- Cryo-electron microscopy was used to determine the 3.74 angstrom resolution structure of the Propionibacterium acnes bacteriophage capsid. - The capsid protein displays a HK97-like fold with characteristic features including an N-arm, E-loop, central beta-sheets, and long alpha-helix. - Individual subunits are stabilized through isopeptide bonds in a manner similar to the chainmail structure seen in HK97 capsids.

1. PROJECT OBJECTIVE
ABSTRACT RESULTS
BACKGROUND
METHODS
CryoEM reveals Siphoviridae morphology
Overall structure of the P. acnes phage capsid
Sidechain residues are visible within density map
Isopeptide bond provides a stabilization mechanism
Skewed subunits of the hexameric unit
P. acnes phage major capsid protein subunit displays a HK97-like fold
PROJECT CONCLUSIONS
Superimposition of capsid protein subunits
FUTURE DIRECTIONS
REFERENCES
ACKNOWLEDGEMENTS
Propionibacterium acnes bacteriophages are long-tailed, dsDNA Siphophages
that infect and lyse bacteria correlated with the cosmetic skin disease acne. Re-
cently, the idea of using phage therapy to counteract acne has emerged as a
possible alternative therapeutic option to topical antibiotics. However, therapeutic
use of these phages to counteract antibiotic-resistant strains of P. acnes bacteria
still requires considerable research, particularly concerning structural stability and
potential modification points. Here, we report a 3.74 Å resolution, icosahedrally
averaged three-dimensional structure of a P. acnes phage capsid head resolved
by cryo-electron microscopy. The reconstructed electron density map allows clear
visualization of amino acid sidechain residues, as well as covalent isopeptide
interactions between neighboring subunits of the capsid protein gp6. An atomic
model of gp6 traced from the density map reveals a HK97-like capsid protein,
which has also previously been discovered in a number of dsDNA viruses. Sub-
tle differences between the seven subunits that compose the asymmetrical unit
were observed in the E-loops when overlain in UCSF Chimera. Evidence of a co-
valent crosslinking hexamers similar to the chainmail interaction found in HK97
hexamers suggests a common structural mechanism to maintain capsid stability
in harsh environments. Overall, this study resolves the structure of a P. acnes
phage for bioengineering purposes and provides more insight into the evolution-
ary development of dsDNAviruses from a structural standpoint. Moreover, since P.
acnes phage genomes demonstrate conserved genes coding for structural com-
ponents, we predict that this structure is representative of all P. acnes phages.
Determine the structure of a Propionibacterium acnes bacteriophage capsid at high resolution using the single particle analysis technique of
cryo-electron microscopy and construct a three-dimensional atomic model of the asymmetrical unit for comparison to known viral structures.
Propionibacterium acnes bacteriophage capsid protein structure determined by cryoEM
Joshua Chiou1
, Xing Zhang1
, Robert Modlin1,3
, and Z. Hong Zhou1,2
1 Department of Microbiology, Immunology, and Molecular Genetics , University of California, Los Angeles, CA 90095
2 California NanoSystems Institute, University of California, Los Angeles, CA 90095
3 Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA 90095
• Phage therapy is a proposed theraputic using P. acnes phages to treat acne.
Further research is required to optimize treatment.1
• P. acnes phage genomes display a high degree of homology, especially to-
wards the left arm which contains structural proteins.2
• The first atomic resolution characterization of a dsDNA viral capsid protein
subunit revealed the HK97 fold.3
• Six of these subunits are stabilized by isopeptide bonds between Lys169 of
one subunit and Asn356 of a neighboring subunit to form a hexamer.3
HK97 hexamers form a covalent chainmail interaction.3
• The HK97 fold has been found in viruses infecting all three domains of life
(bacteria, archaea, and eukaryotes).4
• Based on mass spectrometry, capsid protein gp6 has been speculated to form
complexes of 6-8 CP subunits, which is similar to the observation in HK97.5
Figure 1. Left: Representative micrograph from which capsid particles used for
reconstruction were selected. Mature P. acnes virions display typical Siphoviridae
characteristics,includinganicosahedralcapsidheadandalongnon-contractiletail.
Right: Close-up image of a single P. acnes phage. The long tails are typical-
ly bent indicating flexibility, though it is straight here to demonstrate length.
Figure 2. Icosahedrally av-
eraged P. acnes phage cap-
sid using Frealign8.
displays
triangulation number T=7,
similar to other phages. Ico-
sahedral facets are clearly
visible and can be drawn by
taking three pentamers as
vertices. Pentamers contain
five CP subunits, while hex-
amers contain six.The pen-
tamers are colored in pink
and extend furthest radially
from the center. Hexamers
are colored in green. The
capsidiscomposedoftwelve
pentamers and sixty hexam-
ers, which tallies to a total of
420 CP subunits per capsid.
Figure 5. Subunits of the capsid are held in place by covalent isopeptide
bonds between Lys 65 and Asp 278 from different hexamers. The strong den-
sity at this region provides clear evidence that the bond exists at this location.
