We have purified a minimal core human Ino80 complex from recombinant protein expressed
in insect cells. The complex comprises one subunit each of an N-terminally truncated Ino80,
actin, Arp4, Arp5, Arp8, Ies2 and Ies6, together with a single heterohexamer of the Tip49a
and Tip49b proteins. This core complex has nucleosome sliding activity that is similar to that
of endogenous human and yeast Ino80 complexes and is also inhibited by inositol
hexaphosphate (IP6). We show that IP6 is a non-competitive inhibitor that acts by blocking
the stimulatory effect of nucleosomes on the ATPase activity. The IP6 binding site is located
within the C-terminal region of the Ino80 subunit. We have also prepared complexes lacking
combinations of Ies2 and Arp5/Ies6 subunits that reveal regulation imposed by each of them
individually and synergistically that couples ATP hydrolysis to nucleosome sliding. This
coupling between Ies2 and Arp5/Ies6 can be overcome in a bypass mutation of the Arp5
subunit that is active in the absence of Ies2. These studies reveal several underlying
mechanisms for regulation of ATPase activity involving a complex interplay between these
protein subunits and IP6 that in turn controls nucleosome sliding.