title: Complementary interhelical interactions between three buried Glu-Lys pairs within three heptad repeats are essential for Hec1-Nuf2 heterodimerization and mitotic progression.
abstract: Hec1 and Nuf2, core components of the NDC80 complex, are essential for kinetochore-microtubule attachment and chromosome segregation. It has been shown that both Hec1 and Nuf2 utilize their coiled-coil domains to form a functional dimer; however, details of the consequential significance and structural requirements to form the dimerization interface have yet to be elucidated. Here, we showed that Hec1 required three contiguous heptad repeats from Leu-324 to Leu-352, but not the entire first coiled-coil domain, to ensure overall stability of the NDC80 complex through direct interaction with Nuf2. Substituting the hydrophobic core residues, Leu-331, Val-338, and Ile-345, of Hec1 with alanine completely eliminated Nuf2 binding and blocked mitotic progression. Moreover, unlike most coiled-coil proteins, where the buried positions are composed of hydrophobic residues, Hec1 possessed an unusual distribution of glutamic acid residues, Glu-334, Glu-341, and Glu-348, buried within the interior dimerization interface, which complement with three Nuf2 lysine residues: Lys-227, Lys-234, and Lys-241. Substituting these corresponding residues with alanine diminished the binding affinity between Hec1 and Nuf2, compromised NDC80 complex formation, and adversely affected mitotic progression. Taken together, these findings demonstrated that three buried glutamic acid-lysine pairs, in concert with hydrophobic interactions of core residues, provide the major specificity and stability requirements for Hec1-Nuf2 dimerization and NDC80 complex formation.
authors.affiliation: From the Department of Biological Chemistry, School of Medicine, University of California, Irvine, California 92697.
authors.forename: Bryan
authors.initials: B
authors.lastname: Ngo
authors.forename: Chun-Mei
authors.initials: CM
authors.lastname: Hu
authors.forename: Xuning Emily
authors.initials: XE
authors.lastname: Guo
authors.forename: Brittany
authors.initials: B
authors.lastname: Ngo
authors.forename: Randy
authors.initials: R
authors.lastname: Wei
authors.forename: Jiewen
authors.initials: J
authors.lastname: Zhu
authors.forename: Wen-Hwa
authors.initials: WH
authors.lastname: Lee
mesh_terms: D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics*; D020090:Chromosome Segregation / Q000235:genetics*; D003598:Cytoskeletal Proteins; D018507:Gene Expression Regulation, Developmental; D006367:HeLa Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D018386:Kinetochores / Q000737:chemistry; D008870:Microtubules / Q000737:chemistry / Q000235:genetics; D008938:Mitosis; D046912:Multiprotein Complexes; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics*; D055503:Protein Multimerization / Q000235:genetics*; D017434:Protein Structure, Tertiary / Q000235:genetics
chemical_list: D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D046912:Multiprotein Complexes; C120529:NDC80 protein, human; C490197:NUF2 protein, human; D009687:Nuclear Proteins
keywords: Coiled-Coil; Hec1; Heptad Repeat; Kinetochore; Mitosis; NDC80; Nuf2; Protein-Protein Interactions; Site-directed Mutagenesis; Western Blotting