posted on 2021-03-19, 17:33authored byMarija Iljina, Hisham Mazal, Pierre Goloubinoff, Inbal Riven, Gilad Haran
ClpB is a tightly
regulated AAA+ disaggregation machine. Each ClpB
molecule is composed of a flexibly attached N-terminal domain (NTD),
an essential middle domain (MD) that activates the machine by tilting,
and two nucleotide-binding domains. The NTD is not well-characterized
structurally and is commonly considered to serve as a dispensable
substrate-binding domain. Here, we use single-molecule FRET spectroscopy
to directly monitor the real-time dynamics of ClpB’s NTD and
reveal its unexpected autoinhibitory function. We find that the NTD
fluctuates on the microsecond time scale, and these dynamics result
in steric hindrance that limits the conformational space of the MD
to restrict its tilting. This leads to significantly inhibited ATPase
and disaggregation activities of ClpB, an effect that is alleviated
upon binding of a substrate protein or the cochaperone DnaK. This
entropic inhibition mechanism, which is mediated by ultrafast motions
of the NTD and is not dependent on any strong interactions, might
be common in related ATP-dependent proteases and other multidomain
proteins to ensure their fast and reversible activation.