Architecture of the Dam1 kinetochore ring complex and implications for microtubule-driven assembly and force-coupling mechanisms

Abstract

The Dam1 kinetochore complex is essential for chromosome segregation in budding yeast. This ten-protein complex self-assembles around microtubules, forming ring-like structures that move with depolymerizing microtubule ends, a mechanism with implications for cellular function. Here we used EM-based single-particle and helical analyses to define the architecture of the Dam1 complex at 30-A resolution and the self-assembly mechanism. Ring oligomerization seems to be facilitated by a conformational change upon binding to microtubules, suggesting that the Dam1 ring is not preformed, but self-assembles around kinetochore microtubules. The C terminus of the Dam1p protein, where most of the Aurora kinase Ipl1 phosphorylation sites reside, is in a strategic location to affect oligomerization and interactions with the microtubule. One of Ipl1's roles might be to fine-tune the coupling of the microtubule interaction with the conformational change required for oligomerization, with phosphorylation resulting in ring breakdown.

The Composition, Functions, and Regulation of the Budding Yeast Kinetochore

“…The diameter of the ring is 50 nm and appears to interact with microtubules through electrostatic interactions via "arms" that extend from the Dam1 complex (Miranda et al 2005;Westermann et al 2005). These interactions are at least partly mediated through the N terminus of Dam1 and possibly the Duo1 subunit, which also exhibits microtubule-binding activity (Hofmann et al 1998;Cheeseman et al 2001b;Wang et al 2007;Ramey et al 2011). Although there are no atomic structures for any Dam1 components, cryo-EM analyses indicate that the complex does not appear to undergo major rearrangements upon forming a ring around the microtubule (Ramey et al 2011).…”

“…(Miranda et al 2005). Each protein is present at a single copy per complex and 16 complexes can assemble into a ring around microtubules in vitro in either orientation relative to the plus end (Miranda et al 2005;Westermann et al 2005;Wang et al 2007;Ramey et al 2011). However, small oligomers and other larger Dam1 structures can also attach to microtubules, making it unclear which structures are relevant to activity in vivo (Gestaut et al 2008;Grishchuk et al 2008b).…”

“…architecture of many kinetochore assemblies, including the two major microtubule binding complexes in the yeast kinetochore (Miranda et al 2005;Wei et al 2005Wei et al , 2006Wei et al , 2007Westermann et al 2005Westermann et al , 2006Wang et al 2007Wang et al , 2008Maskell et al 2010;Hornung et al 2011).…”

Bi-orienting chromosomes: acrobatics on the mitotic spindle

“…Biochemical reconstitution revealed that about 16 Dam1 complexes oligomerize and form a ring that encircles a microtubule ( Fig. 2b; Miranda et al 2005;Westermann et al 2005;Wang et al 2007). Separate studies suggest that the Dam1 complex can also be present on a microtubule without (Miranda et al 2005;Westermann et al 2005;Wang et al 2007) but also as an oligomer that does not form a ring (Gestaut et al 2008;Grishchuk et al 2008).…”

A non-ring-like form of the Dam1 complex modulates microtubule dynamics in fission yeast

“…Functioning as a heterodecamer with 1:1 stoichiometry (3), its distinct biophysical properties render it an attractive candidate as the coupler (4)(5)(6)(7)(8)(9)(10). In vitro, budding yeast Dam1 (ScDam1) is able to oligomerize, forming a 16-fold-symmetry ring (4, 7, 9) (25-fold by another study; ref.…”