Structurale And Functional Research Of Centromere Protein CENP-I

The accurate separation of sister chromatids during cell division depends on the stable attachment of kinetochore to spindle microtubules.The kinetochore is a macromolecular machine composed of a variety of protein subunits.The molecular assembly and regulation between the subunits directly affects its connection with microtubules.The centromere protein CENP-I is a kinetochore constitutive subunit that plays an important scaffolding role in the recruitment and assembly of other kinetochore subunits.Depletion of the CENP-I leads to chromosome congression and segregation defects.CENP-I,CENP-H,CENP-K and CENP-M can form a relatively stable quaternary complex,which constitutes the core component of the inner layer of kinetochore,and plays an important role in the assembly of kinetochore.However,there are still many unknowns about the structure and function of CENP-I.In the first part of this thesis,we determined the crystal structure of CENP-I N-terminal from Chaetomium thermophilum(ct CENP-I^NT).ctCENP-I^NT consists of 11?helices??1-?11?,and?1-?10 helix are stacked in antiparallel to form a typical HEAT repeats.The last?-helix??11?folds around the HEAT repeats.Due to the elastic variation of the classical HEAT repeat,we studied the effects of external forces on the microstructural changes of ctCENP-I^NT.The results indicate that the force-induced HEAT repeats configuration changes promote the dissociation of?11 helix from the HEAT repeats,suggesting that?11 may be involved in maintaining the HEAT repeats folding state.The crystal structure shows that there is a wide hydrophobic interaction between the?11 helix and the HEAT domain.The three amino acid residues Phe208,Thr215 and Leu219 in the?11 helix are involved in the hydrophobic interaction between the?11 and HEAT repeats.After?11 helix deletion,the HEAT repeats(ct CENP-I^HEAT)showed a highly polymerized state in size exclusion chromatography,indicating that the hydrophobic interaction between?11 helix and HEAT repeats can maintain the stable conformation of the HEAT repeats.Bioinformatics analysis based on primary sequence revealed that Phe208,Thr215,and Leu219 are highly conserved among CENP-I homologous proteins.Amino acid mutations at these sites result in changes in the conformation of the protein.In addition,we explored the molecular assembly mechanism of ctCENP-I^NT and other kinetochore subunits CENP-H/K.The pull-down results showed that ctCENP-I^NT directly binds to CENP-H/K.However,the deletion of?11 or amino acid at key sites affects the folding state of HEAT domain and loses or reduces the binding ability of CENP-H/K.Immunofluorescence imaging revealed that those key conserved amino acid residues on?11 play an important role in the localization of CENP-I on centromeres.The results of this study reveal for the first time that ctCENP-I has a HEAT repeats and we found the key regions that affect the conformational change of the HEAT repeats.These efforts help us to understand CENP-I's function as a scaffolding protein and the regulation of kinetochore assembly.