Structural and functional studies of PCNA from African swine fever virus

Figures

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  Fig 1. Crystal structure of AsfvPCNA. (A) Overall folding of AsfvPCNA observed in Form I structure. Domain I, Domain II, and IDCL linker are colored in cyan, yellow, and red, respectively. (B) Topology of AsfvPCNA. (C) Cartoon representation of the trimeric AsfvPCNA protein. One protomer is colored as in panel A, whereas the other two protomers are colored in wheat and light blue, respectively. (D–F) The detailed interactions involved in AsfvPCNA trimerization. The C-atoms of the neighboring protomers are colored in yellow and light blue, respectively.

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  Fig 2. Comparison of Form I AsfvPCNA with the 7DRH structure. (A) Superposition of Form I AsfvPCNA with the 7DRH structure. (B) Structural superposition showing the orientational difference between Domain I and Domain II in Form I and 7DRH structures. (C) Superposition showing conformational changes in Domain II in Form I and 7DRH structures. (D) B-factor putty presentation of Form I AsfvPCNA structure. (E) Superposition of IDCL linkers in Form I and 7DRH structures. Domain I, Domain II, and IDCL of Form I structure are colored as in Fig. 1A, whereas 7DRH structure is colored in blue in all panels.

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  Fig 3. Comparison of AsfvPCNA with homologous PCNA proteins. (A) Superposition of AsfvPCNA with the ScPCNA structure (PDB_ID: 3K4X), which is colored in blue. (B) Structural superposition showing the conformational differences between the protomers of AsfvPCNA and ScPCNA. (C) Superposition of AsfvPCNA with the SsPCNA structure (PDB_ID: 2HII). The three SsPCNA protomers are colored in blue, green, and magenta, respectively. (D) Structural superposition showing the conformational differences between AsfvPCNA and SsPCNA protomers. In panels A and C, the AsfvPCNA structure is colored as in Fig. 1A.

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  Fig 4. Comparison of AsfvPCNA with sliding clamp structural homologs from other viruses. (A) Comparison of the protomers of UL42 (PDB_ID: 1DML), UL44 (PDB_ID: 1T6L), PF-8 (PDB_ID: 3HSL), BMRF1 (PDB_ID: 2Z0L), and AsfvPCNA. (B) Comparison of the head-to-head dimer of BMRF1 and the head-to-tail trimer of AsfvPCNA. (C) Comparison of the IDCL sequence of AsfvPCNA and the viral sliding clamps. Domain I, Domain II, and IDCL linker are colored in cyan, yellow, and red, respectively.

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  Fig 5. Verification of DNA binding by AsfvPCNA. (A) Surface electrostatic potential of AsfvPCNA trimer. The negative and positive residues are colored in red and blue, respectively. (B) Conformation of positively charged Lys and Arg residues from Domain I and Domain II of two neighboring protomers, which are colored in light blue and yellow, respectively. (C) In vitro EMSA assay showing DNA binding by AsfvPCNA. (D) Comparison of the dsDNA- and single-stranded DNA (ssDNA)-binding curves of AsfvPCNA.

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  Fig 6. AsfvPCNA facilitates DNA ligation by AsfvLIG. (A) Cryo-EM structure of archaeal SsPCNA-LIG-DNA ternary complex. The interacting IDCL of SsPCNA and PIP of SsLIG are colored in blue and wheat, respectively. (B) In vitro DNA ligation assays showing the impacts of AsfvPCNA and ScPCNA on the catalytic activity of AsfvLIG. The percentage of the product is given at the bottom of the image. (C) Superposition of AsfvLIG/dsDNA complex (PDB_ID: 6IMK) and AsfvPCNA onto the SsPCNA-LIG-DNA structure. For clarity, the SsPCNA-LIG-DNA structure was omitted.