Implications for Kinetochore-Microtubule Attachment from the Structure of an Engineered Ndc80 Complex

Abstract

Kinetochores are proteinaceous assemblies that mediate the interaction of chromosomes with the mitotic spindle. The 180 kDa Ndc80 complex is a direct point of contact between kinetochores and microtubules. Its four subunits contain coiled coils and form an elongated rod structure with functional globular domains at either end. We crystallized an engineered "bonsai" Ndc80 complex containing a shortened rod domain but retaining the globular domains required for kinetochore localization and microtubule binding. The structure reveals a microtubule-binding interface containing a pair of tightly interacting calponin-homology (CH) domains with a previously unknown arrangement. The interaction with microtubules is cooperative and predominantly electrostatic. It involves positive charges in the CH domains and in the N-terminal tail of the Ndc80 subunit and negative charges in tubulin C-terminal tails and is regulated by the Aurora B kinase. We discuss our results with reference to current models of kinetochore-microtubule attachment and centromere organization.

A Redundant Function for the N-Terminal Tail of Ndc80 in Kinetochore–Microtubule Interaction in Saccharomyces cerevisiae

“…The N-terminal tail of Ndc80 is enriched for Ipl1/AuroraB phosphorylation sites, and MT binding can be regulated by the kinase (Guimaraes et al 2008;Welburn et al 2010;Lampson and Cheeseman 2011;. MT-binding affinity of yeast and human Ndc80 complexes is decreased when the Ndc80 N-terminal tail is deleted (Wei et al 2005;Ciferri et al 2008). Reducing the positive charge by mutating some residues in the Ndc80 N-terminal tail abolishes MT attachments (Guimaraes et al 2008;Miller et al 2008;.…”

“…Recombinant Ndc80 complex binds MTs in vitro, and the activity is restricted to the head domains of Nuf2 and Ndc80 (Wei et al 2005;Ciferri et al 2008). Yeast has an additional protein complex, Dam1, composed of 10 proteins, that is also required for KT-MT attachment (Westermann et al 2006;Lampert et al 2010;Tien et al 2010).…”

“…The subtle phenotypes associated with ndc80-112 are surprising, given that the N-terminal tail of Ndc80 is essential in human cells and is required for yeast Ndc80 to bind MTs in vitro (Wei et al 2005;Ciferri et al 2008;Miller et al 2008;. One of the differences between kinetochores of yeast and human cells is the Dam1 complex that serves as a processivity factor for the Ndc80 complex, allowing Ndc80 to track the plus ends of microtubules and establish load-bearing connections (Lampert et al 2010; Figure 1 A mitotic delay in ndc80-112 cells.…”

“…T HE evolutionarily conserved Ndc80 complex is composed of Ndc80, Nuf2, Spc24, and Spc25, integral kinetochore (KT) proteins responsible for mediating microtubule (MT) attachments from yeast to humans (Wigge and Kilmartin 2001;McCleland et al 2003;Ciferri et al 2008). Recombinant Ndc80 complex binds MTs in vitro, and the activity is restricted to the head domains of Nuf2 and Ndc80 (Wei et al 2005;Ciferri et al 2008).…”

The perpetual movements of anaphase

“…This data suggest that the Ndc80 complex may correspond to the kinetochore fibrils observed by electron tomography, but that additional factors may be involved in microtubule depolymerization-coupled movement [150]. The use of "bonsai" forms of the Ndc80 complex that retain microtubule binding capacity but are only 15 nm long [151] should prove useful in determining the molecular nature of the kinetochore fibrils.…”