Characterizing Interactions Between Nap1 and its Binding Partners




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Nucleosome Assembly Proteins (Naps) are a well-known family of proteins that regulate the assembly and disassembly of the nucleosome. They act as histone chaperones by facilitating the transport of histones from the cytoplasm to the nucleus as well as regulating their deposition onto nucleosomes. Naps play an important role in regulating chromatin structure by not only interacting with histones but also with other transcriptional machinery. In humans, Naps are involved in cellcycle regulation and their mutations are implicated in several cancers. The proteins in the Nap family share a highly conserved core domain but have less conserved, disordered N- and Cterminal tails. Nap1 is a well-studied member of the Nap-family of proteins as well as the main focus of the research presented in this dissertation. Using comprehensive biochemical and structural characterization, we investigate the molecular details of interactions between Nap1 and histones H2A-H2B and H3-H4, as well as between Nap1 and the Taz2 domain of transcriptional co-activator p300. We report the crystal structure of Caenorhabditis elegans Nap1 (CeNap1). CeNap1 naturally lacks an N-terminal tail and has a very short C-terminal tail. Biochemical characterization of CeNap1 and comparison to full-length and tail-less Saccharomyces cerevisiae Nap1 (ScNap1) reveals a role of the acidic Nap-tails in self- association, histone binding and competition with DNA for H2A-H2B binding. The presence of the tails influences the stoichiometry of the complexes formed between ScNap1 and H2A-H2B to be either 2:1 or 2:2, with only a small population of higher order oligomers. We also show evidence that Nap1 has a different binding mode with H2A-H2B than with H3-H4. We also report the molecular details of the interaction between Homo sapiens Nap1L1 (referred to as Nap1) with the Taz2 domain of Homo sapiens p300. Transcriptional co-activator p300 is a histone acetyltransferase that acetylates histones and prepares the chromatin landscape for transcription. We show that Nap1 interacts with Taz2 at a 2:2 stoichiometry, and the interactions between them are primarily ionic in nature. The Nap1-Taz2 interaction is paralog-specific, as other members of the Nap-family, such as Taf1β do not interact with Taz2. Using the hybrid structural approach, we performed biochemical analyses and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to narrow down and identify a Nap1 binding site on Taz2. We also study the formation of ternary complexes between Nap1, Taz2 and histones H2A-H2B as well as with H1. While there is no ternary complex formed between Nap1, Taz2 and H2A-H2B, we see the formation of a ternary complex between Nap1, Taz2 and H1. Competition assays with Nap1 and DNA show that Nap1 can interact with H2A-H2B bound to DNA in the absence of Taz2, but in the presence of Taz2, Nap1 is unable to interact with DNA-bound H2A-H2B. These results elucidate a previously uncharacterized protein-protein interaction and lays foundation for further research on the Nap1-p300 interaction and its effect on chromatin structure and function.



Stoichiometry, Chromatin, Nucleoproteins, Histones, Mass spectrometry, Light -- Scattering


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