Structural comparison of heterotrimer PCNA from Crenarchaeon <i>Aeropyrum pernix</i> by solution scattering, cryo-EM and crystallography
Sliding clamps are ring-shaped proteins that encircle DNA and confer high processivity on DNA polymerases.In bacteria, the β-clamp protein forms a homodimer, whereas in eukaryotes or euryarchaeotes, proliferating cell nuclear antigen (PCNA) proteins form homotrimers.However, PCNA from Aeropyrum pernix (ApPCNA), a crenarchaeote species, forms a heterotrimer.The actual structure of ApPCNA-mediated sliding clamps and the mechanism by which they slide along DNA is unknown.Previously, we have analysed the crystal structure of ApPCNA1 from the APE_0162 gene [1] , ApPCNA2 from the APE_0441.1,and ApPCNA3 from the APE_2182 genes [2] .The present study aimed to analyse the crystal, solution structure and cryo-electron microscopy (cryo-EM) of the heterotrimeric ring of ApPCNA, examine its interaction with DNA and other proteins, and elucidate the mechanism of PCNA function.Each ApPCNA molecule, which constitutes a heterotrimer, was expressed using the Escherichia coli expression system.The proteins were purified using heat treatment, ammonium sulfate precipitation, and column chromatography.The purified proteins were crystallized using the vapor-diffusion method and the crystals were analysed by X-ray diffraction.To verify the ring shape of ApPCNA2 in solution, the solution structure was analysed using size-exclusion chromatography-small-angle X-ray scattering (SEC-SAXS).A mixture of ApPCNA1-2-3 and ApPCNA2-3 were analysed by SEC-multi-angle light scattering for the presence of a complex, and the solution structure was analysed by SEC-SAXS.The mixture was analysed by cryo-EM, after purified with gel filtration chromatography.The solution structure of the ApPCNA1-2-3 complex is similar to shape of the British Isles islands.ApPCNA2 and ApPCNA3 interact in a similar manner as the PCNA rings of other organisms; however, ApPCNA1 is located such that it did not form a perfect ringshaped structure.The scattering curves of the complex and those of the model edited trimeric ring are almost similar with minor differences.The solution structure of ApPCNA2-3 complex was similar to shape of a naan.This particle contains four subunits rather than trimer.The electron density from cryo-EM forms hexagon.The solution structure was not trimeric ring, containing ApPCNA1-2-3.The N-terminus of ApPCNA1 is approximately 10 residues longer than that of ApPCNA2 and ApPCNA3.This could be why the tripartite complex is not ring shaped.Moreover, Met16 is present downstream of the N-terminal of ApPCNA1.In the future, the effect of N-terminus deletion and binding of the DNA duplex on ApPCNA1 structure should be evaluated.The solution structure of ApPCNA2-3 complex was not trimeric ring too.In crystal structure of ApPCNA3, the C-terminus interacts between adjacent subunits, probably PIP-Box binding site.This interaction may cause ApPCNA2-3 Complex dose not form ring shape.Generally, PCNA rings that consists of homotrimer have 3-fold symmetry, comprise six edges from concave edge between subunits and flat edge that formed PIP-Box binding site.This hexagonal electron density suggests ApPCNA1-2-3 forms trimeric ring in cryo-EM structure.Interestingly, one of the three edges is completely separated.The Fitting model containing ApPCNA1-2-3 hetero subunits suggests, the long N-terminus of ApPCNA1 cause this separated edge.
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