particles are uniformly spherical, thin-walled, and with little surface relief. Bar = 500 Å.
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6
Comparison of Expansion Intermediate IV and Head II Structures
Comparison of three-dimensional models of EI-IV from cryo-EM ([A], [C], [E], and [F]; this study) and the mature Head II solved crystallographically ([B], [D], and [F]; adapted from Wikoff et al., 2000). The resolution of the Head II model was limited to match that of EI-IV. Apart from the difference in curvature of the icosahedral facets, the two structures are very similar. External surface views of the respective pentons and hexons (A and B) are virtually identical, as emphasized in the inset combination views of both capsomers. Arrows mark positions of small protrusions around the vertices on EI-IV that are not present on Head II and which represent undescended E loops (see Results). Interior surfaces (C and D) show similar patterns of indentations beneath the hexons, while the penton cavities appear less open in EI-IV. Spherical sections through the EI-IV density map (E) show well-resolved protrusion densities around the pentons (red arrow, left, radius of 294 Å) and their absence around the hexons (arrowhead, right, radius of 284 Å). Central sections through both structures (F), offset by 0.8° from the 2-fold axis, also reveal the differences in curvature of the capsid walls and the small peripentonal protrusions in EI-IV (left, red arrow) compared to HII (right). Bar = 100 Å.
Molecular Cell  , DOI: ( /j.molcel )

8. Figure 7 Models of Crosslinking in EI-III, EI-IV, and Head II
(A) EI-III model.
(B) EI-IV model.
(C) Head II model.
Upper diagrams show color-coded crosslinks and subunits superimposed on a particle diagram derived from VIrus Particle ExploreR (VIPER) (Reddy et al., 2001). Lower panels have simplified diagrams and show the major oligomers and idealized and actual SDS gels for comparison. The arrangements of subunits and crosslinks are based on the high-resolution structure of HK97 Head II (Wikoff et al., 2000). Ovals represent capsid subunits: hexon subunits in red; penton subunits in blue. Lines represent crosslinks: black lines for links that make closed pentamers seen in gels, red lines for most of the links that make closed hexamers, and blue lines for links that connect penton subunits into closed hexamers. Models are best understood in comparison with Head II (C), where every subunit is crosslinked to two other subunits (some are not shown for simplicity), creating covalently closed hexamer and pentamer rings that are interlocked (chain mail, see legend to Figure 3). Note that the hexons and pentons of particles are not the same as the hexamers and pentamers observed in gels; closed pentamers (circles, black bonds) are derived from subunits of hexons, and every closed hexamer has one subunit derived from a penton subunit. EI-IV (B) has all the crosslinks of Head II, except for one (blue) crosslink from each penton subunit to an adjacent hexon subunit. The result is that no closed hexamers can form, so only open hexamers and closed pentamers are observed in gels. EI-III (A) is a less crosslinked version of EI-IV where there are fewer total crosslinks (∼60%) per particle and the particles may not be homogeneous. The location of the crosslinks shown for EI-III is speculative.
Molecular Cell  , DOI: ( /j.molcel )