Dynamic structural states of ClpB involved in its disaggregation function

Takayuki Uchihashi,Susumu Uchiyama,Hiroki Watanabe,Yosuke Nakazaki,Ryota Iino,Takashi Yamasaki,Kentaro Ishii,Takahiro Maruno,Toshio Ando,Yo-Hei Watanabe,Chihong Song,Kazuyoshi Murata

doi:10.1038/s41467-018-04587-w

Abstract

The ATP-dependent bacterial protein disaggregation machine, ClpB belonging to the AAA+ superfamily, refolds toxic protein aggregates into the native state in cooperation with the cognate Hsp70 partner. The ring-shaped hexamers of ClpB unfold and thread its protein substrate through the central pore. However, their function-related structural dynamics has remained elusive. Here we directly visualize ClpB using high-speed atomic force microscopy (HS-AFM) to gain a mechanistic insight into its disaggregation function. The HS-AFM movies demonstrate massive conformational changes of the hexameric ring during ATP hydrolysis, from a round ring to a spiral and even to a pair of twisted half-spirals. HS-AFM observations of Walker-motif mutants unveil crucial roles of ATP binding and hydrolysis in the oligomer formation and structural dynamics. Furthermore, repressed and hyperactive mutations result in significantly different oligomeric forms. These results provide a comprehensive view for the ATP-driven oligomeric-state transitions that enable ClpB to disentangle protein aggregates.

Highlights

The superfamily of ATPases associated with diverse cellular activities (AAA+) includes a wide variety of members that are involved in membrane fusion, DNA replication, protein degradation, and others[1,2]
The orientation was further confirmed by binding of streptavidin to the top surface of ΔN-thermophilus ClpB (TClpB) biotinylated at the N-terminal side (ΔN-TClpB-Q142C-biotin) (Supplementary Figure 1b−d)
Since the N-terminal deletion does not have a major impact on the disaggregation activity of TClpB11–13 (Supplementary Figure 2), we hereafter use the ΔN-TClpB as wild-type

Summary

Introduction

The superfamily of ATPases associated with diverse cellular activities (AAA+) includes a wide variety of members that are involved in membrane fusion, DNA replication, protein degradation, and others[1,2]. The N-terminal domain is highly mobile and participates in substrate binding but is not essential for the disaggregation activity[11,12,13] Both AAA1 and AAA2 bind and hydrolyze ATP and constitute a core of the hexameric ring[10,14,15]. The AAA1 and AAA2 modules regulate each other in a complex manner as regards ATP-binding and hydrolysis[21,22,23,24,25], and the cooperative ATPase cycle causes structural changes to drive the threading of aggregated proteins[12,17,18], the threading of denatured proteins does not require ATP hydrolysis[26]. Understanding the dynamic nature of oligomeric structure is essential for elucidating the disaggregation mechanism of ClpB

Methods

Results

Conclusion