Co-Folding of the Histone Chaperone DAXX and H3.3/H4

Abstract

Histone chaperones mediate the assembly and disassembly of canonical and variant nucleosomes. Currently there is little known of the conformational changes that occur upon chaperone-histone binding, but these are likely to be key to understanding the mechanism of histone transfer on and off of DNA. For this study, we focus on the death domain-associated protein (Daxx), a chaperone that specifically recognizes the H3.3 histone variant, mediating its deposition into heterochromatin. Using hydrogen/deuterium exchange (H/DX) coupled with mass spectroscopy to measure polypeptide backbone dynamics, we have obtained biochemical evidence of a co-folding mechanism of the Daxx histone-binding domain (HBD) with the H3.3/H4 histone dimer. Monomeric DAXX[HBD] undergoes extremely rapid H/DX, exhibiting the behavior of an unfolded protein. Upon binding to H3.3/H4, both Daxx and the H3.3/H4 subunits are globally stabilized, as measured by H/DX protection of several orders of magnitude. The H/DX rates in the ternary complex are matched at contact points between all three subunits as revealed by crystallography (Elsasser et al., 2012, in press), suggesting that they sample unfolded/folded states with the same kinetics throughout the complex. Importantly, Daxx binding stabilizes the helices of a H3.3/H4 complex in which point mutations disrupt stability in several of the histone fold helices in free H3.3/H4 complexes. In sum, we have found that DAXX stabilizes a pre-nucleosomal folded form of H3.3/H4 that differs from its final nucleosome form or its form free in solution. While the steps in the folding pathway we studied are certainly tied to the mechanism of chromatin assembly, so to is the massive destabilization we predict occurs to DAXX[HBD] upon releasing H3.3/H4 dimers during nucleosome assembly.