The work presented here elucidates important new aspects of the molecular mechanism of ClpS delivery and ClpAP degradation of N-end-rule substrates. Our current view of these processes is shown in the model of , which begins with formation of a low-affinity ternary complex (LATC; ), proceeds to a high-affinity delivery complex (HADC; ), and ends with active substrate handoff from ClpS to ClpA (). As illustrated in , uncoupled sets of binary contacts between ClpS and the N-end-rule substrate and between ClpScore and the ClpA N domain stabilize the LATC. Formation of the HADC involves additional interactions mediated by junction residues of the NTE of ClpS, by His66 of ClpS, by the N-end residue of the substrate, and by the D1 ring of ClpA (). The ClpS•substrate portion of the complex is highly mobile in the LATC, because of the flexible tethering of the N domain to the D1 ring of ClpA, but is constrained in the HADC by additional contacts with the D1 ring.
Model for staged delivery of N-end-rule substrates
The properties of the LATC are based on previous studies of the interaction of ClpS with substrates or the N domain of ClpA. Our current work supports the existence of the HADC and defines many of its properties. For example, we find that degrons containing just the N-end residue and peptide bond enhance ClpS affinity for ClpA6
and are bound far more tightly by ClpS and ClpA6
together than by either individual protein. Moreover, high-affinity binding requires multiple regions of ClpS (including the junction region of the NTE and His66
, which contacts the N-end residue of the substrate), as well as the AAA+ body of ClpA and a suitably long linker between the ClpA D1 ring and N domain. Consistent with the models, we find that the mobility of the ClpS portion of complexes with ClpA is higher when the NTE is absent. Work presented here and previously (Hou et al., 2008
; Wang et al., 2008a
; Schuenemann et al., 2009) shows that mutation of His66
or deletion of the NTE severely compromises ClpS delivery of N-degron substrates to ClpAP, indicating that formation of the HADC is a critical step in substrate delivery.
It is not currently known what parts the AAA+ body of ClpA make contacts with the junction residues of the NTE or with His66 and the N-end residue in the HADC, but our studies set the stage for future experiments to define these interactions in greater molecular detail. FeBABE-cleavage experiments do show that residues near the center of the NTE can contact the D1 AAA+ ring of ClpA6, and given the size of ClpS, other contacts with the AAA+ body of ClpA6 would likely also be restricted to the D1 ring. For example, a residue in the D1 ring of ClpA could contact the His66 of ClpS and stabilize its interaction with the α-amino group of the N-degron, explaining the importance of all of these elements in stabilizing the HADC. Alternatively, the conformation of the His66 side chain could change in the HADC, allowing one set of D1 interactions with His66 and another set of interactions with the N-degron. Interestingly, a sufficiently long linker between the ClpA N domain and AAA+ ring is needed to allow formation of the ClpA contacts mediated by the N-end degron and ClpS binding pocket.
In addition to its role in delivery of N-degron substrates, ClpS binding prevents recognition and degradation of other types of substrates by ClpAP (Dougan et al., 2002
). In the absence of N-end-rule substrates, it would be counterproductive if ClpS bound ClpA too tightly as this would preclude degradation of other substrates. However, we find that ClpS binds ClpA6
~10-fold more tightly when N-degron substrates are present, providing an elegant solution to this problem. Substrate-dependent affinity enhancement would help to ensure the formation of a ClpAPS complex when N-end-rule substrates were available but also keep ClpAP largely free to perform other functions when these substrates were absent.
