Stepwise Unfolding of a β Barrel Protein by the AAA+ ClpXP Protease

Abstract

In the AAA+ ClpXP protease, ClpX uses the energy of ATP binding and hydrolysis to unfold proteins before translocating them into ClpP for degradation. For proteins with C-terminal ssrA tags, ClpXP pulls on the tag to initiate unfolding and subsequent degradation. Here, we demonstrate that an initial step in ClpXP unfolding of the 11-stranded β barrel of superfolder GFP-ssrA involves extraction of the C-terminal β strand. The resulting 10-stranded intermediate is populated at low ATP concentrations, which stall ClpXP unfolding, and at high ATP concentrations, which support robust degradation. To determine if stable unfolding intermediates cause low-ATP stalling, we designed and characterized circularly permuted GFP variants. Notably, stalling was observed for a variant that formed a stable 10-stranded intermediate but not for one in which this intermediate was unstable. A stepwise degradation model in which the rates of terminal-strand extraction, strand refolding or recapture, and unfolding of the 10-stranded intermediate all depend on the rate of ATP hydrolysis by ClpXP accounts for the observed changes in degradation kinetics over a broad range of ATP concentrations. Our results suggest that the presence or absence of unfolding intermediates will play important roles in determining whether forced enzymatic unfolding requires a minimum rate of ATP hydrolysis.

RpoS proteolysis is controlled directly by ATP levels in Escherichia coli

“…3B). This nonlinear effect is similar to what has been observed with other stalling substrates (Martin et al 2008;Nager et al 2011). Thus, RpoS belonged to the category of proteins whose degradation is critically dependent on ATP hydrolysis rates.…”

“…3A). Even at 50 mM ATP, TitinV15P-ssrA degradation was robust and no detectable substrate remained after 16 h. However, a small subset of ClpXP substrates, including the MuA transposase tetrameter and some variants of GFP-ssrA, is not degraded in low ATP conditions (Martin et al 2008;Nager et al 2011; AH Abdelhakim and TA Baker, unpubl.). RpoS fell into this category of substrates, as degradation failed at 50 mM ATP, and after 16 h, nearly 90% of the protein remained (Fig.…”

Architecture and assembly of the archaeal Cdc48⋅20S proteasome

“…Proteins were concentrated by Amicon centrifugation devices (100-kDa cutoff; Millipore) and further purified by size-exclusion chromatography on a Superdex S-200 column (GE Healthcare) in 20 mM Hepes-KOH (pH 7.5), 50 mM NaCl, 1 mM DTT, and 1 mM EDTA. SF GFP-ssrA and Kaede-ssrA were expressed and purified as described (27,28). All proteins were frozen in liquid nitrogen and stored at −80°C.…”

Remodeling of a delivery complex allows ClpS-mediated degradation of N-degron substrates

“…This model also requires that ΔβClpS not be globally denatured and degraded by ClpAP, because it has been established that ClpS is not degraded during substrate delivery (19). There is precedent for this type of β strand extraction by AAA+ unfoldases; for example, we note that ClpXP initially extracts a terminal β strand from a sheet in GFP-ssrA without causing global unfolding (37,38). Moreover, under some conditions, the extracted β strand appears to slip from the pore of the AAA+ unfoldase, allowing refolding to native GFP (38).…”

“…There is precedent for this type of β strand extraction by AAA+ unfoldases; for example, we note that ClpXP initially extracts a terminal β strand from a sheet in GFP-ssrA without causing global unfolding (37,38). Moreover, under some conditions, the extracted β strand appears to slip from the pore of the AAA+ unfoldase, allowing refolding to native GFP (38). For the ClpSdelivery model, we suggest that following transfer of the N-degron, a slipping event could also allow ΔβClpS to refold and thus restore native ClpS.…”