The Structural And Functional Research Of Saccharomyces Cerevisiae CK2

CK2is a ubiquitous and conserved protein kinase in eucaryotic organisms, and important in many biological processes. It is unique by keeping constitutive activity and using both ATP and GTP as phosphor donor. CK2is a heterotetramer like a "butterfly" consisting of catalytic and regulatory subunits. In this study, we present the crystal structures of recombinant scCKα with GMPPNP, ATP and AMPPN complexed with either Mg2+or Mn2+as the coordinated divalent cations. The overall structure of scCK2α shows high similarity with its homologous proteins by consisting of two domains and that co-substrate lies in the cleft between them. Whereas, three characteristic features distinct scCK2a from its homolog. Firstly, scCK2a is unique by containing an insertion region that contributes to keep the constitutive activity conformation of scCK2a catalytic site. Secondly, interaction of Lys45-Glu53and Arg48-Glu53in scC2a leads Lys50to adopt a unique conformation able to stabilize the y-phosphate of co-substrate, which makes the existence of the "essential divalent cation" not that essential. The multiple nucleotide-divalent cation binding modes of the active site of scCK2a is apparently different from the two-divalent cation ion occupied the catalytic active site of zmCK2a or hCK2a. Finally, conformation change of Glu53of scCK2a-AMPPN breaks its interaction with Lys45and Arg48, as a result, the co-substrate-binding pocket becomes more open. This could suggest a clue of a possible ADP/GDP release pathway, because the NE1of Trp in "DWG motif" of CK2α forms a hydrogen bond with the O atom of Leu212, which seems making the ADP release process by means of "DFG-in flip to DFG-out" model in most of eukaryotic protein kinase impossible.Previous researches provided that CK2was a strong transient complex and its subunits could move independently though high affinity (4nM of Kd) among its subunits.The crystal structure of hCK2isoform α2β2demonstrated that the two β-subunits interacted with each other to form a dimer, and the two catalytic subunits bind to the opposite side. Our results displayed that interaction between the subunits of scCK2may be different from hCK2isoform α2β2, though more experimental verification is required.The subunits of CK2could combine with other proteins to form complex with weak affinity compared with inner CK2. Interestingly, this interaction is mediated by regulatory subunits.hSpt16and SSRP1could bind to CK2induced by UV irradiation and this direct interaction results in conformation change of CK2, which could then specifically target p53over other substrates. In the case of Saccharomyces cerevisiae, properly the CK2interact indirectly with Spt16-Pob3through CHD1. There are also reports referring to that CK2associated with Spt16-Pob3, but they did not provide experimental evidence.FACT is a histone chaperone with roles in transcription, replication, and repair. It could promote and subsequently restrict access to DNA as a result of dynamic nucleosome reorganization. Human FACT is a stable heterodimer comprised of hSpt16and SSRP1proteins. Whereas in S. cerevisiae, yFACT consists of Spt16/Cdc68, pob3and Nhp6which is a HMGl-like protein.The yeast and human Spt16proteins are structurally related throughout their lengths. However, the Pob3protein does not contain sequences related to the C-terminal portion of SSRP1, which contains a high-mobility group (HMG) box domain. We cloned and purified the subunits of CK2and yFACT of S. cerevisiae. Our result confirmed that CK2could interact with FACT though their function is still unclear. Meanwhile, we obtained the crystal structure of Pob3-M and Pob3(1-112aa), but Pob3-M does not participate in the interaction of Pob3and CK2. EMSA assay demonstrated that Pob3(1-112aa) could bound dsDNA, which need more studies to determine the significance of its DNA-binding function.