Analyses Of Protein-protein Interactions Of Inner Kinetochore Subunits CENP-H/I/K/M And CENP-O/P/U/Q/R

Correct separation and equal distribution of sister chromosomes during mitosis are important for the cell growth,development and reproduction of an organism.Proper chromosomes segregation depends on the function of a super complex kinetochore,which is localized on both the sides of chromosome.The kinetochore is divided into two layers:inner kinetochore(constitutive centromere associated network,CCAN)and outer kinetochore(Knl1complex,Mis12 complex,and Ndc80 complex;KMN).Based on the interaction and localization order,the subunits of CCAN are grouped into five sub-complexes:CENP-C,CENP-H/I/K/M,CENP-N/L,CENP-T/W/S/X and CENP-O/P/U/Q/R.These sub-complexes form a polyprotein complex.The inner kinetochore localizes on the centromere constitutively during the whole cell cycle and recruits the outer kinetochore at prometaphase.The outer kinetochore associates with microtubules and plays a key role in connecting the centromeres and microtubules.Any deletion or defection in the components of kinetochore directly causes mitotic defects and induces chromosomal mis-segregation and aneuploidy.As a member of CENP-H/I/K/M,CENP-I plays an important role in mitosis.The deletion or defect in CENP-I results in severe mitotic defects.Our research group successfully determined the crystal structure of C.thermophilum CENP-I N-terminus(Ct CENP-I^NT)alone and CENP-H/I/K complex,and revealed the molecular mechanism of CENP-I as an important scaffold protein bound to CENP-H/K.However,the interaction mechanism of CENP-I and CENP-M and intramolecular interaction among CENP-I itself are less discussed.The first part of this thesis focuses on CENP-I.We found a unique unpaired?helix(?11)in the N-terminus of Ct CENP-I(Ct CENP-I^NT),which has a broad hydrophobic interface with N-terminal HEAT domain and supports the proper folding of Ct CENP-I^NTunder in vitro conditions.We found,by sequence alignment and tertiary structure prediction,that Hs CENP-I^NTalso contains a corresponding helix,which offers the binding interfaces to the HEAT domain through conserved hydrophobic interactions.Further cellular and biochemical experiments revealed that the corresponding helix mediates the interaction between CENP-I and CENP-M directly.This finding would help to further reveal the interaction between CENP-I and CENP-M.CENP-O,CENP-P,CENP-U,CENP-Q,and CENP-R are interdependent in functions and locations;they interact with each other to form an elaborate sub-complex,CENP-O Class,which plays important roles in mitosis and spindle checkpoint.Lacking any member of CENP-O Class causes chromosome mis-alignment during the metaphase and mis-segregation during the telophase.Although the structure of yeast COMA complex(CENP-O Class homologs)has been determined,there is a big difference between COMA complex and CENP-O Class.COMA complex showed a rod-like shape in the high-resolution cryo-EM structure,whereas CENP-O Class appeared a bi-lobed shape,which showed two connected spherical domains in the negatively stained TEM images.Therefore,the interaction and assembly mechanism of CENP-O Class are still unclear.Next,we try to reveal the interactions of subunits and assembly model of CENP-O Class.We successfully purified the full-length CENP-O/P complex through an E.coli expression system and found that the N-termini of both CENP-O and CENP-P are important for their in vitro interactions.Furthermore,the N-terminal deletion in CENP-O and CENP-P disrupts the complex formation.We obtained the GST-CENP-U/SUMO-CENP-Q complex,purified the truncated complex of CENP-U^241-360/CENP-Q^101-268,collected some protein crystals after large-scale crystal condition screening,purified full-length CENP-R by denaturation and refolding method.We also identified the CENP-U/Q complex binding to CENP-O/P complex and CENP-R simultaneously by employing the GST pull-down assay,while CENP-O/P complex showed no interaction with CENP-R in vitro.These results imply that the CENP-U/Q complex acts as a scaffold for the assembly of CENP-O Class.Based on the exploratory discovery of interactions,we will further reconstitute CENP-O complex in vitro for structural and functional analyses to reveal the inter-molecular interactions and assembly mechanisms of CENP-O Class.