The budding-yeast RWD protein Csm1 scaffolds diverse protein complexes through a conserved structural mechanism

Abstract

RWD domains mediate protein-protein interactions in a variety of pathways in eukaryotes. In budding yeast, the RWD domain protein Csm1 is particularly versatile, assembling key complexes in the nucleolus and at meiotic kinetochores through multiple protein interaction surfaces. Here, we reveal a third functional context for Csm1 by identifying a new Csm1-interacting protein, Dse3. We show that Dse3 interacts with Csm1 in a structurally equivalent manner to its known binding partners Mam1 and Ulp2, despite these three proteins' lack of overall sequence homology. We theorize that the unique "clamp" structure of Csm1 and the loose sequence requirements for Csm1 binding have led to its incorporation into at least three different structural/signaling pathways in budding yeast.

Keywords: RWD domain; S. cerevisiae; X-ray crystallography; monopolin complex; protein-protein interactions.

Figure 1

Identification of Dse3 as a Csm1‐binding protein. (A) Schematic of known Csm1‐interacting proteins from biochemical and structural information. Nucleolar proteins are shown in violet, meiotic kinetochore proteins in yellow, and others in white. Dashed and solid lines indicate that the given interaction has been detected by either two (dashed) or three or more (solid) separate studies. Colored lines indicate that the given interaction has been structurally characterized and used Csm1 Surface 1 (blue), Surface 2 (green), or N‐terminal coiled‐coil (yellow). (B) Domain schematic of Mam1 and Ulp2, which interact with Csm1 Interface 2, and the uncharacterized protein Dse3. In each case, conserved domains are shaded grey, and Csm1‐interacting regions (putative in the case of Dse3) are shaded green. (C) Sequence alignment of conserved Motif #2 in budding‐yeast Dse3 proteins. Green outline shows the region of S. cerevisiae Dse3 used for reconstitution with Csm1 (Panel D). (D) SDS‐PAGE analysis of the purified His₆‐SUMO‐Dse3^60–80:Csm1^69–181 complex. The His₆‐SUMO tag was removed prior to crystallization.

Figure 2

Structure of the Dse3–Csm1 complex. (A) Structure of Dse3^60–80 (green) bound to a Csm1^69–181 dimer (blue). The Dse3 chain shown is a composite comprising Residues 60–66 of one Dse3 protomer (chain C) and Residues 67–79 of the second Dse3 protomer (chain D). See Figure S1 for additional structural details. (B) Closeup view of the Dse3–Csm1 interaction. (C),(D) Views equivalent to (A) and (B) showing Csm1 (blue) binding Mam1 (yellow). (E) and (F) Views equivalent to (A) and (B) showing Csm1 (blue) binding Ulp2 (violet). (G) Structure‐based sequence alignment of three Csm1 Interface #2 binding regions from Ulp2, Mam1, and Dse3 (ClustalX coloring). (H) Superposition of Dse3 (green), Mam1 (yellow), and Ulp2 (violet) on Csm1, showing key Csm1‐interacting residues shared between the three proteins.