Abstract
SummaryAAA+ proteins form asymmetric hexameric rings that hydrolyze ATP and thread substrate proteins through a central channel via mobile substrate-binding pore loops. Understanding how ATPase and threading activities are regulated and intertwined is key to understanding the AAA+ protein mechanism. We studied the disaggregase ClpB, which contains tandem ATPase domains (AAA1, AAA2) and shifts between low and high ATPase and threading activities. Coiled-coil M-domains repress ClpB activity by encircling the AAA1 ring. Here, we determine the mechanism of ClpB activation by comparing ATPase mechanisms and cryo-EM structures of ClpB wild-type and a constitutively active ClpB M-domain mutant. We show that ClpB activation reduces ATPase cooperativity and induces a sequential mode of ATP hydrolysis in the AAA2 ring, the main ATPase motor. AAA1 and AAA2 rings do not work synchronously but in alternating cycles. This ensures high grip, enabling substrate threading via a processive, rope-climbing mechanism.
Highlights
a single layer of ATPase (AAA)+ proteins couple energy from ATP hydrolysis to mechanical work, which is typically a directional threading activity linked to force generation for unwinding DNA or RNA, protein complex disassembly, protein unfolding, or protein disaggregation
We studied the disaggregase ClpB, which contains tandem ATPase domains (AAA1, AAA2) and shifts between low and high ATPase and threading activities
We determine the mechanism of ClpB activation by comparing ATPase mechanisms and cryoEM structures of ClpB wild-type and a constitutively active ClpB M-domain mutant
Summary
AAA+ proteins couple energy from ATP hydrolysis to mechanical work, which is typically a directional threading activity linked to force generation for unwinding DNA or RNA, protein complex disassembly, protein unfolding, or protein disaggregation They are usually hexamers, which can consist of a single layer of ATPase (AAA) domains or two tiers of tandem ATPase domains that form a ring-shaped oligomer with a central pore. Cycling of AAA domains between different activity states was recently observed directly, in case of the AAA+ rings of the homohexameric archaeal PAN (Majumder et al 2019) and heterohexameric eukaryotic 26S proteasome regulatory subunit (de la Pena et al, 2018; Dong et al, 2019) It is unclear how ATPase and threading activities are coordinated in double-ring AAA+ hexamers composed of tandem AAA domains. How do the two AAA rings communicate, and what is the consequence for the threading mechanism? These questions relate directly to the reasons why some AAA+ proteins have two ATPase rings
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