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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 dsDNA viruses 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 Chiou 1 , Xing Zhang 1 , Robert Modlin 1,3 , and Z. Hong Zhou 1,2 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, including an icosahedral capsid head and a long non-contractile tail. 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 Frealign 8. 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 capsid is composed of twelve 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). Figure 3. Avisual anal- 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.