Figure 6. The hexam-
er unit of the capsid is not
completely symmetrical,
and does not display the
typical 6-fold symmmetry
in the center. Instead, it dis-
plays a skewed arrange-
ment with the central loop
of subunits A and D located
below the capsid surface,
which has previously only
been seen in immature vi-
rion hexamers. The fact
that this hexamer is from
a mature P. acnes phage
capsid challenges our cur-
rent understanding of viral
maturation mechanisms.
Figure 4. The atomic model of the P. acnes phage capsid
protein traces from Aspartate 4 to Glutamate 303. Missing res-
idues at the N-terminus and the C-terminus were unable to
be traced in the density map. Interesting regions within the
fold are highlighted in color. The protein starts at the N-arm,
which extends outwards toward other hexamers. It then comes
down to the extended loop (E-loop), which contains two ma-
jor antiparallel β-sheets and lysine 65, an essential residue
forming the basis of capsid stabilization. The trademark long
α-helix and 5 central β-sheets form the core of the HK97-like
viral protein folds, and is observed here in a P. acnes phage.
The direction of the β-sheets are arranged in a similar up, up,
down, up, up fashion to HK97. The protein domain connec-
tivity also matches HK97, rather than the newly-discovered
topology permutation of BPP-1. Overall, the capsid protein in
P. acnes phage is similar to other HK97-like proteins, which
supports the notion of conserved viral structural proteins.
• Structure of P. acnes phage determined by cryo-electron microscopy at 3.74 Å. At this resolution, sidechains are clearly visible and interactions between individ-
ual subunits that make up the capsid are observable.
• P. acnes phage displays a T=7 capsid, which is consistent with the structure of HK97 and various other dsDNA phages.
• P. acnes phage capsid protein bears a striking resemblance to HK97 capsid protein, leading to its classification as an HK97-like fold. It displays the typical
characteristics of HK97-like folds: N-arm, E-loop, long α-helix and 5 central β-sheets.
• Isopeptide (covalent) bonds stabilize individual subunits to form the capsid. The isopeptide bond found in P. acnes phage is a variation of the one in HK97.
• The subunits that compose the hexamer are skewed, resulting in lack of 6 fold symmetry at the center. This has previously only been seen in immature virions.
• Subunits of the asymmetrical unit display distinct conformational differences. This contrast is especially pronounced at the N-arm, E-loop, and center facing
loops. The lone pentamer-forming subunit shows even more variance relative to the rest of the subunits.
Figure 7. Superimposition of the subunits that comprise the asymmetrical unit
show that most of the conformational differences arise in the N-arm, E-loop, and
center-facing loop. The rest of the protein appears to be conformationally similar.
Immediate Future Directions
• Determine the structure of the P. acnes phage tail at high resolution
• Biologically engineer P. acnes phage to increase capsid stability
• Modify the phage as a targetable delivery mechanism for therapeutics
• Analyze the differences between the skewed arrangement observed in P.
acnes phage and the immmature HK97 virion
Broader Future Directions
• Push for better resolution density maps with technological advances
• Discover differences in structures that resemble the HK97-like viral lineage
• Unravel the mystery behind viral evolution and search for a common ancestor
• Clinical testing for the effectiveness of phage therapy to treat acne
1. Farrar et al. 2007. J. Bacteriol. 189, 4161-4167.
2. Marinelli et al. 2012. MBio 3.
3. WIkoff et al. 2000. Science 289, 2129-2133.
4. Pietilä et al. 2013. Proc. Natl. Acad. Sci. U.S.A. 110, 10604–10609.
5. Lood and Collin. 2011. BMC Genomics 12, 198.
6. Grigorieff, N. 2007. J. Struct. Biol. 157, 117–125.
7. Emsley and Cowtan. 2004. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132.
8. Brünger et al. 1998. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921.
9. Adams et al. 2010. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221.
10. Pettersen et al. 2010. J Comput Chem 25, 1605–1612.
This work was supported in part by grants from the NIH (GM071940 and AI094386 to Z.H.Z). We ac-
knowledge the use of instruments at the Electron Imaging Center for Nanomachines supported by UCLA
and by instrumentation grants from NIH (1S10RR23057, 1S10OD018111) and NSF (DBI-1338135).
Figure3.Avisualanal-
ysis of the sidechains in
the map shows that the
most of the densities
are easily visible, which
is consistent with the
resolution assessed by
FREALIGN at 3.74 Å.
Sidechain densities are
clearer in the β-sheet
region depicted in this
figure than the α-he-
lix shown above. Den-
sities are especially
clear for bulky amino
acid residues such as
lysine and arginine.