Structural And Functional Studies Of Kinetochore CENP-H/I/K Complex In Mitosis

Mitosis is the basic mode of eukaryotic cell proliferation,and plays an important role in the growth and development of multicellular organisms.Accurate segregation of genetic material is crucial for the survival of daughter cells.In mitosis,the alignment and segregation of chromosomes are dependent on the kinetochore.The kinetochores possess three-layer disc-sharped structure on the chromosomes,which is a macromolecular complex composed of dozens of core proteins,and binds directly to the spindle microtubules to ensure the accurate segregation of chromosomes during cell division.In centromeric chromosomes,CENP-A with histone folding can replace H3 in canonical nucleosomes,which initiate the assembly of inner kinetochore and then recruit the outer kinetochore composed of KNL1,NDC80,MIS12 complexes.The outer kinetochore connects chromosomes to spindle microtubules to promote chromosome segregation.The inner kinetochore consists of five sub-complexes: CENP-C,CENP-L/N,CENP-H/I/M,CENP-T/W/S/X and CENP-O/P/U/Q/R.However,the concrete regulation mechanisms is not completely characterized.CENP-H/I/K complex is located in the middle layer of CCAN,which is closely related to upstream and downstream kinetochore subunits,and plays an important role in kinetochore assembly and function.In this thesis,we firstly obtained the N-terminal crystal structure of C.thermophilum CENP-I and in complex with T.terrestris CENP-H/K.It is found that the N-terminal of the ctCENP-I consists of 11 α-helices,the first 5 pairs helices are folded as a typical HEAT-repeat,and the following helix is wrapped back on one side of the HEAT to stabilize its structure.For the structure of CENP-H/I/K complex,The CENP-H acts as a birdge that and mediated the formation of ternary complex.Through a series of amino acid mutations,we clarified the key sites that affect CENP-H/I/K binding,and verified the conservation of these sites on the basis of human proteins.We next demonstrated that the interaction of CENP-H and CENP-K maintained by both N and C-terminus,and the N-terminal complex of CENP-H/K were not involved in the binding to CENP-I.Interestingly,although no direct interaction between CENP-K and CENP-I was observed in the structure,but the formation of CENP-H/K complex enhances the recruitment of CENP-I.Furthermore,we introduced mutations that affected CENP-H/I/K interactions in mitosis cell,and observed deficient centromeric localization of the CENP-H/I/K complex and chromosomes mis-alignment in metaphase.In summary,we determinated the crystal structures of fungal CENP-I and CENP-H/I/K complex,identified the key sites of CENP-H/I/K complex interaction,and further verified the molecular assembly model of CENP-H/I/K complex in cells and important role in the alignment and accurate segregation of chromosomes.These results together provide the basis for understanding the assembly of inner kinetochore,and promotes comprehension of the regulatory mechanism of kinetochore in segregation of chromosomes.