Point mutations in the nucleosome docking domain reduce the physical stability of Htz1’s association with chromatin.

<p><b>[A]-[B]</b> Mutants are less resistant to washing with increased ionic-strength buffers. <b>[A]</b> HA-Htz1 levels in total cell lysates (T), and in insoluble chromatin fractions after washing with buffers containing 100, 200, 300 or 400 mM NaCl were determined by immunoblotting with anti-HA and anti-H4. <b>[B]</b> For each strain, levels of HA-Htz1 normalised to H4 for each washed chromatin sample were compared to the 100 mM wash sample and averaged (n = 3). Error bars indicate standard error of the mean. <b>[C]-[F]</b> Computational modelling of point mutations at equivalent positions in the mouse H2A.Z crystal structure predicts changes in local intermolecular interactions. KiNG software was used to model the effect of mutating residues in the mouse H2A.Z nucleosome crystal structure [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076394#B18" target="_blank">18</a>], at the positions equivalent to yeast serine 111 and isoleucine 109 (glycine 106 and isoleucine 104 respectively). In each panel, blue, grey and cyan lines represent the main chains, hydrogen atoms and side chains respectively, apart from G106, I104 and the residues substituted at these sites, where the main chains are coloured red and the side chains are pale pink. Green and blue dots indicate stabilising van der Waals interactions; yellow, orange and red spikes indicate small intermolecular clashes and pink spikes indicate large clashes. Local interactions are modelled for G106 <b>[C]</b>, but when substituted with proline <b>[D]</b> substantial van der Waals overlaps are seen. I104 makes several stabilising van der Waal’s contacts <b>[E]</b>, which are lost upon substitution with threonine <b>[F]</b> (right hand side of panel). In addition the hydrogen-bonding potential of the threonine hydroxyl group is unsatisfied.