Structural biochemistry of the INO80 chromatin remodeler reveals an unexpected function of its two subunits Arp4 and Arp8

The INO80 complex is a chromatin remodeler involved in diverse
nuclear processes like transcriptional regulation, replication fork
progression, checkpoint regulation and DNA double strand break
repair. In the yeast S. cerevisiae the complex consists of 15
subunits with a total molecular mass of about 1.2 MDa. Knowledge
about the atomic structure and molecular architecture of the entire
complex is scarce. Similarly, an understanding for the roles of the
individual subunits of the complex is mostly lacking. Especially
the function of actin and the actin related proteins Arp4 and Arp8
which are found as monomeric components of INO80 and other
chromatin remodelers is poorly understood. The goal of this study
was to elucidate the functional architecture of the INO80 complex
by using a hybrid methods approach. Different structural techniques
such as X-ray crystallography, small angle X-ray scattering (SAXS)
and electron microscopy (EM) were combined to achieve this goal.
Additionally, various functional assays to study the biochemical
properties of the actin related proteins and their interaction with
actin were employed. In a set of primary experiments expression and
purification protocols for seven individual INO80 components,
namely Arp4, Arp5, Arp8, Ies4, Ies5, Ies6 and Nhp10 could be
established. Additionally, four subcomplexes containing more than
one protein, namely Rvb1-Rvb2, Nhp10-Ies5, Nhp10-Ies5-Ies3 and
Arp5-Ies6 were purified. Thereby two previously unknown
interactions between the INO80 subunits Nhp10 and Ies5, as well as
Arp5 and Ies6 could be identified. Subsequently, the newly
identified complexes of Nhp10-Ies5-Ies3 and Arp5-Ies6 were studied
with SAXS to obtain low resolution solution structures of both. On
top of that the entire INO80 complex was purified endogenously from
S. cerevisiae and studied by EM. Unfortunately, a three dimensional
reconstruction of the remodeler could not be created.
Crystallization attempts on all purified INO80 components were
successful for the complex of Rvb1-Rvb2 and the actin related
protein Arp4. Whereas the structure of Rvb1-Rvb2 could not be
solved due to limited diffraction an atomic structure of ATP bound
Arp4 at 3.4 Å resolution was obtained. Remarkably, Arp4 does not
form filaments despite its high similarity to conventional actin.
The lack of polymerization is confirmed by the SAXS structure of
isolated Arp4 which indicates it to be monomeric and can be nicely
explained on the basis of the crystal structure. Several loop
insertions and deletions at positions which are crucial for contact
formation within the actin filament, especially at the pointed end
of the molecule, prevent Arp4 to engage in filament like
interactions. Furthermore, the crystal structure of Arp4 reveals an
ATP molecule to be constitutively bound to the protein. The lack of
ATPase activity of Arp4 in contrast to actin can be explained with
the help of the crystal structure as well. Several residues in the
nucleotide clamping loops of Arp4 are divergent from actin leading
to a tighter closure and better shielding of the phosphate moieties
of the bound ATP from the environment. Most interestingly, Arp4
dramatically influences actin polymerization kinetics. Different
fluorescence assays and in vitro TIRF microscopy were used to show
that Arp4 is able to inhibit actin polymerization and to
depolymerize actin filaments most likely by complex formation with
monomeric ADP-actin via the barbed end. Its ability to inhibit
actin filament nucleation without sequestering actin while still
allowing ADP to ATP exchange within actin resembles the actin
binding protein profilin. Arp8 was confirmed by SAXS measurements
to be monomeric as well. It is able to sequester actin monomers and
to slowly depolymerize actin filaments. Consistent with the
formation of a discrete Arp4-Arp8-actin complex within the INO80
remodeler the effects of Arp4 on actin polymerization are further
stimulated by Arp8. As both proteins reciprocally enhance their
individual effects on actin it is likely that they help to maintain
actin in a defined monomeric state within the INO80 chromatin
remodeler. The data further suggest a possible assembly between
actin and Arp4 via their barbed ends and a model how the
Arp4-Arp8-actin complex is integrated into the INO80 chromatin
remodeler. Taken together, the findings represent a remarkable
advancement in the understanding of nuclear actin related proteins
and nuclear actin biochemistry in general. Most excitingly, they
indicate a link between chromatin remodeling and nuclear actin
dynamics possibly giving chromatin remodeling complexes a role in
the actin mediated large scale movement of chromatin.