Our working model for substrate delivery culminates with engagement of the substrate N-degron by the ClpA pore (). A key feature of this model is binding of a portion of the ClpS NTE in the ClpA pore (), allowing the translocation/unfolding machinery to pull on ClpS and facilitate transfer of the N-degron from ClpS to ClpA (). Although aspects of the transfer model are speculative, it accounts for many experimental observations. For example, we found that a truncated ClpS variant beginning at NTE-residue 13 mediated efficient substrate degradation, whereas deleting one additional NTE residue dramatically reduced delivery. The presence of the extra residue could allow the NTE to reach a binding site in the ClpA6
pore that was critical for initiating substrate delivery. Indeed, our FeBABE-cleavage results suggest that this central region of the NTE could contact the pore of the ClpA D1 ring. Engagement of the NTE by the ClpA pore is also supported by our finding that appending the ClpS NTE to GFP, a protein which is not normally degraded, results in efficient ClpAP degradation. Despite NTE engagement, our results also show that the folded portion of ClpS resists ClpAP degradation. In combination, these results account for our observation that delivery-competent NTE truncations result in lower ClpA ATPase rates than delivery-incompetent truncations. For example, AAA+ unfoldases hydrolyze ATP more slowly during attempts to unfold a protein (Kenniston et al., 2003
; Wolfgang et al., 2009
), and the lower ATPase rates seen using delivery-competent NTE truncations are therefore consistent with failed ClpA attempts to unfold ClpS.
How could ClpA tugging on ClpS facilitate handoff of N-end rule substrates? Given that the NTE is distant from the ClpS substrate-binding pocket, an attractive model is that translocation-mediated pulling on the NTE deforms ClpScore
, facilitating transfer of the N-end degron to a site in the ClpA pore (). This model requires independent recognition of the N-degron by ClpA, which is supported by the observation that ClpAP alone can recognize and degrade N-end-rule substrates, albeit with relatively low KM
’s compared to values obtained with ClpS (Wang et al. 2007
). Moreover, in resisting unfolding, ClpS could slip from the grasp of ClpA, as observed for other difficult-to-unfold proteins (Kenniston et al., 2005
), clearing the pore as a prelude to substrate degradation (). Experiments with the related ClpXP enzyme also reveal that multiple polypeptide chains can simultaneously occupy the pore (Burton et al., 2001
; Bolon et al., 2004
There are parallels between the model and the delivery of ssrA-tagged substrates to the ClpXP protease by the SspB adaptor. For example, one region of SspB binds the N domain of ClpX, another part of SspB binds to a segment of the ssrA degron, a different part of this degron binds to the ClpX pore, and each binary interaction is substantially weaker than the overall ternary interaction (Levchenko et al., 2000
; Wah et al. 2003
; Bolon et al., 2004
; Martin et al., 2008
). Because the ssrA tag of the substrate is positioned in the pore of the ClpX AAA+ ring in the high-affinity complex, ATP-fueled translocation allows tag contacts with the adaptor to be broken at the same time that degradation is initiated.
Assembly of increasingly stable macromolecular complexes frequently drives biological recognition. This mechanism provides directionality by proceeding downhill to a thermodynamic minimum but also results in an energy well from which spontaneous escape is difficult, creating a problem if the high-affinity complex is not the final product. For example, recombination catalyzed by MuA transposase is driven by increasingly stable protein-DNA complexes, which eventually must be disassembled in an ATP-dependent process by ClpX (Burton and Baker, 2005
). As shown here and previously, adaptor-mediated delivery of substrates to AAA+ proteases also involves a progression from low-affinity to high-affinity complexes. This type of assembly has several advantages. From a kinetic perspective, splitting the overall pathway into discrete bimolecular and unimolecular steps speeds assembly. For example, ClpS with bound N-degron substrate could initially dock with any of the six N domains of ClpA6
. Moreover, these N domains are highly mobile, further increasing the chances for productive collisions. Subsequent assembly steps would then be unimolecular, allowing the use of relatively weak interactions to position the substrate/adaptor near the translocation machinery of ClpA6
We propose that adaptors for AAA+ proteases will fall into two general categories. In one category, exemplified by SspB, enzymatic pulling on the substrate disrupts the HADC and initiates degradation. In the second category, exemplified by ClpS, enzymatic tugging on the adaptor destabilizes the HADC, allowing substrate transfer and degradation. Many adaptors that function by a ClpS-type mechanism are likely to be degradation resistant. For example, Rad23 facilitates interactions between ubiquitinated substrates and the proteasome and is refractory to degradation (Heessen et al., 2005
; Fishbain et al., 2010). However, a ClpS-type mechanism could also work if the adaptor were degraded. Indeed, the MecA adaptor is degraded by ClpCP during substrate delivery (Turgay et al., 1998