Combining Bacterial Surface Display and Photocrosslinking for Analyzing Protein-Protein-Interactions of Human Protein Kinase CK2

Nickelsen A, Nienberg C, Jose J

Abstract

The human protein kinase CK2 is a heterotetrameric, constitutively active Ser/Thr kinase, consisting of two catalytic (CK2α/α’) and two regulatory (CK2β) subunits. CK2 shows a high pleiotropy with an increasingnumber of substrates [1]. Some of them are involved in the development and progression of cancer. The CK2β subunit functions as a modulator of CK2 activity and stability as well as a docking platform for CK2substrates. Photocrosslinking represents a versatile application to discover and characterize protein-protein-interactions (PPI), in particular enabling the detection of also low-affinity and transient interactions [2].Combined with Autodisplay, a surface displaying technology based on the secretion mechanism of autotransporter proteins in Gram-negative bacteria [3], it enables PPI-investigations of CK2 with a relieved separationof unbound proteins and without the need of CK2 purification. In this study purified CK2β and surface displayed CK2β-FP were covalently crosslinked to the catalytic CK2α subunit. Therefore the unnatural amino acidp-azidophenylalanine (pAzF) [4] was incorporated at different positions of CK2β. Afterwards a CuAAC click reaction (Copper(I)-Catalyzed Alkyne-Azide Cycloaddition) [5] and a SPAAC click reaction (Strain-PromotedAlkyne-Azide Cycloaddition) [6] were performed to prove the success of the pAzF-incorporation. With the purified as well as with the surface displayed modified CK2β it was possible to crosslink CK2α by UV lightirradiation of 365 nm. Photocrosslinking with pAzF incorporated in different positions of CK2β on the surface of E. coli can be used for studying PPI of CK2, in particular regarding substrate identification and inhibitordiscovery.