HDX-MS reveals concealed conformations of ISWI during different stages of nucleosome sliding

Abstract

AbstractNucleosome spacing across the genome is regulated by the adenosine 5’-triphosphate (ATP)- dependent nucleosome sliding activity of Imitation Switch, ISWI. ISWI is believed to be auto-inhibited in the resting-state by the binding of its N-and C-terminal regulatory regions to its central ATPase-domain, attaining a “closed” conformation. To slide nucleosomes ISWI must i) transition to the state competent for nucleosome binding, ii) bind to nucleosome and iii) carry the ATP-dependent nucleosome sliding. The conformations attained by full-length ISWI (FL-ISWI) during the entire sliding process have remained inaccessible by the methods used so far. Using Hydrogen/Deuterium-exchange coupled to Mass-Spectrometry (HDX-MS), we monitored conformational dynamics of theDrosophilaFL-ISWI at all the stages of sliding process. HDX-MS data show that in the resting state, ISWI samples an ensemble of conformations showing varying levels of deuterium uptake in many regions including N-and C-terminal regulatory regions, suggesting ISWI intrinsically samples relatively “open-states”. In addition to substantiating previous nucleosome binding studies, HDX-MS reveals that during actual sliding-step, regions of ATPase-domain which bind to the nucleosomal DNA undergo major conformational change. The C-terminal HSS domain switches from the solvent protected stable state to a more dynamic state, implying several interactions established by ISWI with the nucleosome upon binding are relieved during sliding. In sum, this study provides mechanistic insights into how ISWI can switch from an auto-inhibited “closed-state” to an “open-state” competent for nucleosome binding, and reveals the conformation attained by ISWI during the actual nucleosome sliding step. We propose that, like ISWI, intrinsic dynamics may be involved in functioning of other Rec-like ATPase-domain containing protein families.