Structural and Biochemical Studies of Multiple Importin-Histone Interactions




Soniat, Michael Maurice, II

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Multiple Importins can bind the N-terminal tails of histones H3 and H4, and import them into the nucleus to be assembled into the nucleosomes. However, it is not known what sequence elements in the histone tails are recognized by each of the Importins. Through structural and quantitative biochemical analysis, I identified binding determinants in the N-terminal tails of histones H3 and H4 for each of seven different human Importins (Impα, Impβ, Kapβ2, Imp4, Imp5, Imp7, Imp9). Crystal structure of the H3 tail bound to Kapβ2 identified H3 tail residues 11-27 as the important binding element, which resembles a PY-NLS that is missing the canonical proline-tyrosine motif. This same N-terminal basic segment of H3 is also important for binding Impβ, Imp4, Imp5, Imp7, Imp9, and Impα. In addition, a C-terminal IK-NLS-like motif at residues 35-40 of H3 is also used to bind Imp5, Imp7, Imp9 and Impα. Interactions of the H4 tail with the same Importins show a similar trend of relative affinities as the H3 tail, though at least 10-fold weaker. Similar to the H3 tail, the H4 tail also uses one or two basic regions to bind the Importins. I also studied the effects of histone tail acetylation on Importin-histone interactions and showed that acetylation of Lys14 of the H3 tail impairs binding to all six Importins and Impα while acetylation of Lys18 of H3 tail and acetylation of Lys5 and Lys12 of the H4 tail had only mild effects on binding to the Importins. Lastly, I studied Importin binding to the H3/H4 dimer and showed that only one Importin molecule binds each H3/H4 dimer. Furthermore, the Importin-binding trend with the H3/H4 dimer is very different than with the N-terminal tails alone suggesting additional interactions with the histone fold domains of H3 and H4. Overall, I have mapped Importin-binding determinants for the H3 and H4 and revealed acetylation effects on Importin-histone binding.

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