Chasing tails: Cathepsin-L improves structural analysis of histones by HX-MS

ABSTRACT: The N-terminal regions of histone proteins (histone tails) are dynamic elements that protrude from the nucleosome and are involved in many aspects of chromatin organization. Their epigenetic role is well-established, and post-translational modifications present on these regions contribute to transcriptional regulation. Considering their biological significance, relatively few structural details have been established for histone tails, mainly due to their inherently disordered nature. While hydrogen/deuterium exchange mass spectrometry (HX-MS) is well-suited for the analysis of dynamic structures, it has seldom been employed in this context, presumably due to the poor N-terminal coverage provided by pepsin, the dominant protease in HX-MS studies. Inspired from histone-clipping events, we profiled the activity of Cathepsin-L under HX-MS quench conditions and characterized its specificity employing the four core histones (H2A, H2B, H3 and H4). Cathepsin-L demonstrated cleavage patterns that were substrate- and pH-dependent and showed optimum activity in a slightly reducing and non-denaturing environment. Cathepsin-L generated overlapping N-terminal peptides about 20 amino acids long for H2A, H3 and H4 proving its suitability for the analysis of histone tails dynamics. We developed a comprehensive HX-MS method in combination with pepsin and obtained full sequence coverage for all histones. We employed our method to analyze histones H3 and H4. We observe rapid deuterium exchange of the N-terminal tails and cooperative unfolding (EX1 kinetics) in the histone-fold domains of histone monomers in-solution. When in a mononucleosome, we find evidence for inter- or intramolecular interactions within the H4 tail but not for H3.1. Further, EX1 kinetics observed in the respective monomers are absent when in the mononucleosome, indicating stabilization of the histone-folds. The data obtained here may serve as a baseline towards the comparison of nucleosome dynamics harboring different histone variants or post-translational modifications. Overall, this novel strategy opens new avenues for investigating the dynamic properties of histones and nucleosomes that are not apparent from the crystal structures, providing insights into the structural basis of the histone code.

INSTRUMENT(S): Xevo G2-S QTof, Q Exactive

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Jacob D. Jaffe  

PROVIDER: MSV000083315 | MassIVE | Tue Jan 15 08:59:00 GMT 2019

REPOSITORIES: MassIVE