Structural and Biochemical Characterization of the Human Angiogenin-Proliferating Cell Nuclear Antigen Interaction

The molecular details of the interaction between humanangiogenin(hAng) and proliferating cell nuclear antigen (PCNA) have been investigatedby isothermal titration calorimetry (ITC), mutagenesis, and NMR spectroscopy.The two proteins were shown to interact directly through immunoprecipitationstudies of hAng with PCNA in vitro, and their interactionwas quantified by ITC, obtaining information on stoichiometry, enthalpy,entropy, and binding kinetics of the association. The hAng-PCNAassociation is strong, with a K (d) valueof 126 nM. The interaction surface was mapped by NMR spectroscopy,indicating participating residues. A structural model for the PCNA-hAngcomplex was constructed by docking and molecular dynamics simulationsbased on NMR data. The model was validated by mutating the hAng residuesArg5 and Arg101, which seem critical for the complex formation, toglutamate. ITC experiments showed that the angiogenin variants R5Eand R5ER101E displayed 6.5 and 7.8 times higher K (d) values, respectively, than that of the native protein,indicating the correctness of the model. The hAng S28AT36AS37A andhAng S28AT36AS37AS87A variants were also tested as positive controls,further supporting the validity of the model. The crystal structuresof the hAng variants S28AT36AS37A and S28AT36AS37AS87A showed thatthe mutations did not cause any significant conformational change.This study presents evidence for the structural mode of the hAng-PCNAinteraction, revealing valuable information about the angiogenin andPCNA biological roles in the cytoplasm.

Automated summary

The interaction between human angiogenin (hAng) and proliferating cell nuclear antigen (PCNA) was studied using isothermal titration calorimetry (ITC), mutagenesis, and NMR spectroscopy. The two proteins were found to directly interact with each other through in vitro immunoprecipitation studies, and their interaction was quantified using ITC. NMR spectroscopy and molecular dynamics simulations were used to map the interaction surface and construct a structural model for the complex. The validity of the model was confirmed by mutagenesis experiments. Overall, this study provides valuable insights into the structural details and biological roles of hAng and PCNA.