Structure of an actin-related subcomplex of the SWI/SNF chromatin remodeler

Abstract

The packaging of DNA into nucleosomal structures limits access for templated processes such as transcription and DNA repair. The repositioning or ejection of nucleosomes is therefore critically important for regulated events, including gene expression. This activity is provided by chromatin remodeling complexes, or remodelers, which are typically large, multisubunit complexes that use an ATPase subunit to translocate the DNA. Many remodelers contain pairs or multimers of actin-related proteins (ARPs) that contact the helicase-SANT-associated (HSA) domain within the catalytic ATPase subunit and are thought to regulate ATPase activity. Here, we determined the structure of a four-protein subcomplex within the SWI/SNF remodeler that comprises the Snf2 HSA domain, Arp7, Arp9, and repressor of Ty1 transposition, gene 102 (Rtt102). Surprisingly, unlike characterized actin-actin associations, the two ARPs pack like spoons and straddle the HSA domain, which forms a 92-Å-long helix. The ARP-HSA interactions are reminiscent of contacts between actin and many binding partners and are quite different from those in the Arp2/3 complex. Rtt102 wraps around one side of the complex in a highly extended conformation that contacts both ARPs and therefore stabilizes the complex, yet functions to reduce by ∼2.4-fold the remodeling and ATPase activity of complexes containing the Snf2 ATPase domain. Thus, our structure provides a foundation for developing models of remodeler function, including mechanisms of coupling between ARPs and the ATPase translocation activity.

Fig. 1.

Structure of Arp7-Arp9-Snf2^HSA-Rtt102. (A) Surface representation. Viewed from the side that binds Rtt102 (rainbow ribbon, disordered residues dotted). (B) Same as A, viewed from below with ordered segments of Rtt102 shown as a green surface. (C) Schematic of actin/ARP fold showing chain trace and domain location viewed from the front, which corresponds to the left side of A for both Arp7 and Arp9. (D) Actin (PDB code 1YAG) (29), shown as a ribbon representation, rainbow-colored N (blue) to C (red), with ATP shown as spheres, (E) Arp7 and (F) Arp9 in the same style and orientation as D. The Arp9 loop that was shortened to facilitate crystallization is indicated with an asterisk.

Fig. 2.

Role of Rtt102. (A) Rtt102 overexpression partially suppresses the temperature-sensitive phenotype of a C-terminal truncation of Arp7. A strain (YBC1534) with an Arp7 derivative lacking a small portion of the C terminus of Arp7 (arp7ΔC2) is temperature sensitive compared with a WT strain (YBC1533) (33). Strains were transformed either with an empty vector that is maintained at high copy (pRS423) or with pRS423 expressing RTT102. For each assay, two independent transformants were spotted onto selective plates and incubated at the temperatures indicated for either 2 d (Left) or 4 d (Right). (B) Rtt102 attenuates nucleosome remodeling. Pure complexes of the core ARP/ATPase subcomplex of RSC [Sth1(301-1097) and full-length Arp7 and Arp9], containing or lacking Rtt102, were prepared and quantified as described in the Methods section. Remodeling reactions (Methods) measure the exposure by the remodeler of a restriction enzyme site (AluI), which initially resides within a mononucleosome (174 bp). Cleavage was quantified after DNA extraction and gel electrophoresis. The Cy3-labeled DNA was quantified by scanning on a Typhoon Trio (GE). A representative experiment of two replicates is shown.

Fig. 3.

ARP/actin interactions. (A) Schematic representation of actin/ARP domains within actin filaments (orange) (31). (B) Domain organization within the Arp2/3 complex (green) (19). Arp2 domains 1 and 2 are not visible in the Arp2/3 crystal structure but are shown here in positions inferred from the overall actin fold. (C) Arp7–Arp9 domain organization. Note that residues from Arp7 domain 4 interact with Arp9 residues forming an extended loop from domain 3, although this interaction is not apparent in the stylized representation that depicts spheres centered on domains. (D) Front view of Arp7, with surface contacting Arp9 colored purple. (E) Back view of Arp9, with surface contacting Arp7 colored yellow.

Fig. 4.

ARP–HSA interactions. Interactions of Arp7 and Arp9 with the Snf2 HSA domain showing prominent interacting side chains from the HSA helix. Structures were aligned on the entire actin/ARP molecule. The helix direction is indicated with an arrow that points in the N to C direction of the helix. Equivalent views are shown for the actin complex with gelsolin (PDB code 1EQY) (43) and with ciboulet (PDB code 1SQK) (44).