To explain why degradation of GFP-ssrA ceases at low rates of ATP hydrolysis, Martin et al. proposed that ClpXP extracts the ssrA-tagged terminal strand from the GFP β barrel but the intermediate refolds before further unfolding and degradation can occur.7
Our results strongly support this stepwise model for ClpXP unfolding of GFP, which leads to futile cycles of strand extraction and refolding at low concentrations of ATP. For example, we designed a split variant in which the ssrA-tagged 11th
strand was non-covalently bound to the remaining GFP structure and found that ClpXP extracted this strand without denaturing the rest of the protein. Moreover, the rate of strand extraction in this experiment was fast enough to account for the rate of GFP proteolysis, as expected for an on-pathway step in the degradation reaction. Notably, when ClpXP extracted the 11th
strands of another split GFP variant, the remaining portions of GFP did denature. Thus, GFP lacking its C-terminal 11th
strand is reasonably stable but subsequent extraction of the adjacent 10th
strand results in unfolding. GFP-ssrA lacking its C-terminal strand loses 400-nm fluorescence, which depends upon Glu222
in strand 11, but maintains normal 467-nm fluorescence. Importantly, we found that ClpXP produces GFP species with a reduced ratio of 400/467 fluorescence both under low-ATP stalling conditions and high-ATP degradation conditions, as predicted if a strand-extracted intermediate is populated under these conditions.
We designed a circularly permuted variant of superfolder GFP (cp7-SF
GFP-ssrA), which should also form a stable 10-stranded intermediate after extraction of its C-terminal strand.29
Like non-permuted SF
GFP-ssrA, this variant was resistant to ClpXP degradation at low ATP-hydrolysis rates. By contrast, another circularly permuted variant (cp6-SF
GFP-ssrA) unfolded following extraction of its C-terminal strand and did not stall ClpXP. Thus, the ability to resist ClpXP degradation at low ATP concentrations correlates with the ability of the substrate to form a stable unfolding intermediate, even though stalling did not correlate with the global stabilities of different substrates. At saturating ATP, ClpXP degraded stalling GFP variants more slowly than the non-stalling variant. Thus, the presence of a stable unfolding intermediate appears to slow normal degradation. We note, however, that Vmax
for ClpXP degradation of SF
GFP-ssrA was less than half the value of the other stalling substrates (), suggesting that changes in the rates of strand extraction and/or in the rates of unfolding of the 10-stranded barrel also play roles in determining the overall rates of degradation of these proteins.
ClpXP appears to unfold proteins using a power-stroke mechanism.15–17
Specifically, each cycle of ATP hydrolysis by ClpX is thought to result in an attempt to translocate a segment of the substrate polypeptide, thereby pulling the native protein against the narrow axial channel and creating a transient unfolding force. For stable substrates, like the titinI27
domain, hundreds of cycles of ATP hydrolysis can be required before denaturation becomes statistically probable.15
This result suggests that a power stroke must coincide with a stochastic decrease in protein stability to successfully extract the terminal structural element of the substrate. For titinI27
, this initial ClpXP-mediated unfolding event appears to cause global denaturation.7
Studies of ClpXP unfolding of a multidomain filamin substrate assayed by optical-trapping nanometry also support this model.16
For example, in different single-molecule experiments, the dwell time before unfolding of a specific filamin domain varied from a few to more than 100 s, with the latter time being sufficient to hydrolyze several hundred ATP molecules. However, once unfolding of a filamin domain commenced, highly cooperative denaturation was typically complete in less than 1 ms. Subsequent ATPase cycles then resulted in translocation of the unfolded protein in steps of 5–8 amino acids at an average rate of ~30 residues s−1
Our current view of the mechanism by which ClpXP unfolds GFP-ssrA begins with repeated enzymatic tugging on the 11th or C-terminal strand that is attached to the ssrA tag. During this process, enzymatic pulling will occasionally partially or completely dislodge the terminal strand, leaving a native 10-stranded barrel. Multiple cycles of ATP hydrolysis are then required to finish translocation of the extracted strand and preceding turn (~18 residues) and to unfold the remaining 10-stranded structure to allow degradation. The time required for completion of these events will increase as the ATPase rate decreases. Refolding of the extracted strand before unfolding of the 10-stranded barrel would restore the original substrate, necessitating renewed attempts to begin denaturation of the 11-stranded barrel. Strand refolding probably requires slipping of the substrate from the grip of ClpXP and is therefore more likely to occur at low ATPase rates when a higher fraction of enzymes are in an ATP-free state. Indeed, good fits of the observed ATP dependence of GFP degradation by ClpXP required a strand-refolding rate proportional to 1 minus the fractional ATP-hydrolysis rate.
The protein-unfolding activities of different AAA+ proteases display considerable variation.31
For example, the HslUV and FtsH proteases fail to degrade GFP proteins with suitable C-terminal recognition tags, whereas Lon degrades such substrates about 200-fold more slowly than ClpXP or ClpAP.32–36
For the proteases that degrade these GFP variants poorly, it is presently unclear if these AAA+ enzymes fail to dislodge the C-terminal strand or if they fail in a subsequent step in unfolding and degradation. Our results suggest that this question could be resolved by assaying 400-nm and 467-nm fluorescence of appropriate GFP variants during unfolding attempts by Lon, HslUV, and FtsH. Moreover, preliminary experiments suggest that appropriately tagged versions of some of our circularly permuted GFP proteins will be useful model substrates for Lon and HslUV, allowing convenient fluorescence-based assays of the unfolding and degradation activities of these AAA+ proteases. We note that ClpXP extraction of the terminal elements of split proteins also provides a powerful new tool with which to investigate the sequence determinants of protein structure and function. GFP-fusion proteins are commonly used to study protein localization and turnover in vivo
, but interpretations can be complicated if partial degradation generates free GFP. This problem could potentially be overcome by fusing proteins to a circularly permutated GFP that can be completely degraded.