Variants of the Sir4 Coiled-Coil Domain Improve Binding to Sir3 for Heterochromatin Formation in Saccharomyces cerevisiae

Heterochromatin formation in the yeast Saccharomyces cerevisiae is characterized by the assembly of the Silent Information Regulator (SIR) complex, which consists of the histone deacetylase [Sir2][1] and the structural components [Sir3][2] and [Sir4][3], and binds to unmodified nucleosomes to provide gene silencing. [Sir3][2] contains an AAA+ ATPase-like domain, and mutations in an exposed loop on the surface of this domain abrogate [Sir3][2] silencing function in vivo , as well in vitro binding to the [Sir2][1]/[Sir4][3] subcomplex. Here, we found that the removal of a single methyl group in the C-terminal coiled-coil domain (mutation T1314S) of [Sir4][3] was sufficient to restore silencing at the silent mating-type loci [HMR][4] and [HML][5] to a [Sir3][2] version with a mutation in this loop. Restoration of telomeric silencing required further mutations of [Sir4][3] (E1310V and K1325R). Significantly, these mutations in [Sir4][3] restored in vitro complex formation between [Sir3][2] and the [Sir4][3] coiled-coil, indicating that the improved affinity between [Sir3][2] and [Sir4][3] is responsible for the restoration of silencing. Altogether, these observations highlight remarkable properties of selected amino-acid changes at the [Sir3][2]-[Sir4][3] interface that modulate the affinity of the two proteins. [1]: http://www.yeastgenome.org/locus/S000002200/overview [2]: http://www.yeastgenome.org/locus/S000004434/overview [3]: http://www.yeastgenome.org/locus/S000002635/overview [4]: http://www.yeastgenome.org/locus/S000029655/overview [5]: http://www.yeastgenome.org/locus/S000029214/overview