Division of labor between the pore-1 loops of the D1 and D2 AAA+ rings coordinates substrate selectivity of the ClpAP protease

Abstract

ClpAP, an ATP-dependent protease consisting of ClpA, a double-ring hexameric unfoldase of the ATPases associated with diverse cellular activities superfamily, and the ClpP peptidase, degrades damaged and unneeded proteins to support cellular proteostasis. ClpA recognizes many protein substrates directly, but it can also be regulated by an adapter, ClpS, that modifies ClpA’s substrate profile toward N-degron substrates. Conserved tyrosines in the 12 pore-1 loops lining the central channel of the stacked D1 and D2 rings of ClpA are critical for degradation, but the roles of these residues in individual steps during direct or adapter-mediated degradation are poorly understood. Using engineered ClpA hexamers with zero, three, or six pore-1 loop mutations in each ATPases associated with diverse cellular activities superfamily ring, we found that active D1 pore loops initiate productive engagement of substrates, whereas active D2 pore loops are most important for mediating the robust unfolding of stable native substrates. In complex with ClpS, active D1 pore loops are required to form a high affinity ClpA•ClpS•substrate complex, but D2 pore loops are needed to “tug on” and remodel ClpS to transfer the N-degron substrate to ClpA. Overall, we find that the pore-1 loop tyrosines in D1 are critical for direct substrate engagement, whereas ClpS-mediated substrate delivery requires unique contributions from both the D1 and D2 pore loops. In conclusion, our study illustrates how pore loop engagement, substrate capture, and powering of the unfolding/translocation steps are distributed between the two rings of ClpA, illuminating new mechanistic features that may be common to double-ring protein unfolding machines.

AAA+ protease-adaptor structures reveal altered conformations and ring specialization

“…Moreover, the portion of the ClpS NTE in the D1 ring of class- III structures is bound in the same fashion as our class- I and class- II structures. This structural snapshot of ‘divided’ NTE engagement between the D1 and D2 pore-1 loops reinforces biophysical and biochemical experiments that reveal a division of labor between the two AAA+ modules of ClpA (Kress et al, 2009; Kotamarthi et al, 2020; Zuromski et al, 2021). Multiple studies of other double-ring remodeling/unfoldase enzymes, including ClpB, Hsp104, ClpC, Cdc48/p97/VCP, and the ribosomal assembly factor Rix7, report the separation of substrate binding/recognition functions in one ring from the role of the second ring as the principal motor performing mechanical work (Hattendorf and Lindquist, 2002; Mogk et al, 2003; Wang et al, 2011; Doyle et al, 2012; Bodnar and Rapoport, 2017a, 2017b; Lo et al, 2019).…”

“…526–531). Pore-1 loops of AAA+ unfoldases and protein-remodeling machines contain a key, conserved aromatic side chain (usually tyrosine; underlined in K Y R and G Y VG) that contacts the substrate polypeptide in the channel and functions in the binding, unfolding, and translocation of target proteins (Schlieker et al, 2004; Weibezahn et al, 2004; Hinnerwisch et al, 2005a; Martin et al, 2008; Doyle et al, 2012; Iosefson et al, 2015; Lopez et al, 2020; Zuromski et al, 2021). The ClpS NTE was bound by many K Y R and G Y VG pore-1 loops and also by the D1 pore-2 loops of ClpA.…”

“…Despite the degron-like interactions of the NTE with ClpA, ClpS is not degraded (Dougan et al, 2002; Román-Hernández et al, 2011). Interestingly, mutation of Pro 24 -Pro 25 to Ala 24 -Ala 25 near the ClpS NTE-core junction generates a ClpS variant that can be degraded by ClpAP (Rivera-Rivera, 2015; Zuromski et al, 2021). In our structures, Pro 24 -Pro 25 binds near the top of the ClpA channel, and the ClpS core domain is flexibly positioned directly above the pore.…”

“…More broadly, pore-loop tucking may be used during the process of enzyme pausing and/or unloading by ClpA and other AAA+ unfoldase motors. For example, the ClpA D1 ring functions as a ‘back up’ motor to prevent pausing when the principal D2-ring motor fails (Kotamarthi et al, 2020; Zuromski et al, 2021). Transiently breaking contacts with the polypeptide via pore-loop tucking in only the D2 ring would allow the weaker D1 ring to continue unfolding/translocation without working against the stalled D2 motor.…”

“…The D2 ring, a member of the HCLR AAA+ clade, is the principal ATPase motor responsible for unfolding and translocating substrates, including proteins with high thermodynamic stabilities (Kress et al, 2009;Kotamarthi et al, 2020;Zuromski et al, 2020). In contrast, the D1 ring, a classic AAA+ clade member, assists the D2 ring as an auxiliary motor, improves enzyme processivity, and plays a major role in substrate recognition (Kress et al, 2009;Kotamarthi et al, 2020;Zuromski et al, 2020Zuromski et al, , 2021. ClpS differentially regulates the activities of the D1 and D2 rings (Kress et al, 2009;Zuromski et al, 2021) via interactions of its NTE that we characterize here.…”